(1995). "A CHALLENGE TO CHYMOSIN." Dairy Industries International 60(2): 27-27. Addeo, F., G. Garro, et al. (1995). "GEL-ELECTROPHORESIS AND IMMUNOBLOTTING FOR THE DETECTION OF CASEIN PROTEOLYSIS IN CHEESE." Journal of Dairy Research 62(2): 297-309. The whole N fraction of six samples of hard and semi-hard pressed cheese s was analysed using PAGE, polyacrylanzide gel isoelectric focusing and immunobl otting with polyclonal antibodies against beta- and alpha(s1)-casein. The origin of some electrophoretic bands corresponding to peptides produced from the enzym ic degradation of the casein fractions was established. A number of these peptid es were also present in the in vitro hydrolysates of casein with plasmin and chy mosin. Thus, it was also possible to determine which casein was the source of ea ch peptide and which enzymes were active in cheese. Compared with the traditiona l Coomassie staining procedures, immunoblotting is more sensitive and specific, making the interpretation of each electrophoretic profile easy. Thus, it was als o possible to obtain a clear picture of the state of each casein fraction in a c heese variety. Two main peptides were isolated from the pH 4.6-insoluble N fract ion of Parmigiano-Reggiano using DEAE-cellulose chromatography and identified, f rom the amino acid sequence of the N- and C-terminal ends, as gamma(3)-casein ( (beta-casein(f108-209)) and alpha(s1)-PL1 (alpha(s1)-casein(f80-199). In both ca ses, a Lys-X bond was hydrolysed, indicating the action of a trypsin-like enzyme in beta- and alpha(s1)-casein hydrolysis during the ripening of this variety of hard pressed cheese. Addeo, F., P. Martin, et al. (1984). "SUSCEPTIBILITY OF BUFFALO AND COW K-CASEIN S TO CHYMOSIN ACTION." Milchwissenschaft-Milk Science International 39(4): 202-2 05. Addeo, F., J. P. Pelissier, et al. (1980). "SPECIFIC ACTION OF MILK-CLOTTING ENZ YMES ON WATER BUFFALO CASEINS .1. EFFECT OF CHYMOSIN ON BETA-CASEIN." Journal of Dairy Research 47(3): 421-426. Addis, M., G. Piredda, et al. (2005). "Effect of the use of three different lamb paste rennets on lipolysis of the PDO Pecorino Romano Cheese." International Da iry Journal 15(6-9): 563-569. Two rennet pastes were prepared from lambs on different diets, and then, together with a commercial lamb rennet paste, were used to produce protected de signation of origin (PDO) Pecorino Romano cheeses. Chymosin activity and lipolyt ic activity were higher in the prepared rennets than in the commercial rennet. L ipolysis was greatest in cheese produced with one of the prepared rennets. Both quantitative and qualitative differences in free fatty acids were found in the c heeses. The taste of cheeses made with the prepared rennets was preferred by an expert sensory panel and also considered more piquant than that of cheese made w ith commercial rennet. The results indicate that both the diet of the lambs and slaughtering conditions should be regulated to produce rennet that preserves the traditional characteristics of PDO Pecorino Romano cheese. Both lipolytic and p roteolytic properties of rennet pastes should be standardised for the preparatio n of such cheeses. (c) 2005 Elsevier Ltd. All rights reserved. Addis, M., G. Piredda, et al. (2008). "The use of lamb rennet paste in tradition al sheep milk cheese production." Small Ruminant Research 79(1): 2-10. The clotting of milk in cheese making is a key passage obtained by the u se of enzymes or rennet. Several types of rennet are commercially available, the y differ both on their origin (animal, vegetable, microbial and recombinant from genetically modified microorganism) and their physical state (liquid, powder or paste). Usually rennet is derived from the abomasa (fourth stomach or vell) of unweaned calves. Commercial preparations of calf rennet, available as liquid or powdered form, contains chymosin and a different number of proteases such as: pe psin A, gastricsin or pepsin B. This rennet do not contain lipolytic enzymes, wh ich are denatured during the activation process of chymosin and pepsin zymogens.

In Mediterranean countries, is common the use of lamb or kid rennet paste, alwa ys obtained from the abomasa of these small ruminants. Lamb rennet paste, beside s milk-clotting and proteolytic enzymes (chimosin and pepsins), also contains a complex system of lipolytic enzymes. Lamb rennet paste is used in the production of some PDO traditional sheep milk cheeses, such as Idiazabal and Roncal in Spa in, Fiore Sardo, Pecorino Romano, Canestrato Pugliese in Italy and Feta in Greec e. The aim of this review is to summarize the current knowledge in lamb rennet p aste including methods of preparation, enzymatic composition and factors influen cing it, analytical aspects related to the enzymatic composition, proteolysis, l ipolysis and sensory characteristics of cheeses. (C) 2008 Elsevier B.V. All righ ts reserved. Agarwal, S., M. Costello, et al. (2005). "Gas-flushed packaging contributes to c alcium lactate crystals in Cheddar cheese." Journal of Dairy Science 88(11): 377 3-3783. Gas-flushed packaging is commonly used for cheese shreds and cubes to pr event aggregation and loss of individual identity. Appearance of a white haze on cubed cheese is unappealing to consumers, who may refrain from buying, resultin g in lost revenue to manufacturers. The objective of this study was to determine whether gas flushing of Cheddar cheese contributes to the occurrence of calcium lactate crystals (CLC). Cheddar cheese was manufactured using standard methods, with addition of starter culture, annatto, and chymosin. Two different cheese m ilk compositions were used: standard ( lactose: protein = 1.47, protein: fat = 0 .90, lactose = 4.8%) and ultrafiltered (UF; lactose: protein = 1.23, protein: fa t = 0.84, lactose = 4.8%), with or without adjunct Lactobacillus curvatus. Curds were milled when whey reached 0.45% titratable acidity, and pressed for 16 h. A fter aging at 7.2 degrees C for 6 mo, cheeses were cubed ( 1 x 1 x 4 cm) and eit her vacuum-packaged or gas-flushed with carbon dioxide, nitrogen, or a 50: 50 mi xture of carbon dioxide and nitrogen, then aged for an additional 3 mo. Heavy cr ystals were observed on surfaces of all cubed cheeses that were gas-flushed, but not on cheeses that were vacuum-packaged. Cheeses without Lb. curvatus exhibite d L(+)-CLC on surfaces, whereas cheeses with Lb. curvatus exhibited racemic mixt ures of L(+)/D(-)-CLC throughout the cheese matrices. The results show that gas flushing ( regardless of gas composition), milk composition, and presence of non starter lactic acid bacteria, can contribute to the development of CLC on cheese surfaces. These findings stress the importance of packaging to cheese quality. Agrawal, P. and A. N. Hassan (2007). "Ultrafiltered milk reduces bitterness in r educed-fat Cheddar cheese made with an exopolysaccharide-producing culture." Jou rnal of Dairy Science 90(7): 3110-3117. The objectives were to reduce bitterness in reduced-fat Cheddar cheese m ade with an exopolysaccharide (EPS)producing culture and study relationships amo ng ultrafiltration (UF), residual chymosin activity (RCA), and cheese bitterness . In previous studies, EPS-producing cultures improved the textural, melting, an d viscoelastic properties of reduced-fat Cheddar cheese. However, the EPS-positi ve cheese developed bitterness after 2 to 3 mo of ripening due to increased RCA. We hypothesized that the reduced amount of chymosin needed to coagulate UF milk might result in reduced RCA and bitterness in cheese. Reduced-fat Cheddar chees es were manufactured with EPS-producing and nonproducing cultures using skim mil k or UF milk (1.2x) adjusted to a casein:fat ratio of 1.35. The EPS-producing cu lture increased moisture and RCA in reduced-fat Cheddar cheese. Lower RCA was fo und in cheese made from UF milk compared with that in cheese made from control m ilk. Ultrafiltration at a low concentration rate (1.2x) produced EPS-positive, r educed-fat cheese with similar RCA to that in the EPS-negative cheese. Slower pr oteolysis was observed in UF cheeses compared with non-UF cheeses. Panelists rep orted that UF EPS-positive cheese was less bitter than EPS-positive cheese made from control milk. This study showed that UF at a low concentration factor (1.2x ) could successfully reduce bitterness in cheese containing a high moisture leve l. Because this technology reduced the RCA level (per g of protein) to a level s imilar to that in the control cheeses, the contribution of chymosin to cheese pr

oteolysis would be similar in both cheeses. Agrawal, P. and A. N. Hassan (2008). "Characteristics of reduced fat Cheddar che ese made from ultrafiltered milk with an exopolysaccharide-producing culture." J ournal of Dairy Research 75(2): 182-188. In a previous study, ultrafiltration (UF) at 1.2 x reduced residual chym osin activity and bitterness in exopolysaccharide (EPS)-positive reduced fat Che ddar cheese. The objective of this research was to study the effect of this leve l of concentration on the textural and functional characteristics of the reduced fat cheese. Ultrafiltration (1.2x) did not affect the hardness, cohesiveness, a dhesiveness, chewiness, and gumminess of EPS-positive cheese. The 6-month old UF cheeses were springier than non-UF cheeses. However, the springiness of the EPS -positive cheese made from UF milk was much lower than that of the EPS-negative cheeses. Texture of the EPS-negative cheese was more affected by UF than that of the EPS-positive cheese. Differences were seen in the extent of flow between UF and non-UF cheeses at 1 and 3-months but not after 6 months ripening. Ultrafilt ration increased the elastic modulus in the 6-month old EPS-positive cheeses. Hi gher body and texture scores were given to EPS-positive cheeses than the EPS-neg ative ones. Sensory panelists found the body of the UF and non-UF cheeses to be similar. Aguilar, C. F., M. P. Newman, et al. (1993). "THE USE OF PROTEIN HOMOLOGS IN THE ROTATION FUNCTION." Acta Crystallographica Section A 49: 306-315. The success of molecular replacement depends, in part, on the degree of similarity of the target and search molecules. We have systematically investigat ed this effect in cross-rotation functions for members of the aspartic proteinas e family of enzymes. The influence of various parameters on peak heights was inv estigated for six search models using \F(obs)\ data for two target enzymes. The beneficial effects of high-resolution data and a large radius of integration are most pronounced when target and search molecules have high-percentage identitie s. Correction for small differences in domain-domain orientation (typically 4-8degrees) between search and target structures leads to only a marginal improveme nt in the rotation-function peak height. There is an almost linear relationship between the structural distance, D, a parameter used in cluster analysis to defi ne differences between three-dimensional protein structures, and the height of t he cross-rotation-function peaks. Aichinger, P. A., M. Michel, et al. (2003). "Fermentation of a skim milk concent rate with Streptococcus thermophilus and chymosin: structure, viscoelasticity an d syneresis of gels." Colloids and Surfaces B-Biointerfaces 31(1-4): 243-255. Fermentation of a reconstituted skim milk concentrate (8% protein) was i nvestigated to elucidate the effects of various fermentation parameters on the s tructural, rheological and visual (wheying-off) properties of the resulting gels (pH 4.60). Fermentation trials were performed with non-exocellular polysacchari de-producing strains of Streptococcus thermophilus at various fermentation tempe ratures and at various chymosin levels. Oscillatory vane rheometry carried out o n the intact gels (at 4 degreesC) showed that the level of chymosin had a signif icant impact on the gel strength (storage modulus G'). This can be explained by the arrangement of casein micelles into more compact aggregates and the enhanced fusion of aggregated casein micelles as revealed by transmission electron micro scopy for the gels fermented with chymosin. Wheying-off of the stirred gels as m easured by a centrifugation test (at 4 degreesC) and pore size of the intact gel structures investigated by scanning electron microscopy both increased with inc reasing level of chymosin and increasing fermentation temperature (resulting in an increase in acidification rate). A higher level of syneresis (wheying-off) is explained by the larger pore size, since larger pores present a lower resistanc e to the outflow of whey from the gel. (C) 2003 Elsevier B.V. All rights reserve d. Aikawa, J., M. Nishiyama, et al. (1992). "PROTEIN ENGINEERING OF THE MILK-CLOTTI

