Food Chemistry 126 (2011) 46–53

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Food Chemistry

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Purification and characterisation of a novel protease from Cordyceps sinensisand determination of the cleavage site motifs using oriented peptide librarymixtures

Bo Bi, Xinyu Wang, Hezhen Wu, Qun Wei ⇑Department of Biochemistry and Molecular Biology, Beijing Normal University, Beijing Key Laboratory, Beijing 100875, PR China

a r t i c l e i n f o

Article history:Received 22 April 2010Received in revised form 21 August 2010Accepted 14 October 2010

Keywords:ProteasePurificationCleavage site motifsOriented peptide libraryCordyceps sinensis

0308-8146/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.10.057

⇑ Corresponding author. Tel./fax: +86 10 58807365E-mail address: weiq@bnu.edu.cn (Q. Wei).

a b s t r a c t

A novel protease, from the edible fungus Cordyceps sinensis, was purified and characterised. Its cleavagesite motifs were determined by oriented peptide library mixtures and validated by synthetic peptides andnatural proteins.

The protease was purified to homogeneity using anion-exchange chromatography, sieve chromatogra-phy, native PAGE and reversion phase chromatography. Its molecular weight, estimated by SDS–PAGE,was approximately 43 kDa. The results of MS-MS, MALDI-TOF MS and de novo sequencing demonstratedthat it was a completely new protease.

We used oriented peptide library mixtures to determine cleavage site motifs. Cleavage requires lysineat P1 and proline or lysine at P30. P2 and P10 also show some preference. A series of synthetic peptides andnatural proteins were used to validate the substrate specificity. The protease has special substrate pref-erences different from other proteases. It also has excellent biochemical properties, which make it able towithstand harsh conditions and suitable for industrialisation and commercialisation.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Proteases are enzymes that catalyse the breakdown of proteinsby hydrolysis of peptide bonds. Protease are ubiquitous in biolog-ical systems, and perform diverse vital functions, such as regulat-ing protein processing and intracellular protein levels andremoving abnormal or damaged proteins from the cell. They are in-volved in many roles such as development, physiology, defenceand stress responses. Proteases are also widely used in scientificfields, e.g. protein chemistry and protein engineering, and in differ-ent industrial processes, such as food, pharmaceutical, peptide syn-thesis and detergent (Gupta, Beg, & Lorenz, 2002; Kumar & Takagi,1999). They constitute one of the most important groups of en-zymes and account for 60% of the total world enzyme production(Rao, Tankasale, Ghatge & Desphande, 1998b).

Based on active site and their mechanism of catalysis, proteasescan be divided into seven groups by the MEROPS database (theMEROPS database is an information resource for peptidases andthe proteins that inhibit them. http://merops.sanger.ac.uk): serineproteinases, cysteine (thiol) proteinases, aspartic proteinases,metalloproteinases, threonine proteases, glutamic acid proteasesand unknown proteases (Rawlings, Morton, Kok, Kong, & Barrett,

ll rights reserved.

.

2008). Serine proteases, named for the nucleophilic Ser residue atthe active site, are probably the most thoroughly investigated en-zyme system. Approximately 50 serine protease families are cur-rently classified by MEROPS, including almost one-third of allproteases. Serine proteases show a wide range of activities, andthey are involved in numerous biological processes, such as diges-tion, blood clotting, development, complement activation, patho-genesis, apoptosis, immune response and secondary metabolism(Barrett, 2004; Hedstrom, 2002).

Aspartic proteases, which have two highly conserved aspartatesin the active site, are important in health and disease. Some ofthem are involved in pathogens like HIV, Candida albicans orPlasmodium falciparum (Kempf, Sham, & Marsh, 1998). Cysteineproteases have a common catalytic mechanism that involves anucleophilic cysteine thiol in a catalytic triad. Many importantcommercially available proteases are cysteine proteases, such aspapain, bromelain and caspases. Metalloproteinases have metalions (zinc or cobalt) bound at the active site. Threonine proteaseshave N-terminal threonine in the active site (Löwe et al., 1995).Glutamic acid proteases were not described until the discovery ofthe importance of glutamate and glutamine residues in the activesites of proteases (Fujinaga, Cherney, Oyama, Oda, & James,2004). They are found only in bacteria, fungi and archaea.

