research article heterologous, expression, and...

Research ArticleHeterologous Expression and Characterization ofThermostable Glucoamylase Derived from Aspergillus flavusNSH9 in Pichia pastoris

Kazi Muhammad Rezaul Karim12 Ahmad Husaini1 Md Anowar Hossain3

Ngieng Ngui Sing1 Fazia Mohd Sinang1 Mohd Hasnain Md Hussain1

and Hairul Azman Roslan1

1Department of Molecular Biology Faculty of Resource Science and Technology Universiti Malaysia Sarawak94300 Kota Samarahan Sarawak Malaysia2Institute of Nutrition and Food Science University of Dhaka Dhaka 1000 Bangladesh3Department of Biochemistry and Molecular Biology University of Rajshahi Rajshahi 6205 Bangladesh

Correspondence should be addressed to Ahmad Husaini haahmadunimasmy

Received 2 May 2016 Revised 17 June 2016 Accepted 19 June 2016

Academic Editor Pengjun Shi

Copyright copy 2016 Kazi Muhammad Rezaul Karim et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

A novel thermostable glucoamylase cDNA without starch binding domain (SBD) of Aspergillus flavus NSH9 was successfullyidentified isolated and overexpressed in Pichia pastorisGS115The complete open reading frame of glucoamylase from Aspergillusflavus NSH9 was identified by employing PCR that encodes 493 amino acids lacking in the SBD The first 17 amino acids werepresumed to be a signal peptideThe cDNAwas cloned into Pichia pastoris and the highest expression of recombinant glucoamylase(rGA) was observed after 8 days of incubation period with 1 methanol The molecular weight of the purified rGA was about78 kDa and exhibited optimum catalytic activity at pH 50 and temperature of 70∘C The enzyme was stable at higher temperaturewith 50 of residual activity observed after 20min at 90∘C and 100∘C Low concentration of metal (Mg++ Fe++ Zn++ Cu++ andPb++) had positive effect on rGA activity This rGA has the potential for use and application in the saccharification steps due to itsthermostability in the starch processing industries

1 Introduction

Glucoamylase (14-120572-D-glucan glucohydrolase EC 3213GA) is an exoacting enzyme that yields 120573-D-glucose fromthe nonreducing ends of starch and related oligo- andpolysaccharide chains by hydrolyzing120572-14 and120572-16 linkages[1] This enzyme is able to completely hydrolyze starch ifincubated for extended periods of time and hence calledthe saccharifying enzyme Glucoamylase is an importantgroup of enzymes in starch processing in the food industriesas it is used for the production of glucose and fructosesyrup from liquefied starch [2] It is also used in bakingjuice beverage pharmaceuticals confectionery and many

fermented foodstuffs industries for commercial production[3] It is the most important type of industrial enzymebecause of its widespread uses together with 120572-amylases anddebranching enzymes in the saccharification of starch toyield soluble sugars which are widely used by many foodindustries and in the production of bioethanol [4]

From a structural point of view glucoamylase enzymesconstitute the bulk of family GH15 of glycoside hydrolases[5] Glucoamylase (GA) can be derived mostly in microor-ganisms and also in animals and plants A large number ofmicrobes including bacteria yeast and fungi are capableof producing glucoamylase Filamentous fungi constitute themajor source of all microorganisms However the exclusive

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 5962028 10 pageshttpdxdoiorg10115520165962028

2 BioMed Research International

productions of glucoamylase in the enzyme industry areachieved by Aspergillus niger [6] Aspergillus oryzae andRhizopus oryzae [7]

Fungi often produce more than one form of enzyme thatmay have different molecular weights amino acid composi-tion glycosylation ability to digest soluble and raw starchesand stability [8] The majority of fungal glucoamylase is mul-tidomain consisting of a catalytic domain at the N-terminusand a starch binding domain (SBD) at the C-terminus [9]which is separated by an O-glycosylated linker region rich inserine and threonine residues But with exception glucoamy-lases with a single domain structure are also available in Aniger [10]Rhizopus oryzae [11] andA oryzae [12] Two differ-ent forms of glucoamylase with and without starch bindingdomain are produced from one gene or two different genesFor example A niger and A awamori var kawachi producetwo forms of glucoamylase (GA) from the same gene [13]while A oryzae produces two GA from two different genes[14]