NG ASPARTIC PROTEINASES." Scandinavian Journal of Clinical & Laboratory Investig ation 52: 51-58. Calf Chymosin and a fungal protease from Mucor pusillus (Mucor rennin) a re members of the aspartic proteinases used as milk-coagulants in cheese industr y. A system for production of recombinant chymosin as inclusion bodies in Escher ichia coli cells and its refolding into the active form was established. Another expression system for production of Mucor rennin in Saccharomyces cerevisiae wa s also established. Mucor rennin was efficiently excreted from the yeast host as a heavily glycosylated form. Glycosylation affected both the secretion and the enzyme properties. Site-directed mutagenesis of the Tyr residue at position 75 i n chymosin and Mucor rennin revealed its crucial role in catalytic function of t he aspartic proteinases. The results also suggested possibility to improve pract ical properties of the milk-clotting enzymes by site-directed mutagenesis. Aikawa, J., Y. N. Park, et al. (2001). "Replacements of amino acid residues at s ubsites and their effects on the catalytic properties of Rhizomucor pusillus pep sin, an aspartic proteinase from Rhizomucor pusillus." Journal of Biochemistry 1 29(5): 791-794. Site-directed mutagenesis was carried out to investigate the functional roles of amino acid residues of Rhizomucor pusillus pepsin (RMPP) in substrate-b inding and catalysis, Mutations of two amino acid residues, E13 in the S3 subsit e and N219 in the S3/S4 subsites, caused marked changes in kinetic parameters fo r two substrate peptides with different sequences. Further site-directed mutagen esis at E13 suggested that E13 plays a critical role in forming the correct hydr ogen bond network around the active center. In the crystal structure of Rhizomuc or miehei pepsin (RMMP), which is an aspartic proteinase produced by Rhizomucor miehei and shows 81% amino acid identity to RMPP, the O epsilon atom of N219 for ms a hydrogen bond with the N-H of isovaline in pepstatin A, a statine-type inhi bitor, at the P3 position, suggesting that the loss of the hydrogen bond causes an unfavorable arrangement of the P3 residue. Among the mutants constructed, the E13A mutant showed a 5-fold increase in the ratio of clotting versus proteolyti c activity without significant loss of clotting activity. This mutant may presen t a promising candidate for a useful milk coagulant. Albenzio, M., A. Santillo, et al. "Biochemical patterns in ovine cheese: Influen ce of probiotic strains." Journal of Dairy Science 93(8): 3487-3496. This study was undertaken to evaluate the effect of lamb rennet paste co ntaining probiotic strains on proteolysis, lipolysis, and glycolysis of ovine ch eese manufactured with starter cultures. Cheeses included control cheese made wi th rennet paste, cheese made with rennet paste containing Lactobacillus acidophi lus culture (LA-5), and cheese made with rennet paste containing a mix of Bifido bacterium lactis (BB-12) and Bifidobacterium longum (BB-46). Cheeses were sample d at 1, 7, 15, and 30 d of ripening. Starter cultures coupled with probiotics st rains contained in rennet paste affected the acidification and coagulation phase s leading to the lowest pH in curd and cheese containing probiotics during ripen ing. As consequence, maturing cheese profiles were different among cheese treatm ents. Cheeses produced using rennet paste containing probiotics displayed higher percentages of alpha(S1)-I-casein fraction than traditional cheese up to 15 d o f ripening. This result could be an outcome of the greater hydrolysis of a-casei n fraction, attributed to higher activity of the residual chymosin. Further evid ence for this trend is available in chromatograms of water-soluble nitrogen frac tions, which indicated a more complex profile in cheeses made using lamb paste c ontaining probiotics versus traditional cheese. Differences can be observed for the peaks eluted in the highly hydrophobic zone being higher in cheeses containi ng probiotics. The proteolytic activity of probiotic bacteria led to increased a ccumulation of free amino acids. Their concentrations in cheese made with rennet paste containing Lb. acidophilus culture and cheese made with rennet paste cont aining a mix of B. lactis and B. longum were approximately 2.5 and 3.0 times hig her, respectively, than in traditional cheese. Principal component analysis show ed a more intense lipolysis in terms of both free fatty acids and conjugated lin

oleic acid content in probiotic cheeses; in particular, the lipolytic pattern of cheeses containing Lb. acidophilus is distinguished from the other cheeses on t he basis of highest content of health-promoting molecules. The metabolic activit y of the cheese microflora was also monitored by measuring acetic, lactic, and c itric acids during cheese ripening. Cheese acceptability was expressed for color , smell, taste, and texture perceived during cheese consumption. Use of probioti cs in trial cheeses did not adversely affect preference or acceptability; in fac t, panelists scored probiotic cheeses higher in preference over traditional chee se, albeit not significantly. Albert, A., T. L. Blundell, et al. (1998). Protein engineering aspartic proteina ses - Site-directed mutagenesis, biochemical characterisation, and X-ray analysi s of chymosins with substituted single amino acid substitutions and loop replace ments. Aspartic Proteinases - Retroviral and Cellular Enzymes. M. N. G. James. 4 36: 169-177. Alcocer, M. J. C., C. S. M. Furniss, et al. (2003). "Comparison of modular and n on-modular xylanases as carrier proteins for the efficient secretion of heterolo gous proteins from Penicillium funiculosum." Applied Microbiology and Biotechnol ogy 60(6): 726-732. Genes encoding three enzymes with xylanase activity from the filamentous fungus Penicillium funiculosum are described. Two of the encoded xylanases are predicted to be modular in structure with catalytic and substrate-binding domain s separated by a serine and threonine-rich linker region; the other had none of these properties and was non-modular. In order to develop P. funiculosum as a ho st for the secreted production of heterologous proteins, each of the xylanases w as assessed for use as a carrier protein in a fusion strategy. We show that one of the modular xylanases (encoded by xynA) was an effective carrier protein but the other (encoded by xynB) and the non-modular xylanase (encoded by xynC) were not effective as secretion carriers. We show that the P-glucuronidase (GUS) prot ein from Escherichia coli is secreted by P. funiculosum when expressed as an XYN A fusion but that the secreted GUS protein, cleaved in vivo from XYNA, is glycos ylated and enzymatically inactive. Al-Otaibi, M. M. and R. A. Wilbey (2004). "Effect of temperature and salt on the maturation of white-salted cheese." International Journal of Dairy Technology 5 7(1): 57-63. White-salted cheeses were prepared from ultrafiltered (UF) cows' milk an d salted to give final salt-in-moisture (SM) levels of 2.5, 3.2 and 4.0%. The ch eeses were stored at 5degreesC and 10degreesC for up to 15 weeks. The microflora was dominated by lactic acid bacteria (LAB) but some mould growth was evident w ithin 15 weeks at all SM levels and both temperatures. Levels of water-soluble n itrogen (WSN), attributed to chymosin activity, increased significantly with tim e, the rate being inversely proportional to the SM level and increasing with sto rage temperature. Similar effects were noted for trichloroacetic acid-soluble ni trogen (TCA-SN) and free amino acid (FAA) levels, both of which would also be af fected by bacterial protease activity. The proteolytic activity was reflected by changes in the hardness and fracturability of the cheeses. Al-Otaibi, M. M. and R. A. Wilbey (2005). "Effect of chymosin and salt reduction on the quality of ultrafiltrated white-salted cheese." Journal of Dairy Researc h 72(2): 234-242. This study demonstrated that both chymosin and salt-in-moisture (SM) wer e important factors for proteolysis in the manufacture of ultrafiltrated white-s alted cheese, with significant effects on water-soluble nitrogen and nitrogen so luble in trichloroacetic acid. In contrast, the levels of free amino acids were not significantly affected by chymosin and salt treatments. The cheeses made usi ng high levels of chymosin with low SM had lower levels of residual (s1)and -casein at the end of ripening. On texture profile analysis, the hard ness and fracturability of the cheeses significantly increased with SM and decre