At present, microbial proteases constitute approximately 40% ofthe total worldwide production of enzymes (Godfrey & West,1996). Although a large proportion of commercially available

B. Bi et al. / Food Chemistry 126 (2011) 46–53 47

proteases is derived from bacteria, especially the genus Bacillus(Mehrota, Pandey, Gaur, & Darmwal, 1999), the increasing demandfor proteases with specific properties has led biotechnologists toexplore newer sources of proteases. However, few proteases fromfungi have been purified and characterised. Fungal proteases havean added advantage over bacterial proteases in that large quanti-ties of enzyme are obtained from the biological source. Microbe-free broth can easily be obtained by conventional filtration of fungi,whereas cost-intensive filtration technology is required for isola-tion of the bacterial enzyme. Therefore, the potential use of fungalsources is now being increasingly realised (Phadatare, Deshpande,& Srinivasan, 1993; Samal, Karan, & Stabinsky, 1990).

Cordyceps sinensis, a member of the ergot family of fungi, is atraditional and precious Chinese medicinal herb which has beenused throughout Asia for centuries. Recent work with Cordycepssinensis has focused on tumor suppression properties, immuneactivation, radiation mitigation effect (Lin & Chiang, 2008; Wu,Zhang, & Leung, 2007), and sources of biochemicals with interest-ing biological and pharmacological properties, such as cordycepin,a drug helpful in tumor suppression and human organ transplants(Noh et al., 2009; Thomadaki, Scorilas, Tsiapalis, & Havredaki,2008).

In this paper we report, for the first time, purification and char-acterisation of a completely new protease produced by the rareedible fungus Cordyceps sinensis. We determined its cleavage mo-tifs using oriented peptide library mixtures and then compared itwith other proteases which have similar cleavage motifs.

2. Materials and methods

2.1. Protease activity analysis

Protein was measured by the classical method (Lowry,Rosebrough, Farr, & Rondal, 1951) with bovine serum albumin(BSA) as standard. The concentration of protein during purificationstudies was calculated from the standard curve.

Cleavage of substrates by the protease was assayed in 25 ll of20 mM Tris–HCl (pH 9.0) containing 1.0 mg/ml of natural substrateor 5.0 mM peptide at 30 �C for 30 min. The reaction was termi-nated by addition of 25 ll of 0.02 M NaOH. The substrate decreaseswere analyzed by Purifier with a reverse-phase column. Naturalsubstrate decreases were analyzed with SOURCE 15RPC and syn-thesised peptides with SOURCE 5RPC. The natural substrates weredetected at 215 nm and 280 nm, and synthesised peptides at215 nm. Cleavage of substrates was estimated from the diminutionof peak area of substrates or from the new peak of peptides.

One unit of protease was equivalent to the amount of enzymerequired to release 1 lmole of peptide (ARKQYP)/ml/min understandard assay conditions. Each substrate peptide was titratedfrom 0.4 mM to 10 mM. Kinetic parameters (Kcat and Km) weredetermined by fitting the velocity (initial rates at < 5.00% of totalsubstrate hydrolysis) versus substrate concentration data to theMichaelis–Menten equation. The initial velocity and steady-stateconditions for the enzyme reaction were established for each pep-tide substrate.

2.2. Protease purification

The material was mashed thoroughly and soaked with ten vol-umes of Tris–HCl buffer (20 mM Tris, pH 7.4) for 24 h at 4 �C. Super-natant of soak was obtained after centrifugation at 18000 rpm for30 min.

Purification was carried out at 4 �C. Crude extracts were firstpurified by anion-exchange chromatography on a HiTrap Q XL col-

umn (Pharmacia Biotechnology, 1 ml), which had been equili-brated with 20 mM Tris–HCl buffer, pH 7.4. After loading thesample, the column was washed with the same buffer until theoptical density of the effluent at 280 nm was zero. The bound pro-teins were then eluted with a linear gradient of sodium chloride(1 M) in the range 0–100% of the equilibrating buffer. The fractionswith high protease activity were collected and pooled, then con-centrated by lyophilisation. The residue was dissolved in 20 mMTris–HCl buffer, pH 7.4, and volume was made up to 1 ml.

The active fraction from the anion-exchange column was centri-fuged at 12 000 rpm for 15 min and then filtered through a0.22 mm filter. The sample was applied to the sieve chromatogra-phy on a Superdex 200 prep grade column (GE Healthcare, 120 ml),which had been equilibrated with 500 mM NaCl in 20 mM Tris–HClbuffer, pH 7.4. The fractions with high protease activity were col-lected and pooled, then concentrated by lyophilisation.