ased during ripening. Increases in chymosin significantly contributed to the ove rall weakening of the structure throughout ripening. Bitter flavour was detected after 12 weeks in the cheese made with the higher chymosin level and lower SM, which could be the result of accumulation of -casein fractions. The senso ry data indicated that the hedonic responses for low chymosin with low SM cheese s were good and acceptable in flavour, which may be due to the moderate levels o f proteolysis products. Al-Otaibi, M. M. and R. A. Wilbey (2006). "Effect of chymosin reduction and salt substitution on the properties of white salted cheese." International Dairy Jou rnal 16(8): 903-909. A whey salts mixture was used as a partial substitute for sodium chlorid e to provide a modified Na:K ratio (1:3.4) in the manufacture of white salted ch eese using ultrafiltration. Reduction of chymosin addition from 20 to 8 mu L kg( -1) of cheese was also investigated. Variation of salt and chymosin levels did n ot result in any significant differences in composition and physicochemical prop erties. The rates of proteolysis in terms of water-soluble nitrogen (WSN) and ni trogen soluble in 12% trichloroacetic acid (TCA-SN) were affected by chymosin le vels but not by salt treatment. Urea-PAGE electrophoretic analysis of caseins fr om the cheeses manufactured using three levels of chymosin and two salt types sh owed that the hydrolysis of alpha(s1)-casein was higher than for beta-caseins bu t the differences between the cheeses were not significant (P > 0.05). The chymo sin level did not have a significant effect (P > 0.05) on hardness and fracturab ility, suggesting that any variation in hardness due to the initial hydrolysis w as being confounded by other variables. Cheeses including the whey salts product were harder and more fracturable (P < 0.01) than the cheese treated with NaCl o nly. Both hardness and fracturability values decreased (P < 0.05) over the matur ation period. The scores for bitterness were low; neither the effects of salt no r chymosin levels were significant (P > 0.05). (c) 2005 Elsevier Ltd. All rights reserved. Amourache, L. and M. A. Vijayalakshmi (1984). "AFFINITY-CHROMATOGRAPHY OF KID CH YMOSIN ON HISTIDYL-SEPHAROSE." Journal of Chromatography 303(1): 285-290. Andersen, B. (1933). "The methodology of regulating pepsin and chymosin activity in gastric contents." Biochemische Zeitschrift 262: 99-118. Andersson, H. and A. Andren (1990). "INFLUENCE OF CHROMATOGRAPHICALLY PURE BOVIN E CHYMOSIN AND PEPSIN-A ON CHEESE CURD SYNERESIS." Journal of Dairy Research 57( 1): 119-124. Andersson, H., A. Andren, et al. (1989). "AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY F OR DETECTION OF CHYMOSIN IN DAIRY-PRODUCTS." Journal of Dairy Science 72(12): 31 29-3133. Andreeva, N., J. Dill, et al. (1992). "CAN ENZYMES ADOPT A SELF-INHIBITED FORM RESULTS OF X-RAY CRYSTALLOGRAPHIC STUDIES OF CHYMOSIN." Biochemical and Biophys ical Research Communications 184(2): 1074-1081. Andreeva, N. S. (1992). "SOME ASPECTS OF STRUCTURAL STUDIES ON ASPARTIC PROTEINA SES." Scandinavian Journal of Clinical & Laboratory Investigation 52: 31-38. This paper gives a brief overview over the differences and similarities in the structure of aspartic proteinases presently available. Comparison of the three-dimentional stucture of different aspartic proteinases by a common intramo lecular coordinate system have been performed. The intramolecular movable subdom ains have been localized and the role of motion in substrate binding and zymogen activation is discussed. Andreeva, N. S. (2006). "State-of-the-art and problems of X-ray diffraction anal ysis of biomacromolecules." Crystallography Reports 51(6): 988-993.

The state-of-the-art of X-ray diffraction studies of biomacromolecules i s briefly characterized, and the challenge imposed by science is discussed. Thes e studies are characterized by a wide scope and extensive use. This field of sci ence is of great interest and is developed in many countries. The main purpose i s to solve practical problems in medicine consisting in the design of drugs agai nst various diseases. X-ray diffraction analysis of enzymes brought the pharmace utical industry to a new level, thus allowing the rational design of drugs again st formerly untreatable diseases. Modern X-ray diffraction studies of biomacromo lecules laid the basis for a new science called structural biology. This method allows one to solve fundamental problems of physical chemistry for a new state o f matter existing in living systems. Here, science poses numerous problems in an alysis of X-ray diffraction data on biological macromolecules. Many of theses pr oblems are in their infancy. Andreeva, N. S. and I. V. Pechik (1995). "Comparison of three-dimensional struct ures of flexible protein molecules." Molecular Biology 29(5): 650-657. An original technique was developed for comparing 3D protein structures, which obviates superposition and is based on the use of intramolecular coordina te systems (ICS). With atomic coordinates refined at high resolution, and with o bservance of certain requirements for reference points used to construct the ICS , the method provides a fuller picture of the differences in the mutual position s of atoms in the compared protein molecules than the conventional approaches. I t also allows a substantial reduction of the calculations in pair comparison of a large number of homologous structures. Converted into the ICS, the protein ato mic coordinates prove to be automatically ''overlaid'' when working with graphic devices; simultaneously converted are the atomic coordinates for bound water, w hich makes it possible to reveal its conserved positions. Application of the met hod is exemplified with the data for ribonuclease A and its complexes with deoxy nucleosides, as well as for pepsin versus pepsinogen. Andren, A., L. Bjorck, et al. (1980). "QUANTIFICATION OF CHYMOSIN (RENNIN) AND P EPSIN IN BOVINE ABOMASA BY ROCKET IMMUNOELECTROPHORESIS." Swedish Journal of Agr icultural Research 10(3): 123-130. Andren, A., L. Bjorck, et al. (1981). "EFFECT OF SUPPLEMENTARY MILK-FEEDING ON C ONTENT OF CHYMOSIN IN THE ABOMASAL MUCOSA OF CONCENTRATE-FED CALVES." Swedish Jo urnal of Agricultural Research 11(1): 11-15. Andren, A., L. Bjorck, et al. (1981). "LEVELS OF CHYMOSIN AND PEPSIN IN BOVINE A BOMASAL MUCOSA." Netherlands Milk and Dairy Journal 35(3-4): 365-366. Andren, A., P. J. Dekoning, et al. (1983). "CHANGES IN IMMUNOLOGICALLY AND CATAL YTICALLY ACTIVE-SITES OF CHYMOSIN, BOVINE PEPSIN AND PORCINE PEPSIN." Netherland s Milk and Dairy Journal 37(1-2): 11-20. Andren, A. and C. Vonreedtz (1990). "EFFECTS OF CHROMATOGRAPHICALLY PURE BOVINE CHYMOSIN AND PEPSIN-A ON CHEESE CURD FIRMNESS." Journal of Dairy Research 57(1): 109-117. Anema, S. G. (1997). "The effect of chymosin on kappa-casein-coated polystyrene latex particles and bovine casein micelles." International Dairy Journal 7(8-9): 553-558. Bovine kappa-casein was adsorbed onto negatively charged polystyrene lat ex particles of various sizes and the changes in the hydrodynamic diameters of t he particles were monitored by photon correlation spectroscopy. The latex partic le diameters were increased on addition of small amounts of kappa-casein, and pl ateaued once a maximum adsorption was attained. The size increase was about 25 n m regardless of the initial size of the latex particle, indicating that the surf ace arrangement of kappa-casein was similar on all latex sizes. Treatment of the casein micelles in skim milk and the kappa-casein-coated latex particles with c

hymosin initially decreased the particle diameter by about 10 nm after which the particle diameter rapidly increased as the aggregation reaction proceeded. The action of chymosin on both the casein micelles and the kappa-casein-coated latex particles was retarded by increasing the NaCl concentration. The similarity bet ween the kappa-casein-coated latex particles and the casein micelles in milk, es pecially their behaviour towards chymosin, indicates that these latex particles may be a simple model system for studying some casein micelle properties under c ontrolled conditions. (C) 1998 Elsevier Science Ltd. All rights reserved. Anema, S. G., S. K. Lee, et al. (2007). "Effect of pH at heat treatment on the h ydrolysis of kappa-casein and the gelation of skim milk by chymosin." Lwt-Food S cience and Technology 40(1): 99-106. Skim milk was adjusted to pH values between 6.5 and 7.1 and heated at 90 degrees C for times from 0 to 30 min. After heat treatment, the samples were re -adjusted to the natural pH (pH 6.67) and allowed to re-equilibrate. High levels of denatured whey proteins associated with the casein micelles during heating a t pH 6.5 (about 70-80% of the total after 30 min of heating). This level decreas ed as the pH at heating was increased, so that about 30%, 20% and 10% of the den atured whey protein was associated with the casein micelles after 30 min of heat ing at pH 6.7, 6.9 and 7.1, respectively. Increasing levels of K-casein were tra nsferred to the serum as the pH at heating was increased. The loss of K-casein a nd the formation of para-K-casein with time as a consequence of the chymosin tre atment of the milk samples were monitored by sodium dodecyl sulphate polyacrylam ide gel electrophoresis (SDS-PAGE). The loss of K-casein and the formation of pa ra-K-casein were similar for the unheated and heated samples, regardless of the pH at heating or the heat treatment applied. Monitoring the gelation properties with time for the chymosin-treated milk samples indicated that the heat treatmen t of the milk markedly increased the gelation time and decreased the firmness (G ) of the gels formed, regardless of whether the denatured whey proteins were ass ociated with the casein micelles or in the milk serum. There was no effect of pH at heat treatment. These results suggest that the heat treatment of milk has on ly a small effect on the primary stage of the chymosin reaction (enzymatic phase ). However, heat treatment has a marked effect on the secondary stage of this re action (aggregation phase), and the effect is similar regardless of whether the denatured whey proteins are associated with the casein micelles or in the milk s erum as nonsedimentable aggregates. (c) 2005 Swiss Society of Food Science and T echnology. Published by Elsevier Ltd. All rights reserved. Aquilanti, L., V. Babini, et al. "Bacterial dynamics in a raw cow's milk Caciott a cheese manufactured with aqueous extract of Cynara cardunculus dried flowers." Letters in Applied Microbiology 52(6): 651-659. Aims: To investigate the bacterial dynamics of a Caciotta cheese traditi onally manufactured in the Montefeltro area (Central Italy) with raw cow's milk and an aqueous extract of dried flowers from Cynara cardunculus as a coagulating agent. Methods and Results: Conventional methods and a combined PCR-DGGE approa ch, relying on culture-dependent and -independent analyses, were used to investi gate the cheese bacterial community, with a special focus on lactic acid bacteri a. A heterogeneous population, including enterococci, lactococci, lactobacilli, food spoilage and other banal micro-organisms, was found. Significance and Impac t of the Study: The study contributed to highlighting the influence of different technological parameters on bacterial dynamics of a raw milk Caciotta cheese co agulated with vegetable rennet. Conclusions: None of the species found in the ve getable rennet became dominant during the cheese-making and a prevailing role of the adventitions microbita coming from the raw milk and the dairy environment w as highlighted. Archer, D. B., D. J. Jeenes, et al. (1994). "STRATEGIES FOR IMPROVING HETEROLOGO US PROTEIN-PRODUCTION FROM FILAMENTOUS FUNGI." Antonie Van Leeuwenhoek Internati onal Journal of General and Molecular Microbiology 65(3): 245-250. Despite the naturally high capacity for protein secretion by many specie