After concentration, active fractions were further purified byNative PAGE (10%) and collected by electro-elution (BIO-RADWhole Gel Eluter). Then, the fractions were desalted by a desaltingcolumn (GE Healthcare, 5 ml) and concentrated by lyophilisation.

Finally, the fractions were dissolved in 2% acetonitrile and puri-fied by reversion phase chromatography on a SOURCE 15RPC col-umn (GE Healthcare, 1.66 ml). The column had been equilibratedwith 2% acetonitrile. After loading the sample, the column waswashed with the same buffer until the optical density of the efflu-ent at 280 nm was zero. The bound proteins were then eluted witha linear gradient of 70% acetonitrile in the range 0–100% of theequilibrating buffer. The fractions with high protease activity werecollected and pooled, then concentrated by lyophilisation.

2.3. SDS–PAGE and amino acid sequence analysis

Molecular weight determination was performed by SDS–PAGEaccording to the method of Laemmli (Laemmli, 1970). The SDS–PAGE gel of the purified protease was detected by MS-MS, MALDI-TOF and de novo sequencing (by the Instrumental Analysis Centerof the Academy of Military Medical Sciences and Hunan NormalUniversity). And the protein N-terminal amino acid sequence wasdetermined by the Edman degradation method (by the InstrumentalAnalysis Center of the Academy of Military Medical Sciences).

2.4. Effects of pH, temperature, metal ions, oxidising agents, reducingagents, detergents and enzyme inhibitors on enzyme activity

Effects of pH, temperature, metal ions, oxidising agents, reduc-ing agents, detergents and enzyme inhibitors on purified enzymeactivity were determined by the standing procedure and assayedunder standard assay conditions.

2.5. Protease cleavage motifs prediction

We determined the cleavage motifs using an oriented peptidelibrary mixture according to a reported method (Turk, Huang, Piro,& Cantley, 2001). To validate the cleavage motifs, we used a seriesof synthetic peptides and several natural proteins for authentica-tion. Six peptides were synthesised according to the informationfrom the peptide library method. Purified protease was incubatedwith synthetic peptides and the remaining enzyme activity wasestimated under standard assay conditions. Natural proteins wereincubated with purified protease for 30 min at 30 �C. The reactionwas terminated by addition of SDS–PAGE loading buffer. AfterSDS–PAGE, the cleavage products were electrophoretically trans-ferred to a PVDF membrane, and the protein N-terminal amino acidsequence was determined by the Edman degradation method.

48 B. Bi et al. / Food Chemistry 126 (2011) 46–53

2.6. Properties comparison of proteases with similar cleavage sites

Cleavage patterns and physicochemical properties of proteaseswith similar cleavage sites listed by MEROPS were compared withthe protease.

3. Results

3.1. Protease purification

The purification results of the protein are summarised in Table1. After the final purification step, the enzyme was purified 23.5-fold with a recovery of 21.2%. The specific activity of the finallypurified enzyme was about 42262 U/mg protein. On storage at -20 �C, the percentage of these products gradually increased (datanot shown), and no loss of activity was observed. The purified en-zyme was homogeneous by SDS–PAGE and its molecular mass wasestimated to be 43 kDa (Fig. 1A). Two-dimensional electrophoresisindicated the pI of the enzyme was 5.3 (data not shown).

3.2. Amino acid sequence analysis

Purified proteins were analyzed by Edman sequencing, MS-MS,MALDI-TOF MS and de novo sequencing. The N-terminus of the pro-tein could not be determined, probably because of the presence of ablocking group at the N-terminus. The results of MS-MS and MALDI-TOF MS showed no similar sequences in the data base. Five peptidesequences were identified by de novo sequencing, separately:DNLMRAVGALLR, APATPPSGGHCSGLAR, EQTAD(OXH)DTYPADGSRSR, ACSSAGAKQPHP(OXM)CGKSRR, SVVLAHGSTGDVLKDGSTNAAAPK. After analyzing these internal sequences with the NCBI BLASTpprogramme, we found that they exhibited low similarities, withknown proteins from the protein database (score 6 33.7, Evalue P 2.4). Five sequences produced by BLASTp search with thehighest scores were not related.

3.3. Effects of pH, temperature, metal ions, oxidising agents, reducingagents, detergents and protease inhibitors on protease activity

Later studies indicated that the new protein cleaved some otherproteins, and we presume that it is a proteolytic enzyme. In orderto determine the nature of the protease, enzyme activity was mea-sured in the presence of different enzyme inhibitors (Table 2). Theactivity of the enzyme was effectively blocked (almost > 93%) bythe serine protease inhibitor PMSF. Serine protease inhibitor DFPalso inhibited the activity by 65%. Another serine protease inhibi-tor, aprotinin, had a weak effect on the protease. Chymotrypsin-and trypsin-like protease inhibitors, such as TAME, leupeptin andSBTI, had no effect on the protease. Aspartic protease inhibitor(Pepstatin A) also had no effect on the protease.

The enzyme was obviously inhibited by the chelating agentEDTA (5 mM) and EGTA (5 mM), with 53 and 63% of its originalactivity being lost, respectively (Table 2), and the addition of CaCl2

increased protease activity by 202% of the control, indicating theimportance of Ca2+ in enzyme stabilisation. Other metal ions did

Table 1Purification scheme of the protease from Cordyceps sinensis.

Purification steps Total activity (U)a Total protein (mg

Crude extract 333451 108HiTrap Q XL column 240202 24.5Superdex 200 prep grade column 116615 10.4Native PAGE 98991 2.35SOURCE 15RPC column 70695 1.03

a One unit of protease was equivalent to amount of enzyme required to release 1 lm

not show much effect on this protease. Non-ionic surfactants, suchas Tween-80 and Triton X-100, increased protease activity. Thestrong anionic surfactant, SDS, at 1% (w/v) caused almost completeinhibition (97%). In addition, the protease retained 40% of its activ-ity after incubation for 30 min at 4 �C in the presence of 3% (v/v)H2O2 (Table 2).

The optimum pH and temperature of the enzyme were 9.5 and30 �C, respectively (Fig. 1B and C). It was active at alkaline pH (8.0–11.0) and under 50 �C. The protease only loses 45% of activity after30 min of incubation at 50 �C, and even retained 10% of activityafter 30 min at 60 �C.

3.4. Protease cleavage motifs prediction

We used oriented peptide library mixtures (Turk et al., 2001) todetermine the cleavage motif of the protease. The cleavage sitemotif for a protease involves residues both N- and C-terminal tothe scissile bond, defined as . . . P3-P2-P1-P10-P20-P30. . ., and cleav-age occurs between the P1 and P10 residues. Our first library hadthe sequence Ac-XXXXXXXXXXXX, where X indicated a degenerateposition, and the N-termini were acetylated. After partial digestionby purified protease, the digested mixture was subjected to N-terminal sequencing by Edman degradation. The undigestedpeptides and N-terminal fragments remained blocked and didnot contribute to the sequenced pool; only the C-terminal to thescissile bond was determined (Fig. 2A).

The relative amounts of each amino acid present in a given cycleindicated preference for the residue at a particular site, so the firstsequencing cycle afforded information about the p10 position, thesecond cycle about the P20 position, and so on. The results for posi-tions P10 to P30 are shown in Fig. 2B-2. Clearly, the P30 position hasstrong preference for proline and lysine. The P10 position has somepreference for glutamine, serine and alanine. The P20 positionshows low preference compared with the other two positions.

We used the most abundant amino acid of each position to de-sign the second library. This secondary library had the sequenceMXXXQYPKH(K-biotin), where the fixed QYP sequence corre-sponded to the P10-P30 positions k-biotin was e-(biotinamidohexa-noyl)lysine; the C termini were acetylated and the N termini wereunblocked. This time the cleavage site was between X and Q. Afterpartial digestion, the undigested peptides and C-terminal frag-ments that retained the biotin tag were removed by avidin (0 �C).The remaining N-terminal fragments were subjected to N-terminalsequencing, and the selectivities were determined from the rela-tive abundance of each amino acid in a given sequencing cycle,as before (Fig. 2A). The second library afforded information aboutthe P3, P2 and P1 positions, and results are shown in Fig. 2B-1.The P1 position has an extremely strong preference for lysine,the P2 position inclined to select arginine and lysine. The P3 posi-tion showed low selectivity.

To validate the cleavage motif, we prepared a series of hexapep-tides, based on the amino acid preference, and determined the cat-alytic parameters for cleavage of each peptide (Table 3). All sixhexapeptides could be cleaved by protease, and the kcat/Km of theoptimal peptide was found to be greater than those of other

) Specific activity (U/mg) Recovery (%) Purification (fold)

3101 100 19800 72 3.211213 35 3.642124 29.7 13.672816 21.2 23.5

ole of peptide(ARKQYP)/ml/min under standard assay conditions.