s of filamentous fungi, secteted yields of many heterologous proteins have been comparatively low. The strategies for yield improvement have included the use of strong homologous promoters, increased gene copy number, gene fusions with a ge ne encoding a naturally well-secreted protein, protease-deficient host strains a nd screening for high yields following random mutagenesis. Such approaches have been effective with some target heterologous proteins but not others. Approaches used in heterologous protein production from filamentous fungi are discussed an d a perspective on emerging strategies is presented. Ardo, Y., H. Lilbaek, et al. (2007). "Identification of large phosphopeptides fr om beta-casein that characteristically accumulate during ripening of the semi-ha rd cheese Herrgard." International Dairy Journal 17(5): 513-524. The peptide composition of a cheese reflects its characteristic ripening process, and in this study, large hydrophobic phosphopeptides that accumulate i n semi-hard cheese, Herrgard, were identified. Anion exchange chromatography, re verse phase (RP) HPLC, liquid chromatography mass spectrometry and N-terminal am ino acid sequencing were used. Milk from homozygotic cows was used to prepare be ta-casein A1 and A2, and peptides produced from their hydrolysis by plasmin were analysed to support identification of the peptides in cheese. Eight large phosp hopeptides released by plasmin hydrolysis of beta-casein were identified in the semi-hard cheese, i.e., fractions (f29-105, f29-107, f1-105, f1-107) A1 and A2. Four other peptides that accumulated in the cheese, co-eluted on RP-HPLC with th e large primary plasmin-derived phosphopeptides and contributed significantly to two characteristic large peaks, i.e., one peak for each of the two genetic vari ants A1 and A2 of two beta-casein fractions, mainly (f29-93) but also (f30-93). (c) 2006 Elsevier Ltd. All rights reserved. Areces, L. B., M. B. D. Bonino, et al. (1992). "PURIFICATION AND CHARACTERIZATIO N OF A MILK CLOTTING PROTEASE FROM MUCOR-BACILLIFORMIS." Applied Biochemistry an d Biotechnology 37(3): 283-294. An acid protease having milk clotting activity has been isolated from Mu cor bacilliformis cultures. The enzyme was basically purified by ionic exchange chromatography. An average yield of 29 mg purified product was obtained from 100 mL crude extract. As purity criteria, SDS-PAGE, reverse-phase HPLC, and N-termi nal analysis were performed. The protease is a protein composed of a single poly peptide chain with glycine at the N-terminus. The mol wt is approx 32,000, and i ts amino acid composition is very similar to those of other fungal proteases. As expected, its clotting activity was drastically inhibited by pepstatin A action . On the other hand, its instability against heat treatment and its clotting/pro teolytic activity ratio indicate that it may be considered as a potential substi tute for bovine chymosin. Arnau, J., L. P. Jepsen, et al. (1991). "INTEGRATIVE TRANSFORMATION BY HOMOLOGOU S RECOMBINATION IN THE ZYGOMYCETE MUCOR-CIRCINELLOIDES." Molecular & General Gen etics 225(2): 193-198. Transformation of a Mucor circinelloides Leu- strain with the plasmid pA D45, harbouring the wild-type allele (leuA +) and a chymosin gene, led to the id entification of mitotically stable transformants after one to three vegetative g rowth cycles on non-selective medium. Southern analysis of the stable transforme d strains demonstrated that the vector is integrated, as an intact molecule, int o the resident Mucor leuA locus. Retransformation of Escherichia coli with genom ic DNA restricted with enzymes having no or only a single recognition site withi n the inserted sequence did not permit isolation of plasmids or fragments carryi ng the leuA or chymosin gene. Arruda, L. K., L. D. Vailes, et al. (1995). "MOLECULAR-CLONING OF A MAJOR COCKRO ACH (BLATTELLA-GERMANICA) ALLERGEN, BLA-G-2 - SEQUENCE HOMOLOGY TO THE ASPARTIC PROTEASES." Journal of Biological Chemistry 270(33): 19563-19568. Inhalation of allergens produced by the German cockroach (Blattella germ anica) elicits IgE antibody formation and the development of asthma in genetical

ly predisposed individuals. We compared the allergenic importance of two cockroa ch (CR) allergens, Bla g 1 and Bla g 2, and determined the complete amino acid s equence of the major 36-kDa allergen, Bla g 2. A survey of 106 sera from CR alle rgic patients showed the prevalence of IgE antibodies to Bla gl and Bla g 2 to b e 30.2% and 57.6%, respectively. Immediate skin tests on 7 selected patients gav e positive reactions using 10(-3) mu g/ml either allergen, whereas controls show ed no response to 10 mu g/ml. Natural Bla g 2 was purified and the sequence of t he NH2 terminus and tryptic peptides, comprising 36% of the molecule, was determ ined. The cDNA for Bla g 2 was cloned from a B. germanica expression library and encoded a 24 amino acid signal peptide and a 328-amino acid mature protein, whi ch showed sequence homology to aspartic proteases. Bla g 2 showed the highest de gree of identity to mosquito (Aedes aegypti) lysosomal aspartic protease (30.8%) , with similar identity to pepsin, cathepsins D and E, renin, and chymosin, Bla g 2 mRNA and protein were detected in B, germanica, but not in Periplaneta ameri cana, the other principal domiciliary CR species in the U. S. High concentration s of Bla g 2 were found in CR digestive organs (esophagus, gut, and proventricul us). The results show that Ela g 2 is a major species-specific allergen of B. ge rmanica and suggest that the allergen functions as a digestive enzyme in the coc kroach. Aryana, K. J. and Z. U. Haque (2001). "Microstructure of Jarlsberg type and Edam cheeses as influenced by maturation." Journal of Food Quality 24(4): 337-350. This study investigated the microstructure of a Jarlsberg type (Vallegre t) and Edam cheeses as impacted by maturation. Microstructure was studied by tra nsmission and scanning electron microscopy. Vallegret and Edam cheeses were matu red for eleven and six months respectively at 4C Matured Vallegret cheese showed the presence of fibrous structures whereas the one week immature Vallegret chee se did not. Immature (one week) Vallegret and Edam cheeses both showed the prese nce of tiny porous structures and fine cracks which reduced significantly as the cheeses matured. Protein layers at the fat-protein interface were absent in one week immature Vallegret and Edam cheeses. However, protein layers were observed in both cheeses on maturation. Maturation markedly affected the microstructures of these cheeses. Asakura, T., H. Watanabe, et al. (1995). "RICE ASPARTIC PROTEINASE, ORYZASIN, EX PRESSED DURING SEED RIPENING AND GERMINATION, HAS A GENE ORGANIZATION DISTINCT F ROM THOSE OF ANIMAL AND MICROBIAL ASPARTIC PROTEINASES." European Journal of Bio chemistry 232(1): 77-83. The gene organization and nucleotide sequence of an aspartic proteinase (AP) of plant origin were first disclosed by cDNA and genomic DNA cloning of a r ice AP (oryzasin). The deduced amino acid sequence of oryzasin 1 was significant ly similar to those of other APs (34-85%), with highest similarity (85%) to barl ey AP (HvAP). Oryzasin 1, as well as HvAP, is distinct from animal and microbial APs in that the plant APs contain a unique 104-amino-acid insertion in the C-te rminal region. The oryzasin 1 gene spans approximately 6.6 kbp and is composed o f 14 exons and 13 introns. The exon-intron organization of the oryzasin 1 gene i s totally different from those of genes for animal and microbial APs such as hum an cathepsin D, rat renin, bovine chymosin, aspergillopepsin A of Aspergillus aw amori, proteinase A of Saccharomyces cerevisiae and rhizopuspepsin of Rhizopus n iveus, despite the fact that oryzasin 1 shows overall sequence similarity to the se APs. Asakura, T., H. Watanabe, et al. (1997). "Oryzasin as an aspartic proteinase occ urring in rice seeds: Purification, characterization, and application to milk cl otting." Journal of Agricultural and Food Chemistry 45(4): 1070-1075. An aspartic proteinase in rice seeds (oryzasin) was purified by (NH4)(2) SO4 fractionation, DEAE-celluose anion exchange chromatography, Sephadex G-100 g el filtration, Mono Q anion exchange chromatography, and pepstatin-affinity chro matography. SDS-PAGE showed the affinity-purified enzyme to have two molecular f orms, 57 and 53 kDa, together with their probable autolysates appearing as two s

mall bands at 35 and 25 kDa. Compared with the other three bands, the 57 kDa ban d reacted strongly on western blot analysis. The affinity-purified oryzasin pH o ptimum for hydrolysis is 3.0 and is completely inhibited by pepstatin but not af fected by other proteinase inhibitors such as EDTA, leupeptin, PMSF, and E-64. T he milk-clotting activity of oryzasin was investigated using the crude enzyme ob tained by precipitation at 30% and 60% (NH4)(2)SO4 saturation. The enzyme clotte d a skim milk solution at pH 6.3, yielding the same kappa-casein digest pattern as those of chymosin and pepsin producing a 12 kDa band. Awad, S., A. Hassan, et al. "Impact of exopolysaccharide-containing base Cheddar cheese on reduced fat process cheese." Milchwissenschaft-Milk Science Internati onal 65(2): 173-176. Reduced fat process cheeses were manufactured using a 50:50 mixture of 2 days old exopolysaccharide (EPS) positive or negative reduced fat Cheddar chees e and 6 months old (EPS) negative base reduced fat Cheddar cheese (BRFCC). Moist ure and fat were standardized to 49 and 21%, respectively. Reduced fat process c heeses made from BRFCC containing no EPS were firmer, more chewy and gummy, less deformable, and had higher viscoelastic moduli than cheeses made from EPS posit ive BRFCC. Process cheese made with EPS-positive BRFCC has lower hand firmness a nd higher chewdown degree of breakdown than did cheese made with the EPS negativ e BRFCC. In this process, it was not possible to relate modifications in process cheese properties only to the presence of EPS, as higher primary proteolysis oc curred in the EPS-positive BRFCC than in the control cheese, even during the fir st two days of ripening, due to the ability of EPS to retain water and consequen tly increase residual chymosin activity in the former cheese. In conclusion, EPS affected process cheese characteristics directly by interfering with protein-pr otein interactions and/or indirectly by inducing primary proteolysis in the base cheese. Awad, S., A. N. Hassan, et al. (2005). "Application of exopolysaccharide-produci ng cultures in reduced-fat Cheddar cheese: Composition and proteolysis." Journal of Dairy Science 88(12): 4195-4203. Proteolysis during ripening of reduced fat Cheddar cheeses made with dif ferent exopolysaccharide (EPS)-producing and nonproducing cultures was studied. A ropy strain of Lactococcus lactis ssp. cremoris (JFR1) and capsule-forming non ropy and moderately ropy strains of Streptococcus thermophilus were used in maki ng reduced-fat Cheddar cheese. Commercial Cheddar starter was used in making ful l-fat cheese. Results showed that the actual yield of cheese made with JFR1 was higher than that of all other reduced-fat cheeses. Cheese made with JFR1 contain ed higher moisture, moisture in the nonfat substance, and residual coagulant act ivity than all other reduced-fat cheeses. Proteolysis, as determined by PAGE and the level of water-soluble nitrogen, was also higher in cheese made with JFR1 t han in all other cheeses. The HPLC analysis showed a significant increase in hyd rophobic peptides (causing bitterness) during storage of cheese made with JFR1. Cheese made with the capsule-forming nonropy adjunct of S. thermophilus, which c ontained lower moisture and moisture in the nonfat substance levels and lower ch ymosin activity than did cheese made with JFR1, accumulated less hydrophobic pep tides. In conclusion, some EPS-producing cultures produced reduced-fat Cheddar c heese with moisture in the nonfat substance similar to that in its full-fat coun terpart without the need for modifying the standard cheese-making protocol. Such cultures might accumulate hydrophobic (bitter) peptides if they do not contain the system able to hydrolyze them. For making high quality reduced-fat Cheddar c heese, EPS-producing cultures should be used in conjunction with debittering str ains. Awad, S., Q. Q. Luthi-Peng, et al. (1998). "Proteolytic activities of chymosin a nd porcine pepsin on buffalo, cow, and goat whole and beta-casein fractions." Jo urnal of Agricultural and Food Chemistry 46(12): 4997-5007. The proteolytic specificity and activity of a recombinant chymosin (Maxi ren) and porcine pepsin on buffalo, cow, and goat whole casein (CN) and beta-CN