Fig. 1. Characterisation of the purified protease from Cordyceps sinensis. (A) SDS–PAGE of the purified protease. M is molecular mass markers; lane 1 is purified protease. (B)Effect of pH on enzyme activity and stability. Enzyme samples were incubated in the respective buffers at 30 �C and percent residual activity was calculated. (C) Effect oftemperature on protease activity and stability. The temperature was determined by assaying enzyme activity at various temperatures at pH 9.5. The results shown were themeans ± s.e.m. of three independent experiments.

Table 2Effects of enzyme inhibitors, oxidising agents, detergents and metal ions on theenzyme activity.

Additions Concentration (mM) Relative enzyme activitya (%)

None – 100PMSF 10 7DFP 0.01 35Aprotinin 0.1(mg/ml) 93SBTI 0.1(mg/ml) 100Pepstatin A 1 100Leupeptin 1 100TAME 1 100EDTA 5 47EGTA 5 38b-Mercaptoethanol 10 96Tween 80 1% (v/v) 138Triton X-100 1% (v/v) 126SDS 1% (w/v) 3H2O2 3% (v/v) 40Ca2+(CaCl2) 5 202Mn2+(MnCl2) 5 71Mg2+(MgCl2) 5 99Zn2+(ZnSO4) 5 80

a Enzyme activity measured in the absence of any inhibitor was taken as 100%.

B. Bi et al. / Food Chemistry 126 (2011) 46–53 49

peptides. In each case, changing the predicted optimal residue to asuboptimal residue decreased the kcat/Km for cleavage.

We also prepared several natural proteins to validate the cleav-age motif. BSA, CNB, CNa and GST can be cleaved by protease(Fig. 2C), and CaM cannot be cleaved. We determined some ofthe cleavage sites, using Edman sequencing (Table 4). All the cleav-

age sites determined by Edman sequencing have lysine at P1 posi-tion and proline or lysine at the P30 position.

3.5. Properties comparison of proteases with similar cleavage sites

The cleavage site specificity of a protease is critical, but it is verycomplex for many proteases, and is difficult to define. However,the specificity of the new protease from Cordyceps sinensis is mucheasier to describe. Compared with other proteases, which havestrong preferences for lysine at the P1 position listed by MEROPS,such as gingipain K (EC 3.4.22.47), lysyl peptidase (EC 3.4.21.50),plasmin (EC 3.4.21.7), trypsin 1 (EC 3.4.21.4) and tryptase alpha(EC 3.4.21.59), this new protease has some differences. The cleav-age pattern of gingipain K and lysyl peptidase is -/-/-/K + -/-/-/-;plasmin, trypsin 1 and tryptase alpha are -/-/-/KR + -/-/-/-. It onlyrequires K or K/R at the P1 position, and other positions have no ef-fect on the cleavage activation. The new protease shows strongpreference, not only at the P1 position, but also the P30 position.The cleavage requires proline or lysine at the P30 position. Besides,arginine or lysine at the P2 position and glutamine, serine or ala-nine at the P10 position will possibly make cleavage much easier.

The optimum pH of gingipain K, lysyl peptidase, plasmin, tryp-sin 1 and tryptase alpha is between 8 and 9. The optimum temper-ature is between 37 and 50 �C. The new protease has a morealkaline pH optimum (pH 9.5) and wider pH range (8.0–11.0). Itsoptimum temperature is 30 �C and it is applicable over a widerange of temperature. Gingipain K, lysyl peptidase, plasmin andtrypsin 1 are destroyed or lose activity rapidly above 50 �C, butthe new protease only loses 45% of activity after 30 min of incuba-tion at 50 �C, and even retains 10% of activity after 30 min at 60 �C.