were studied by analyzing the degradation products. The results suggest that the hydrolysis of whole casein of buffalo and goat by chymosin was similar to that of cow casein resulting in alpha(s1)-I and beta-I, -II, and -III as degradation fragments of alpha(s1)- and beta-CN. The exception was goat beta-I which was res istant to further hydrolysis by chymosin but not to porcine pepsin at pH 5.4-6.2 . Increasing NaCl concentration to greater than or equal to 5% reduced the prote olysis of beta-CN in all three species, but not that of alpha(s1)-CN. The fragme nts of beta-I, -II, and -III produced from beta-CN of the three species gave ide ntical results with PAGE. alpha(s1)-I and its degradation fragments had in all t hree species, regardless of the different electrophoretic mobilities on PAGE, th e same sequence of appearance. The results indicate that chymosin and porcine pe psin attacked in buffalo and goat caseins the same regions as known for cow alph a(s1)- and beta-CN. Awad, S., Q. Q. Luthi-Peng, et al. (1999). "Proteolytic activities of Suparen an d Rennilase on buffalo, cow, and goat whole casein and beta-casein." Journal of Agricultural and Food Chemistry 47(9): 3632-3639. The proteolytic specificity and activity of Mucor miehei protease (Renni lase) and Endothia parasitica protease (Suparen) on buffalo, cow, and goat whole casein and beta-casein (CN) were studied by analyzing the degradation products. The results suggest that Rennilase hydrolyzes casein of the three species in a manner similar to that of chymosin, resulting in the formation of alpha(s1)-I an d beta-I, -II, -III as initial degradation fragments of alpha(s1)- and beta-CN. alpha(s1)-I was also the initial breakdown product of alpha(s1)-CN by Suparen. C ontrary to Rennilase, Suparen showed a higher affinity toward beta-CN and hydrol yzes beta-CN, giving rise to degradation products characterized by mobility lowe r than that of beta-CN. Increasing NaCl concentration (>3%) reduced the proteoly sis of beta-CN of the three species by Rennilase but not by Suparen. The hydroly sis of alpha(s1)-CN and alpha(s1)-I by the two enzymes was enhanced in the prese nce of NaCl. Awad, S., Q. Q. Luthi-Peng, et al. (2000). "Proteolytic activity of starter bact eria on buffalo casein peptides produced by coagulants of different origins." Mi lchwissenschaft-Milk Science International 55(9): 492-495. In the present study, the cell wall extracts of Lactococcus lactis ssp. lactis (Lc. lactis), Lactococcus lactis ssp. cremoris HP (Lc. cremoris HP) and L actobacillus casei ssp. casei(Lb. casei) were used to reveal their proteolytic a ctivities towards buffalo casein peptides obtained with chymosin, porcine pepsin , Rennilase and Suparen. The proteolytic activity of the strains on casein pepti des at pH 5.4 and 15 degrees C for up to 10 days was monitored by RP-HPLC, SDS-P AGE, as well as Cd-ninhydrin method. The results indicate that the activity of p roteases and peptidases of the strains tested depends on the identity of casein fragment determined by the coagulant used. The enzymes of Lc. lactis and Lc. cre moris HP acted primarily on casein peptides of MW > 19 kDa. The enzymes of Lb. c asei showed higher proteolytic activity and broader specificity towards a wide r ange of casein peptides. The enzymes of Lb. casei were the most proteolytic and of Lc cremoris HP the least when determining the liberation of free amino acids from casein peptides. The formation and elimination of the bitter peptides depen d on the choice of coagulant as well as the starter strain. The bitterness in Su paren- and Rennilase-peptides could be reduced by the enzymes of all 3 strains t ested. Azarnia, S., N. Robert, et al. (2006). "Biotechnological methods to accelerate c heddar cheese ripening." Critical Reviews in Biotechnology 26(3): 121-143. Cheese is one of the dairy products that can result from the enzymatic c oagulation of milk. The basic steps of the transformation of milk into cheese ar e coagulation, draining, and ripening. Ripening is the complex process required for the development of a cheese's flavor, texture and aroma. Proteolysis, lipoly sis and glycolysis are the three main biochemical reactions that are responsible for the basic changes during the maturation period. As ripening is a relatively

expensive process for the cheese industry, reducing maturation time without des troying the quality of the ripened cheese has economic and technological benefit s. Elevated ripening temperatures, addition of enzymes, addition of cheese slurr y, attenuated starters, adjunct cultures, genetically engineered starters and re combinant enzymes and microencapsulation of ripening enzymes are traditional and modern methods used to accelerate cheese ripening. In this context, an up to da te review of Cheddar cheese ripening is presented. Azuma, N., S. Kaminogawa, et al. (1984). "PROPERTIES OF GLYCOMACROPEPTIDE AND PA RA-K-CASEIN DERIVED FROM HUMAN K-CASEIN AND COMPARISON OF HUMAN AND BOVINE K-CAS EINS AS TO SUSCEPTIBILITY TO CHYMOSIN AND PEPSIN." Agricultural and Biological C hemistry 48(8): 2025-2031. Baankreis, R., S. vanSchalkwijk, et al. (1995). "The occurrence of two intracell ular oligoendopeptidases in Lactococcus lactis and their significance for peptid e conversion in cheese." Applied Microbiology and Biotechnology 44(3-4): 386-392 . Two intracellular oligopeptide-preferring endopeptidases have been detec ted in Lactococcus lactis. A neutral thermolysin-like oligoendopeptidase (NOP) h as been purified to homogeneity and an alkaline oligoendopeptidase has been part ially purified. The specificity of the oligoendopeptidases towards important int ermediary cheese peptides, produced by chymosin action on the caseins, clearly d iffers from that of the cell-envelope proteinase (CEP). NOP is active under cond itions prevailing in cheese and contributes to initial proteolysis in a young ch eese. It probably plays a crucial role in the degradation of an important bitter peptide in cheese, the beta-casein 193-209 fragment. The relatively low activit y of the alkaline endopeptidase is further suppressed in cheese by the highly co mpetitive actions of NOP and CEP. Badasso, M. O., V. Dhanaraj, et al. (2004). "Crystallization and X-ray analysis of the Y75N mutant of Mucor pusillus pepsin complexed with inhibitor." Acta Crys tallographica Section D-Biological Crystallography 60: 770-772. Y75N mutant Mucor pusillus pepsin has been overexpressed in yeast, purif ied and cocrystallized with the iodine-containing human renin inhibitor CP-11397 2 {(2R, 3S)-isopropyl 3-[(L-prolyl-p-iodo-L-phenylalanyl-S- methyl-cysteinyl) am ino-4]-cyclohexyl-2-hydroxybutanoate} for X-ray crystallography. Tetragonal comp lex crystals with space group P4(3)2(1)2 were produced by the hanging-drop vapou r-diffusion method and diffracted to 3.0 Angstrom. The crystals exhibited unit-c ell parameters a=b=182.5, c=99.1 Angstrom and contained four molecules in the as ymmetric unit. A 96% complete data set was collected at 298 K using Cu Kalpha Xrays from a rotating-anode generator. Solution of the crystal structure of Y75N mutant M. pusillus pepsin is under way by molecular replacement using the molecu lar coordinates of wild-type M. pusillus pepsin as a model. Badiefar, L., G. Ahmadian, et al. (2009). "Optimization of conditions for expres sion and activation of a splice variant of prochymosin lacking exon 6 in Escheri chia coli." International Journal of Dairy Technology 62(2): 265-271. A splice form of prochymosin lacking exon-6 was subcloned and expressed. Expression was optimized by changing various parameters. The maximum amount of protein expressed by this method was 44.61% of total cellular protein, compared to the 45.4% predicted by the Qualitek-4 software. The expressed protein was the n purified using ion exchange chromatography. Activation of the recombinant proc hymosin was carried out by changing different parameters. Optimal conditions for activation involved reduction of pH to 5 and 4 h of incubation with the acidic solution, followed by neutralization and 2 h of incubation under neutralized con ditions. The activity of the subsequent enzyme sample was 26 units/mL. Badiefar, L., M. Khodabandeh, et al. "A comparison of the production efficiencie s of full-length and truncated forms of prochymosin." International Journal of D airy Technology 64(2): 288-293.