Fig. 2. Cleavage site motif prediction. (A) Overview of the oriented peptide library method. X indicates a degenerate position. Ac indicates that the termini are acetylated. K-biotin is e-(biotinamidohexanoyl)lysine. (B) Cleavage site motif determined by oriented peptide library. B-1 is the amino acid preference of P3, P2, P1 positions. B-2 is theamino acid preference of P10 , P20 , P30 positions. Amino acid percentage was determined by Edman degradation and MS. The results shown were chosen from one experimentof three independent experiments. (C) Cleavage of natural substrates by purified protease. The substrates of C-1, C-2, C-3 and C-4 were BSA, CNB, CNa and GST, separately. Mindicates the molecular mass markers; Lane 1 is the substrate without protease; Lane 2 is the substrate incubated with purified protease. CNB (ratus) is the b subunit ofcalcineurin. CNa (ratus) is the core component (1–347 amino acid sequence) of calcineurin (PP2b). GST (E. coli) is the abbreviation of glutathione S-transferase.

50 B. Bi et al. / Food Chemistry 126 (2011) 46–53

4. Discussion

In the present study we purified a protein from the edible fun-gus Cordyceps sinensis. To identify the protein, we used Edmansequencing, MS-MS, MALDI-TOF MS and de novo sequencing. The

N-terminal of the protein is blocked. The MS/MS and MALDI-TOFMS fragmentation spectra did not match any peptide sequence inthe database. Five peptide sequences were determined by de novosequencing and were analyzed with the NCBI BLASTp programme.But the highest probability score is only 33.7, and the lowest

Table 3Cleavage of synthetic substrates by purified protease.

Peptide Sequence Ratio of kcat/Kma

1 ARK;QYP 12 ARK;QYK 0.947 ± 0.0053 ARK;SYP 0.701 ± 0.0064 ARK;SYK 0.850 ± 0.0115 ARK;AYP 0.741 ± 0.0136 ARK;AYK 0.618 ± 0.006

a kcat/Km ratios were calculated relative to the kcat/Km value obtained with peptide1 (see text), no measurable activity. ; indicates the cleavage site.

B. Bi et al. / Food Chemistry 126 (2011) 46–53 51

E value is 2.4, which indicates very little similarity and does notidentify the unknown peptides. Besides, five sequences, producedby BLASTp search with the highest scores, are not even related.These results indicate that the protein we isolated and purified isa completely novel protein.

The novel protein can cleave some other proteins when incu-bated with them. It is presumed to be a proteolytic enzyme. It isinhibited by general serine peptidase inhibitors, such as DFP andPMSF, which indicates that it is possibly a serine protease. TAME,leupeptin and SBTI had no effect on the protease, which indicatesthat the protease may not be a trypsin- or chymotrypsin-like pro-tease. Because of the absence of inhibition by SBTI, which is abun-dantly present in many protein-rich foods, this protease may havepotential in the food industry, where it may be effectively used inthe processing of protein-rich food.

This protease can be activated by Ca2+, non-ionic surfactants,such as Tween-80 and Triton X-100, and is tolerant of certain oxidis-ing agents and various other inhibitors. Its ability to withstand theharsh conditions of organic solvents and various chemical denatur-

Table 4Edman sequencing of the natural substrate cleavage products.

Substrate Sequence

GSTa 1 MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDK61 GDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGA121 DFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLY

181 KRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPCNab

1 MSEPKAIDPKLSTTDRVVKAVPFPPSHRLTAKEVFDNDG61 ALRIITEGASILRQEKNLLDIDAPVTVCGDIHGQFFDLMK121 DRGYFSIECVLYLWALKILYPKTLFLLRGNHECRHLTEY

181 AFDCLPLAALMNQQFLCVHGGLSPEINTLDDIRKLDRF241 NEKTQEHFTHNTVRGCSYFYSYPAVCDFLQHNNLLSIL301 SLITIFSAPNYLDVYNNKAAVLKYENNVMNIRQFNCSP

CNBc1 GNEASYPLEMCSHFDADEIKRLGKRFKKLDLDNSGSLSVE

61 FDTDGNGEVDFKEFIEGVSQFSVKGDKEQKLRFAFRIYD121 NNLKDTQLQQIVDKTIINADKDGDGRISFEEFCAVVGG

BSA 1 MKWVTFISLLLLFSSAYSRGVFRRDTHKSEIAHRFKDLGE

61 DEHVKLVNELTEFAKTCVADESHAGCEKSLHTLFGDELC

121 ERNECFLSHKDDSPDLPKLKPDPNTLCDEFKADEKKFW181 ANKYNGVFQECCQAEDKGACLLPKIETMREKVLASSA

241 RLSQKFPKAEFVEVTKLVTDLTKVHKECCHGDLLECAD301 CCDKPLLEKSHCIAEVEKDAIPENLPPLTADFAEDKDVC

361 HPEYAVSVLLRLAKEYEATLEECCAKDDPHACYSTVFD421 LGEYGFQNALIVRYTRKVPQVSTPTLVEVSRSLGKVGT

481 NRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPDET

541 DTEKQIKKQTALVELLKHKPKATEEQLKTVMENFVAF601 STQTALA

CaM 1 MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSL61 NGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGN121 EVDEMIREADIDGDGQVNYEEFVQMMTAK

nd, not determined – , no measurable cleavage. Dot indicates lysine (K). Underline indRestricted by peptide concentration and sensitivity of Edman sequencing, the cleavagdetermined.

a GST (E. coli) is the abbreviation of glutathione S-transferase. The cleavage site of theb CNa (ratus) is the core component(1–347 aa) of calcineurin (PP2b).c CNB (ratus) is the b subunit of calcineurin.

ants may allow various applications to be explored. The protease isactive at alkaline pH. Generally, the commercial proteases frommicroorganisms have maximum activity in the alkaline pH rangeof 8.0–12.0 (Erikson, 1996; Rao et al., 1998b). Compared with otherproteases, which have similar cleavage sites, such as gingipain K, ly-syl peptidase, plasmin, trypsin 1 and tryptase alpha, the protease wepurified has a wider pH range, a more alkaline optimum pH, loweroptimum temperature and higher temperature tolerance. Theseproperties make it suitable for industrial and commercial use.

One of the most important characteristics of a protease is itsproteolytic specificity. There are many methods for determiningprotease specificity (Diamond, 2007), such as substrate phage dis-play libraries, positional-scanning peptide libraries, and mixture-based peptide libraries. The most commonly used method, (phagedisplay libraries), is time consuming, and laborious and does notreveal the precise cleavage sites (Boulware & Daugherty, 2006;Chen, Kridel, Howard, Li, Godzik & Smith, 2001; Kerr et al., 2005;Matthews & Wells, 1993; Smith, Shi, & Navre, 1995). Positional-scanning peptide libraries only provide sequence specificity N-terminal to the scissile bond and cannot be used with proteasesthat require amino acid residues C-terminal to the cleavage site,such as serine and cysteine proteases (Backes, Harris, Leonetti,Craik, & Ellman, 2000; Harris, Backes, Leonetti, Mahrus, Ellman &Craik,, 2000; Rao, Tankasale, Ghatge & Deshpande, 1998a). To over-come these shortcomings, we used oriented peptide library mix-tures, the improved mixture-based peptide libraries. These canbe screened in a rapid and cost-efficient manner and provide datafor both the primed and unprimed positions by first establishingthe prime-side specificity of cleaved peptide pools, and thendesigning a second peptide library, with all members anchoredby the preferred prime-side residues.

Determined cleavage site

WRNKKFELGLEFPNLPYYIDVLDIRYGVSRIAYSKDFETLKVDALDVVLYMDPMCLDAFPKLVCFK

KSDLEVLFQGPLGS

YLK;SSK

KPRVDILKAHLMKEGRLEESVLFEVGGSPANTRYLFLGDYVFTFKQECKIKYSERVYDACMD

KEPPAYGPMCDILWSDPLEDFGRAHEAQDAGYRMYRKSQTTGFPHPYWLPNFM

VVK;AVP

EFMSLPELQQNPLVQRVIDI

MDKDGYISNGELFQVLKMMVGLDIHKKMVVDV

SVK;GDK

EHFKGLVLIAFSQYLQQCPF

KVASLRETYGDMADCCEKQEP

GKYLYEIARRHPYFYAPELLYYRQRLRCASIQKFGERALKAWSVA

DRADLAKYICDNQDTISSKLKEKNYQEAKDAFLGSFLYEYSRR

KLKHLVDEPQNLIKQNCDQFEKRCCTKPESERMPCTEDYLSLIL

YVPKAFDEKLFTFHADICTLP

VDKCCAADDKEACFAVEGPKLVV

nd

GQNPTEAELQDMINEVDADGGYISAAELRHVMTNLGEKLTDE

–

icates the predicted cleavage motif (K at P1 position and P or K at P30 position).e of some sequences fitting predicted cleavage motif mentioned above were not

fragment is determined by both the exact MW and N-terminal sequencing.