Chymosin is one of the enzymes with many applications in the food indust ry. Its recombinant type has been designed and constructed in different forms. I n this investigation, the production efficiency of two types of complete and exo n-6 less prochymosin was assessed. It was found that the average efficiencies of the optimum growth conditions calculated for the two constructs harbouring pETprochymosin and pET-exon-6 less prochymosin were significantly different (P valu e < 0.05) at 192.02 +/- 3.57 and 348.87 +/- 7.76, respectively. Also during ferm entation, the level of the exon-6 less prochymosin production was much higher th an the other construct as demonstrated by SDS-PAGE. Bailey, D., J. B. Cooper, et al. (1993). "X-RAY-CRYSTALLOGRAPHIC STUDIES OF COMP LEXES OF PEPSTATIN-A AND A STATINE-CONTAINING HUMAN RENIN INHIBITOR WITH ENDOTHI APEPSIN." Biochemical Journal 289: 363-371. H-189, a synthetic human renin inhibitor, and pepstatin A, a naturally o ccurring inhibitor of aspartic proteinases, have been co-crystallized with the f ungal aspartic proteinase endothiapepsin (EC3.4.23.6). H-189 [Pro-His-Pro-Phe-Hi s-Sta (statyl)-Val-Ile-His-Lys] is an analogue of human angiotensinogen. Pepstat in A [Iva(isovaleryl)-Val-Val-Sta-Ala-Sta] is a blocked pentapeptide which inhib its many aspartic proteinases. The structures of the complexes have been determi ned by X-ray diffraction and refined to crystallographic R-factors of 0. 1 5 and 0. 16 at resolutions of 0. 1 8 nm (1.8 angstrom) and 0. 2 nm (2.0 angstrom) res pectively. H-189 is in an extended conformation, in which the statine residue is a dipeptide analogue of P1 and P1' as indicated by the conformation and network of contacts an hydrogen bonds. Pepstatin A has an extended conformation to the P2' alanine residue, but the leucyl side chain of the terminal statine residue b inds back into the S1' subsite, and an inverse gamma-turn occurs between P1', an d P3'. The hydroxy moiety of the statine at P1 in both complexes displaces the s olvent molecule that hydrogen-bonds with the catalytic aspartate residues (32 an d 215) in the native enzyme. Solvent molecules originally present in the native structure at the active site are displaced on inhibitor binding (12 when pepstat in A binds; 16 when H-189 binds). Bakri, M. and U. S. Ashworth (1973). "PURIFICATION OF CHYMOSIN (RENNIN)." Journa l of Dairy Science 56(5): 623-623. Baliu, E. and H. M. Wang (1995). "INCREASING THE YIELD OF RECOMBINANT BOVINE CHY MOSIN BY ASPERGILLUS-NIGER VAR AWAMORI BY MODIFICATION OF THE EXPRESSION VECTOR. " Abstracts of Papers of the American Chemical Society 209: 190-BIOT. Banerjee, A. C., A. Kundu, et al. (2003). Genetic manipulation of filamentous fu ngi. New Horizons in Biotechnology. S. Roussos, C. R. Soccol, A. Pandey and C. A ugur: 193-198. Biotechnology, a multidisciplinary science, has many applications. A maj or application in chemical and pharmaceutical industries involves the use of enz ymes, mostly from microbial sources, in the production process of many useful pr oducts. Various compounds including many proteins and enzymes are produced throu gh microbial fermentation. Filamentous fungi, with their many virtues and long h istory of industrial use, have lately become targets for gene manipulation for p roducing strains with improved yield and better expression-secretion system for homologous and heterologous eukaryotic gene products. To genetically modify fila mentous fungi for enhanced expression of a desired gene and secretion of its pro duct, it requires a suitably designed gene-construct with correctly chosen and a ligned sequences of the gene of interest and its controlling elements, as well a s an efficient gene-transfer system for transforming the host fungus. An importa nt feature of the transformation of filamentous fungi is that the transforming D NA stably integrates into the host genome. Substantial research for understandin g the molecular aspects of filamentous fungal expression systems is necessary fo r its biotechnological application in an industrial context. Banks, J. M. (1992). "YIELD AND QUALITY OF CHEDDAR CHEESE PRODUCED USING A FERME

NTATION-DERIVED CALF CHYMOSIN." Milchwissenschaft-Milk Science International 47( 3): 153-156. The yield and flavour characteristics of Cheddar cheese produced using f ermentation-derived calf chymosin in place of calf rennet was examined. There wa s no disadvantage in terms of yield on using calf chymosin as the clotting agent and the flavour of the cheese was equivalent to that produced using a standard calf rennet. Bansal, N., M. A. Drake, et al. (2009). "Suitability of recombinant camel (Camel us dromedarius) chymosin as a coagulant for Cheddar cheese." International Dairy Journal 19(9): 510-517. Cheddar-type cheeses were manufactured using fermentation-produced camel or calf chymosin. There were no significant differences in the composition and pH between the cheeses made with either coagulant. The extent of primary proteol ysis was significantly lower in cheeses made with camel chymosin than in cheeses made with calf chymosin. There were large quantitative differences between the peptide profiles of cheeses: however, the levels of amino acids were similar exc ept for isoleucine, histidine and lysine. The cheeses made with camel chymosin w ere characterized by lower intensities of sulphur and brothy flavours and showed less bitter taste; however, the cheeses made with calf chymosin had greater bre akdown of texture, higher smoothness and mouthcoating and were more cohesive and adhesive. The results of this study suggest that camel chymosin appears to be s uitable for making Cheddar cheese with lower levels of proteolysis but with good flavour. (C) 2009 Elsevier Ltd. All rights reserved. Bansal, N., P. F. Fox, et al. "Inhibition of rennet activity in cheese using equ ine blood serum." Dairy Science & Technology 90(6): 673-685. The objective of this study was to inactivate chymosin in Cheddar-type c heese curd using equine blood scrum This blood serum was added (0 1-2%, v/v) to cheesemilk at 15 degrees C after completion of the first phase of renneting and before aggregation of the rennet-altered casein micelles to inhibit the residual coagulant in Cheddar-type cheeses Throughout ripening the level of pH 4 6-solub le nitrogen expressed as a percentage of total nitrogen was significantly higher in the control cheeses than in experimental cheeses and was about twice that of the experimental cheeses after ripening for 180 days During ripening, there was almost no hydrolysis of alpha(si)-casein in the cheeses made from milk containi ng 0 25-2% blood serum Throughout ripening there were large quantitative differe nces between the peptide profiles of control and experimental cheeses The result s of this study suggest that the addition of equine blood serum to cheesemilk (0 25-2%) was very effective at inhibiting the residual chymosin activity in Chedda r-type cheeses during ripening, tit( activity of plasmin remained unaffected by the added blood serum This study describes an easy and effective method for prod ucing cheese curd free from residual coagulant activity, which will help to stud y the separate roles of the coagulant and other proteolytic enzymes in cheese ri pening Bansal, N., P. F. Fox, et al. "Inhibition of rennets by blood serum." Internatio nal Journal of Dairy Technology 63(3): 381-386. Incubation of calf chymosin with the serum from blood of various mammali an species before addition to milk reduced its milk-clotting activity at 30 degr ees C; the inhibitory potency of the sera was in the order horse > goat > cattle > donkey = human. For all species, inhibition of calf chymosin increased on inc reasing both the incubation time and the level of blood serum. Horse blood serum inhibited other milk coagulants in the order porcine pepsin > Cryphonectria par asitica proteinase = calf chymosin > Rhizomucor miehei proteinase = bovine pepsi n. Methylamine-treated blood serum did not inhibit calf chymosin, suggesting tha t alpha(2)-macroglobulin may be the principal inhibitor of rennet in blood serum . Bansal, N., P. F. Fox, et al. (2007). "Aggregation of rennet-altered casein mice

lles at low temperatures." Journal of Agricultural and Food Chemistry 55(8): 312 0-3126. The rennet-induced coagulation of bovine milk at 10 degrees C was invest igated. The rate of change of absorbance at 600 nm was higher in milk renneted a t 30 degrees C than that at 10 degrees C. The amount of casein sedimented on cen trifuging skim milk at 5000g for 1 h at 10 degrees C increased with time after r enneting. The viscosity of milk at 10 degrees C at low shear rates did not chang e significantly until 10 h after rennet addition, but it increased markedly afte r 20 h. Smaller particles in milk at 10 degrees C disappeared slowly over 36 h a fter rennet addition and aggregated into larger particles. These results suggest ed that casein micelles in milk aggregate at low temperatures. Reasons for the s low aggregation of milk renneted at 10 degrees C were investigated by inhibiting chymosin activity by pepstatin A. It is likely that beta-casein, or its hydroly sis, plays a role in aggregation of rennet-altered casein micelles at low temper atures. Bansal, N., P. F. Fox, et al. (2007). "Factors affecting the retention of rennet in cheese curd." Journal of Agricultural and Food Chemistry 55(22): 9219-9225. The coagulant retained in cheese curd is a major contributor to proteoly sis during ripening. The objective of this study was to quantify the effects of several milk-related factors and parameters during cheese manufacture on the ret ention of coagulant in cheese curd. The amount of coagulant retained in curd was determined by its activity on a synthetic heptapeptide (Pro-Thr-Glu-Phe-[NO2-Ph e]-Arg-Leu) using reversed-phase HPLC. The retention of chymosin in cheese curd increased significantly when the pH of milk was reduced at rennet addition below pH 6.1, the pH at whey drainage below pH 5.7, or the average casein micelle siz e in milk and when the ionic strength of milk was increased. The casein content of milk and the quantity of chymosin added to milk had no significant, effect on the retention of chymosin in curd; the quantity of coagulant bound per gram of casein remained unchanged. Bansal, N., P. F. Fox, et al. (2008). "Factors that affect the aggregation of re nnet-altered casein micelles at low temperatures." International Journal of Dair y Technology 61(1): 56-61. The effects of temperature, CaCl2 concentration, pH and ionic strength o f milk on the aggregation of casein micelles in milk renneted at 15 degrees C we re studied using particle size analysis determined by laser-light scattering. Th e rate of aggregation of rennet-altered casein micelles became significantly slo wer on reducing the temperature of renneting from 30 to 10 degrees C. At 15 degr ees C, the rate of aggregation of rennet-altered casein micelles increased signi ficantly on adding CaCl2, on reducing the pH of renneted milk or on adding NaCl (up to 50 mM). These results indicate that particle size analysis can be used su ccessfully to study the aggregation of rennet-altered casein micelles. Bansal, N., P. F. Fox, et al. (2009). "Comparison of the level of residual coagu lant activity in different cheese varieties." Journal of Dairy Research 76(3): 2 90-293. The coagulant retained in cheese curd is a major contributor to proteoly sis during ripening. The objective of this study was to quantify residual coagul ant in 9 cheese varieties by measuring its activity on a synthetic heptapeptide (Pro-Thr-Glu-Phe-[NO2-Phe]-Arg-Leu) assayed using reversed-phase HPLC. The level of residual coagulant activity was highest in Camembert cheese, probably due to its low pH at whey drainage and the high moisture content of the cheese, follow ed in order by Feta=Port du Salut= Cheddar>Gouda>Emmental = Parmigiano Reggiano= low-moisture part-skim Mozzarella =Mozzarella di Bufala Campana. The high cooki ng temperature (50-54 degrees C) used during the manufacture of Emmental and Par migiano Reggiano cheeses and the cooking and stretching step in hot water during the manufacture of Mozzarella cheese may be the reasons for the lowest residual coagulant activity in these cheeses. The level of residual coagulant activity w as higher in Feta cheese made from milk concentrated by ultrafiltration than in