52 B. Bi et al. / Food Chemistry 126 (2011) 46–53

The first library affords information about the P10, P20 and P30

positions, and the second library affords information about theP1, P2 and P3 positions. There are strong preferences for lysineand proline/lysine at the P1 and P30 positions respectively. TheP10 position tends to select glutamine, serine and alanine. The P2position tends to select arginine and lysine. The P20 and P3 posi-tions show low preference.

Based on these results, we speculate that the P1 and P30 posi-tions are extremely critical for the cleavage motif, while the P2and P10 positions are not as critical as the P1 and P30 positions,but still have some importance. P3 and P20 positions are dispens-able for the cleavage motifs.

The results of experiments with synthetic hexapeptides andnatural proteins validated our speculation. Based on cleavage siteprediction, the synthetic hexapeptides should be cleaved by theprotease. The results of experiments confirmed the predictionand the one with the predicted optimal residue had the highestkcat/Km value.

Natural proteins such as GST, BSA, CNa and CNB, can be cleavedby protease. All the cleavage sites that we detected by Edmansequencing have lysine in the P1 position, which is perfectly con-sistent with the results of former experiments. But lysine in theP1 position is not the only requirement for cleavage activation.For example, as shown in Table 4, the number of lysine in the pep-tide sequence of CaM is 8, but CaM cannot be cleaved by theprotease.

According to the results of former experiments, the P30 positionis also extremely critical for the cleavage motif. We confirmed thatthe motifs satisfy our prediction, K at the P1 position and P or K atthe P30 position (Table 4). The number of lysine is 21 in the aminoacid sequence of GST, but only sequences KYLK and KSSK fit withour predicted cleavage motif. The P10 position has a preferencefor serine according to our speculation and the protease may pref-erentially cleave KSSK. Edman sequencing confirmed that KSSK iscleaved by the protease.

In the amino acid sequence of CNa, the total number of lysineresidues is 20, but only KAVP and KEPP fit with our predictedcleavage motif. The P10 position has a preference for alanineaccording to our speculation and the protease may preferentiallycleave KAVP. Edman sequencing confirmed that KAVP is cleaved.Considering incomplete digestion and the absence of small sizebands, we predict that there should be two major peptide bandsand a minor band besides the CNa protein band. This predictionis consistent with the actual electrophoresis result (Fig. 2C). Like-wise, KRFP, KGDK and KEQK of CNB, which has 15 lysines, fit withour predicted cleavage motif, and KGDK is confirmed to be cleaved.Because of the predicted cleavage sites, KGDK and KEQK are almostcoincident, and the bands of small size are not shown in the elec-trophoresis gel. Our predicted bands are almost consistent with theelectrophoresis result. The large number of motifs fits our predic-tion in the amino acid sequence of BSA and incomplete digestionwould produce numerous bands and lead to dispersion, which isalso consistent with the electrophoresis result.

According to all the results of cleavage motif determinationexperiments above, we conclude that the P1 and P30 positionsare critical for the cleavage motif. The cleavage can occur only withlysine at P1 and proline or lysine at P30. Any other amino acid in thetwo positions will preclude cleavage. The P2 and P10 positions havesome selectivities. Possibly arginine or lysine at the P2 position andglutamine, serine or alanine at the P10 position will make cleavageeasier. The protease may preferentially cleave the one which hasglutamine/serine/alanine in the P10 position or arginine/lysine inthe P2 position when sequences satisfy P1 and P30 positionrequirements.

Restricted by peptide concentration and sensitivity of Edmansequencing, the cleavage of some sequences fitting our predicted

cleavage motif, mentioned above, were not determined. The pref-erences of positions P2 and P10 should be validated with moresequence determinations in future experiments.

The specificity of the new protease is different from otherproteases which have strong preferences for lysine at the P1 posi-tion. Its cleavage pattern is -/-/-/K + -/-/PK/-, instead of -/-/-/K + -/-/-/- or -/-/-/KR + -/-/-/-. Because of the high frequency of ly-sine, other proteases which have strong preferences for lysine inthe P1 position would cut proteins into very tiny fragments, butthe protease we purified would yield larger fragments. This makesit a potential candidate for laboratory use.

In short it can be concluded that the novel protease purifiedfrom Cordyceps sinensis, which has a completely new cleavage pat-tern, is suitable for industrialisation and commercialisation. Fur-ther work on this protease, involving cloning and expression, iscurrently underway.

Acknowledgements

This work was supported by grants from the National ScienceFoundation of China and international cooperation project.

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