conventional Feta. Bansal, P. S., P. A. Grieve, et al. (2006). "Chemical synthesis and structure el ucidation of bovine kappa-casein (1-44)." Biochemical and Biophysical Research C ommunications 340(4): 1098-1103. The caseins (alpha(s1), alpha(s2), beta, and kappa) are phosphoproteins present in bovine milk that have been studied for over a century and whose struc tures remain obscure. Here we describe the chemical synthesis and structure eluc idation of the N-terminal segment (1-44) of bovine K-casein, the protein which m aintains the micellar structure of the caseins. K-Casein (1-44) was synthesised by highly optimised Boc solid-phase peptide chemistry and characterised by mass spectrometry. Structure elucidation was carried out by circular dichroism and nu clear magnetic resonance spectroscopy. CD analysis demonstrated that the segment was ill defined in aqueous medium but in 30% trifluoroethanol it exhibited cons iderable helical structure. Further, NMR analysis showed the presence of a helic al segment containing 26 residues which extends from Pro(8) to Arg(34). This is the first report which demonstrates extensive secondary structure within the cas ein class of proteins. (c) 2006 Elsevier Inc. All rights reserved. Baranyi, M., L. Hiripi, et al. (2007). "Isolation and some effects of functional , low-phenylalanine kappa-casein expressed in the milk of transgenic rabbits." J ournal of Biotechnology 128(2): 383-392. Patients suffering certain metabolic diseases (e.g. phenylketonuria) nee d a low-phenylalanine diet throughout their lives. Transgenic rabbits were creat ed to express low-phenylalanine kappa-casein in their milk. The aim was to demon strate for the first time the feasibility of producing a modified milk protein i n addition to normal milk proteins. A gene construct containing the coding regio n of the rabbit K-casein gene was modified by site-specific oligonucleotide dire cted mutagenesis. Four of the five phenylalanine amino acids present in the matu re protein were mutated and the gene construct was used to create two transgenic rabbit lines. The transgenic rabbits produced the recombinant kappa-casein at a high level in their milk causing a reduction in the average size of the casein micelles. The low-phenylalanine kappa-casein was digestible with chymosin and it was separated from its native counterpart and from the other milk proteins by a one-step HPLC method on a reversed-phase column. In the future, low-phenylalani ne casein produced in transgenic animals could be used as dietary replacements t o meet the special requirements of certain consumer groups. (c) 2006 Elsevier B. V. All rights reserved. Barbano, D. M. and R. R. Rasmussen (1992). "CHEESE YIELD PERFORMANCE OF FERMENTA TION-PRODUCED CHYMOSIN AND OTHER MILK COAGULANTS." Journal of Dairy Science 75(1 ): 1-12. Fat recovery, protein recovery, and cheese yield performance of a fermen tation-produced chymosin was compared with other commonly used milk coagulants. In trial 1, performance of fermentation-produced chymosin was compared with prot eases from Mucor miehei and Mucor pusillus. In trial 2, fermentation-produced ch ymosin was compared with calf rennet and adult bovine pepsin. In each trial, thr ee vats of Cheddar cheese were made simultaneously from the same milk, using the same starter culture, with the three different coagulants. This was replicated 12 times in trial 1 and 9 times in trial 2. Generally, higher fat and protein lo sses in the whey were observed for proteases from Mucor miehei and Mucor pusillu s than for fermentation-produced chymosin or calf rennet. Adult bovine pepsin ha d higher fat losses in the whey, but not higher protein losses in the whey, than fermentation-produced chymosin or calf rennet. In trial 1, fermentation-produce d chymosin had a higher cheese yield efficiency than proteases from Mucor miehei and Mucor pusillus (.54 and .74%, respectively) with a protected least signific ant difference of .34%. In trial 2, fermentation-produced chymosin (100% chymosi n) and calf rennet (94% chymosin) had virtually identical cheese yield efficienc ies, but adult bovine pepsin had a lower (.39%) cheese yield efficiency with a p rotected least significant difference of .27%.

Baron, M., G. Tiraby, et al. (1992). "EFFICIENT SECRETION OF HUMAN LYSOZYME FUSE D TO THE SH BLE PHLEOMYCIN RESISTANCE PROTEIN BY THE FUNGUS TOLYPOCLADIUM-GEODES ." Journal of Biotechnology 24(3): 253-266. Tolypocladium geodes strain NC50 was transformed by different integratin g vectors bearing both a synthetic gene encoding human lysozyme (HLz) and the Sh ble phleomycin resistance marker, either in separate expression cassettes or in transcriptional or translational fusion configurations. Clones derived from all vectors were able to secrete HLz. The highest productivities in shake flasks (u p to 150 mg l-1 in 5 days) were obtained when HLz was fused at the C-terminal en d of the Sh ble protein. The fusion protein is efficiently secreted and release of active lysozyme occurs by extracellular proteolytic cleavage in the junction peptide. Barros, R. M., C. I. Extremina, et al. (2003). "Hydrolysis of alpha-lactalbumin by cardosin A immobilized on highly activated supports." Enzyme and Microbial Te chnology 33(7): 908-916. In the present research effort, production of derivatives of cardosin A (a plant protease) encompassing full stabilization of its dimeric structure has been achieved, via covalent, multi-subunit immobilization onto highly activated agarose-glutaraldehyde supports. Boiling such enzyme derivatives in the presence of sodium dodecyl sulfate and beta-mercaptoethanol did not lead to leaching of enzyme, thus providing evidence for the effectiveness of the attachment procedur e. Furthermore, the cardosin A derivatives prepared under optimal conditions pre sented ca. half the specific activity of the enzyme in soluble form, and were su ccessfully employed at laboratory-scale trials to perform (selective) hydrolysis of alpha-lactalbumin (alpha-La), one of the major proteins in bovine whey. Hydr olysates of alpha-La were assayed for by the OPA method, as well as by FPLC, SDS -PAGE and HPLC. Thermal inactivation of the immobilized cardosin A was also asse ssed at 40, 50 and 55 degreesC; at these temperatures, no thermal denaturation t ook place during incubation for 48 h. The highest degree of hydrolysis was attai ned by 5 h reaction, at 55 degreesC and pH 5.2. SDS-PAGE of alpha-La hydrolysate s displayed bands corresponding to low molecular weight peptides. Our results su ggest that cardosin A in immobilized form is a good candidate to bring about pro teolysis in the dairy industry, namely in whey processing. (C) 2003 Elsevier Inc . All rights reserved. Bartlett, F., N. F. Haard, et al. (1984). "EFFECT OF HEAT-STABLE PROTEASES OF PS YCHROTROPHIC PSEUDOMONADS ON THE MILK CLOTTING BY CHYMOSIN." Canadian Institute of Food Science and Technology Journal-Journal De L Institut Canadien De Science Et Technologie Alimentaires 17(3): R28-R28. Bassani, G., P. Fucinos, et al. "Candida rugosa lipase Lip1-polyethyleneglycol i nteraction and the relation with its partition in aqueous two-phase systems." Co lloids and Surfaces B-Biointerfaces 75(2): 532-537. The interaction between a lipase from Candida rugosa (Lip1) and polyethy leneglycols of different molecular masses was studied using fluorescence and cir cular dichroism approaches in order to be applied to the analysis of the enzyme partition mechanism in aqueous two-phase systems of polyethyleneglycol-potassium phosphate. The decrease of the partition coefficients with the polyethyleneglyc ol molecular mass showed that the enzyme partition is driven by the excluded vol ume effect and not by the enzyme-polymer interaction. The polymer did not affect the secondary and tertiary structure of the enzyme nor its biological activity. The lipase from Candida rugosa lyophilizate was partitioned in favour of the po lyethyleneglycol rich phase; PEG 2000 being the system which showed the better e nzyme recovery (78.26%) with a purification factor of 2.3. This method could be applied as a first step to isolate the enzyme from a culture medium with good re covery and without modifying the enzymatic capacity and the molecular structure. (C) 2009 Elsevier B.V. All rights reserved.

Bauer, R., M. Hansen, et al. (1995). "THE STRUCTURE OF CASEIN AGGREGATES DURING RENNETING STUDIED BY INDIRECT FOURIER TRANSFORMATION AND INVERSE LAPLACE TRANSFO RMATION OF STATIC AND DYNAMIC LIGHT-SCATTERING DATA, RESPECTIVELY." Journal of C hemical Physics 103(7): 2725-2737. Aggregation of casein micelles after addition of the proteolytic enzyme chymosin has been studied by static and dynamic light scattering at three differ ent concentrations of casein corresponding to dilutions 1:100, 1:500, and 1:1000 of native milk. The static light scattering data have been analyzed by an indir ect Fourier transformation method which gives the distance distributions as a fu nction of timer From these curves radius of gyration and an average number of ca sein micelles in the aggregates have been derived as a function of time. The dyn amic light scattering experiments give the hydrodynamic radius as a function of time after the addition of rennet. The initial radius of gyration for the intact casein micelles is 140 nm. The corresponding hydrodynamic radius is also 140 nm . This shows that the casein micelles are not solid spheres. Inspection of a plo t of relative mass versus radius of gyration for the aggregates appearing after the addition of chymosin shows that two processes take place. First extended lin ear aggregates are built up to a relative mass of the aggregates of about 10 and then restructuring of aggregates occurs such that increasingly compact objects are formed. Whereas the first process exhibits a relatively fast growth in size, the aggregates grow slowly in size during the second process. Further evidence of the formation of linear aggregates followed by more dense aggregates was obta ined by forming the ratio between the radius of gyration and the hydrodynamic ra dius. This ratio increases to values of about 2.5 (indicating that linearly exte nded molecules are present followed) by a decrease to about 1. The log-log plot of mass versus radius of gyration ils linear up to relative masses of about 10 w ith a slope of about 2. This extends up to sizes of 1 mu m in diameter. The slop e then increases to values indicating branching and thereby the formation of mor e compact aggregates. For relative masses below 10 and sizes below 1 mu m sedime ntation is unlikely to occur and information about the mechanism of aggregation can be obtained. The aggregation number as a function of time has been analyzed in terms of Smoluchowski's equations with a rate constant including both functio nality and a changing barrier height as a function of the extent of proteolysis. The functionality obtained from Smoluchowski's equations is about 2.1. (C) 1995 American Institute of Physics. Beckers, J. F. (1999). "Inactive members of the aspartic proteinase family, the ruminants pregnancy-associated glycoproteins for serological diagnosis." Annales De Medecine Veterinaire 143(4): 253-+. During the last decade, investigations were carried out in the Faculty o f Veterinary Medicine in order to characterize proteins or glycoproteins synthes ized in the ruminant placenta. Recently, as a result of this research, a large f amily of pregnancy-associated glycoproteins was discovered. Using molecular biol ogy techniques, they were found to be members of the superfamily of the aspartic proteinases which also contains pepsinogen, cathepsines D&E, chymosin, renin et c. Synthesized in the mono or binucleate cells of the trophonlast, the pregnancy -associated glycoproteins (PAG) do not have proteinase activity. It is likely th ey are synthesized together with molecules involved in the tissue remodeling of the placenta. Their release in large quantities into the maternal blood circulat ion results in mesurable plasma concentrations. Thanks to international collabor ative studies, we have shown that PAG levels are a good indicator of foetoplacen tal well being and that sharp decreases in PAG levels occur just before pregnanc y failures in cows and in goats. In Belgium, the PAG assay is available for Doct ors in Veterinary Medicine in the Province laboratories responsible for animal h ealth including immunodiagnosis for brucellosis, leucosis, IBR, BVD, CAEV, VISNA MEDI etc. Beeby, R. (1976). "COMPARISON BETWEEN AMINO-ACID COMPOSITIONS OF 2 CHYMOSIN-SENS ITIVE POLYPEPTIDES AND C-TERMINAL SEGMENTS OF KAPPA-CASEIN." Journal of Dairy Re search 43(1): 37-43.

Beeby, R. (1979). "PROTEOLYSIS OF CASEIN BY IMMOBILIZED PREPARATIONS OF ALPHA-CH YMOTRYPSIN, CHYMOSIN AND A FUNGAL PROTEASE." New Zealand Journal of Dairy Scienc e and Technology 14(1): 1-11. Beeby, R. (1980). "THE USE OF FLUORESCAMINE AT PH 6.0 TO FOLLOW THE ACTION OF CH YMOSIN ON KAPPA-CASEIN AND TO ESTIMATE THIS PROTEIN IN MILK." New Zealand Journa l of Dairy Science and Technology 15(2): 99-108. Beldarrain, A., N. Acosta, et al. (2000). "Characterization of Mucor pusillus re nnin expressed in Pichia pastoris: enzymic, spectroscopic and calorimetric studi es." Biotechnology and Applied Biochemistry 31: 77-84. Bencini, R. (2002). "Factors affecting the clotting properties of sheep milk." J ournal of the Science of Food and Agriculture 82(7): 705-719. A Formagraph was used to test the effects of some of the exogenous facto rs that can affect the processing properties of milk (pH, soluble calcium, renne t concentration, coagulation temperature), and two of the endogenous factors (pr otein and fat concentration), on the comparative clotting properties of sheep an d cows' milk, namely renneting time (r), rate of firming (k20) and curd consiste ncy (A30). A lower pH decreased r and k20 and increased A30 in both sheep and co ws' milk. The addition of calcium chloride did not affect the clotting propertie s of sheep milk, but in cows' milk it decreased r and k20 and increased A30. Inc reasing the concentration of rennet decreased r and k20 and increased A30 for bo th sheep and cows' milk. Increasing the coagulation temperature from 30 to 38 de greesC decreased r for both sheep and cows' milk, but it decreased k20 and incre ased A30 only in cows' milk. Increasing the protein concentration decreased r in both sheep and cows' milk; it did not affect k20 of sheep milk, but it decrease d that of cows' milk and increased A30 in both milks. Increasing the fat concent ration had little effect on r and k20 in either sheep cows' milk, but it decreas ed A30 in both milks. In general, sheep milk had faster renneting times and rate s of firming and greater curd consistency than cows' milk, and its clotting prop erties tended to be less affected by changes in the clotting conditions. (C) 200 2 Society of Chemical Industry. Benkerroum, N., M. Dehhaoui, et al. "The effect of concentration of chymosin on the yield and sensory properties of camel cheese and on its microbiological qual ity." International Journal of Dairy Technology 64(2): 232-239. The transformation of camel milk into soft cheese by using chymosin and yoghurt starter culture (Streptococcus thermophilus and Lactobacillus bulgaricus ) was investigated. The cheese yield and sensory properties were related to the concentration of chymosin. A yield of 16.74 g/100 mL of milk was obtained with a chymosin concentration of 1.7 mL/L of milk. The cheeses obtained with concentra tions ranging between 1.0 mL and 2.9 mL of chymosin/L of milk scored highly rega rding their sensory properties and had an acceptable microbiological quality. Th is study demonstrated that cheesemaking from camel milk can be made successfully providing that the appropriate chymosin concentration is used; and that 1.7 mL of chymosin/L of milk was optimal. Beppu, T. (1996). "Genes, enzymes and secondary metabolites in industrial microo rganisms - The 1995 Thom Award lecture." Journal of Industrial Microbiology 16(6 ): 360-363. Apparently contrasting approaches, ie genetic engineering and screening of new microorganisms, play essential complementary roles to develop current ind ustrial microbiology, Three topics, production and modification of milk-clotting proteinases by genetic engineering, hormonal control of secondary metabolism in streptomycetes, and screening of bioactive metabolites, are introduced as cases of such a hybrid approach, while symbiotic microorganisms are discussed as an e xample of the vast terra incognita still remaining for the future microbiology.

Berankova, E., P. Rauch, et al. (1989). "DETERMINATION OF CHYMOSIN AND BOVINE PE PSIN-A ACTIVITY IN COMBINED RENNETS ON THE BASIS OF IMMUNOCHEMICAL INHIBITION." Journal of Dairy Research 56(4): 631-637. Berankova, E., J. Sajdok, et al. (1988). "DETERMINATION OF CHYMOSIN AND BOVINE P EPSIN CONTENT OF BOVINE RENNETS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY." Neth erlands Milk and Dairy Journal 42(3): 337-340. Berka, R. M. (1991). "HETEROLOGOUS GENE-EXPRESSION IN FILAMENTOUS FUNGI - RECENT IMPROVEMENTS IN PRODUCTION OF BOVINE CHYMOSIN IN ASPERGILLUS-NIGER VAR AWAMORI. " Abstracts of Papers of the American Chemical Society 202: 234-BIOT. Berka, R. M. (1991). "SYSTEMS AND APPROACHES FOR EXPRESSION AND SECRETION OF HET EROLOGOUS PROTEINS IN THE FILAMENTOUS FUNGUS ASPERGILLUS-NIGER VAR AWAMORI - CUR RENT STATUS." Annals of the New York Academy of Sciences 646: 207-211. Berka, R. M., K. H. Kodama, et al. (1991). "THE DEVELOPMENT OF ASPERGILLUS-NIGER VAR AWAMORI AS A HOST FOR THE EXPRESSION AND SECRETION OF HETEROLOGOUS GENE-PRO DUCTS." Biochemical Society Transactions 19(3): 681-685. Bines, V. E., P. Young, et al. (1989). "COMPARISON OF CHEDDAR CHEESE MADE WITH A RECOMBINANT CALF CHYMOSIN AND WITH STANDARD CALF RENNET." Journal of Dairy Rese arch 56(4): 657-664. Bischofberger, T. and Z. Puhan (1979). "CONTINUOUS EXTRACTION OF CHYMOSIN FROM F RESH CALF STOMACHS WITH A CELL-EXTRACTOR." Milchwissenschaft-Milk Science Intern ational 34(10): 614-617. Blundell, T. L., K. Guruprasad, et al. (1997). "Aspartic proteinases: From the f irst X-ray photos of pepsin crystals to hundreds of 3-D structures." Current Sci ence 72(7): 483-489. Bocking, S. P., M. G. Wiebe, et al. (1999). "Effect of branch frequency in Asper gillus oryzae on protein secretion and culture viscosity." Biotechnology and Bio engineering 65(6): 638-648. Highly branched mutants of two strains of Aspergillus oryzae (IFO4177, w hich produces alpha-amylase, and a transformant of IFO4177 [AMG#13], which produ ces heterologous glucoamylase in addition to alpha-amylase) were generated by UV or nitrous acid mutagenesis. Four mutants of the parental strain (IFO4177), whi ch were 10 to 50% more branched than the parental strain, were studied in stirre d batch culture and no differences were observed in either the amount or the rat e of enzyme production. Five mutants of the transformed parental strain (AMG#13) , which were 20 to 58% more branched than the parental strain, were studied in e ither batch, fed-batch or continuous culture. In batch culture, three of the mut ants produced more glucoamylase than the transformed parental strain, although o nly two mutants produced more glucoamylase and alpha-amylase combined. No increa se in enzyme production was observed in either chemostat or fed-batch culture. C ultures of highly branched mutants were less viscous than those of the parental and transformed parental strains. A linear relationship was found between the de gree of branching (measured as hyphal growth unit length) and culture viscosity (measured as the torque exerted on the rheometer impeller) for these strains. DO T-controlled fed-batch cultures (in which the medium feed rate was determined by the DOT) were thus inoculated with either the transformed parent or highly bran ched mutants of the transformed parent to determine whether the reduced viscosit y would improve aeration and give higher enzyme yields. The average rate of medi um addition was higher for the two highly branched mutants (ca. 8.3 g medium h(1)) than for the parental strain (5.7 g medium h(-1)). Specific enzyme productio n in the DOT controlled fed-batch cultures was similar for all three strains (ap prox. 0.24 g alpha-amylase and glucoamylase [g of biomass](-1)), but one of the highly branched mutants made more total enzyme (24.3 +/- 0.2 g alpha-amylase and

glucoamylase) than the parental strain (21.7 +/- 0.4 g oc-amylase and glucoamyl ase). (C) 1999 John Wiley & Sons, Inc. Bodie, E. A., G. L. Armstrong, et al. (1994). "STRAIN IMPROVEMENT OF CHYMOSIN-PR ODUCING STRAINS OF ASPERGILLUS-NIGER VAR AWAMORI USING PARASEXUAL RECOMBINATION. " Enzyme and Microbial Technology 16(5): 376-382. Parasexual recombination was used to obtain improved chymosin-producing strains and to perform genetic analysis on existing strains. Chlorate resistance was used to select for a variety of spontaneous nitrate assimilation pathway mu tations in strains previously improved for chymosin production using classical s train improved methods including mutation and screening, and selection for 2-deo xyglucose resistance (dgr). Diploids of these improved strains were generated vi a parasexual recombination and were isolated on selective media by complementati on of nitrate assimilation mutations. A preliminary genetic analysis of diploid and haploid segregants indicated that the dgr trait, resulting in overexpression of chymosin, was recessive. Also, mutations in two different dgr genes resulted in an increased level of chymosin production. When these mutations were combine d via parasexual recombination, the resulting haploid segregants produced about 15% more chymosin than either pal ental strain. CHEF gel electrophoresis was use d to determine the chromosomal location of the integrated chymosin DNA sequences , and to verify diploidy in one case where the chromosome composition of two hap loid parents differed. Bornaz, S., N. Guizani, et al. "Effect of Plant Originated Coagulants and Chymos in on Ovine Milk Coagulation." International Journal of Food Properties 13(1): 1 0-22. The coagulation of ewe's milk was studied by using plant source coagulan ts namely the artichoke, Cynara scolymus L. cv. Blanca, and latex from the fig t ree (Ficus carica L.). A turbidimetric method was used to evaluate and compare t he coagulation properties of the novel coagulants with chymosin treated samples. Syneresis capacity and sensory evaluation of resultant cheese samples were stud ied and it was found that both cynara and chymosin produced sigmoidal increase i n turbidity to the milk with three distinct phases. The coagulati