Due to industrial importance glucoamylase encodinggene has been cloned characterized and heterologouslyexpressed which derived the Aspergillus species A niger[15] A awamori var kawachi [16] and A oryzae [12]El-Abyad and his coworker [17] screened 21 species foramylolytic activity and found that A flavus was among themost active amylase producer It is also reported that Aflavus NSH9 was the best amylase producer among fourteenisolates of Aspergillus sp [18] Although glucoamylase fromA flavus has been characterized [18 19] little is knownabout its molecular information The genetic sequencemolecular information substrate specificity and biochemicalcharacteristic of glucoamylase from A flavus are not yetwell known On the other hand Pichia pastoris is one ofthe most used eukaryotic systems for the production ofrecombinant proteins It is easy to culture and can reachhigh cell densities and has strong and regulated promotersand ability to secrete proteins as well as introducing post-translational modifications [20 21] Here we described theisolation cloning and sequence analysis of glucoamylasegene (GA) derived from Aspergillus flavus and its expressionin Pichia pastoris

2 Materials and Methods

21 Maintenance and Culturing of Aspergillus Strain Anewly fungal strain of Aspergillus flavus NSH9 with starchdegrading properties was previously isolated from the sagohumus and kept as collection at the Department ofMolecularBiology Faculty of Resource Science and Technology Uni-versiti Malaysia Sarawak Malaysia For enzyme inductionthe actively growing fungal mycelium was transferred frompotato dextrose agar (PDA) plate to a culture mediumcontaining (in g Lminus1) 20 g raw sago starch 3 g KH

2PO4 1 g

(NH4)2SO4 05 g MgSO

4sdot7H2O and 4 g of yeast extract The

mycelia were collected after 4 days of growth in the culturemedia at room temperature of 28∘C with shaking at 150 rpmand then subsequently used to extract the total RNA

22 Extraction of Total RNA and DNA and First-Strand cDNASynthesis Total RNA was extracted by using TRIzol Reagentaccording to the protocol as described by Schumann et al[22] and the genomic DNA was extracted according to themethod of Cubero et al [23] Total RNAwas purified by beingtreated with DNAse and reverse transcriptions were carriedout in 20120583L reaction mixture containing the following 01ndash5 120583g DNA free RNA as a template 05 120583g oligo (dt)

18 4 120583L

of 5x reaction buffer 2 120583L 10mM dNTP Mix 20U RiblockRNase Inhibitor and 40U Moloney Murine Leukemia Virus(M-MuLV) reverse transcriptase (Thermo Scientific) Thereaction was performed at 37∘C for 60min and at 70∘Cfor 5min The product was either used immediately in thesubsequent PCR analysis or stored at minus80∘C

23 Isolation and Subcloning of Glucoamylase cDNA Glu-coamylase gene fromAspergillus flavusNSH9 was isolated byusing GAAsp-F and GAAsp-R primers that were designedbased on sequences of A flavus NRRL3357 (glucoamy-lase precursor putative mRNA XM 0023749641) obtainedfrom the National Center for Biotechnology Information(NCBI) The PCR was carried out in 50120583L reaction mixturecontaining the following 5 120583L 10x Pol Buffer A (1x finalconcentration) 5mMMgCl

2 02mMdNTP 01ndash05 120583Meach

of the forward and reverse gene specific primers 05 120583gcDNA template and 125U Taq DNA polymerase (EURxGdansk Poland)The full length ofA flavusNSH9 glucoamy-lase cDNA and glucoamylase gene were amplified by PCRusing the following oligonucleotide primers GAAsp F (51015840-CGCATGCGGAACAACCTTCTTT-31015840) and GAAsp R (51015840-CTACCACGACCCAACAGTTGG-31015840) The PCR reactionwas carried out using the following conditions denaturationat 94∘C for 2min for one cycle followed by denaturation at94∘C for 45 sec annealing at 55∘C for 45 sec and elongationat 72∘C for 145min for 35 cycles and a final elongationwas at 72∘C for 5min The purified PCR product was thencloned into pGEMT-Easy Vector (Promega) as described bythe manufacturer protocol and transformed into E coli XL1-Blue Purified plasmid was run on agarose gel and sequencedusing T7 and SP6 primers

24 Cloning of Glucoamylase cDNA into Yeast ExpressionVector ThepPICZ120572C (Invitrogen)was selected as expressionvector and used for the cloning of glucoamylase cDNAwithout signal peptide sequences A putative signalsequence was predicted by the Signal-3L program [24]and glucoamylase cDNA without the signal peptide wasamplified using the following list of primers forward primer(51015840-AGCGAATTCAGATTACAAG GACGACGACGAT-AAGCCGTCCTTCCCTATCCAT-31015840) and reverse primer(51015840-ACGACTCTAGAGCCCACGACCCAACAGTTGG-31015840)The designed primers contained an EcoR1 site and Xbal sitein the forward and reverse primers (bold and underline)respectively In addition the forward primer was designed toinclude FLAG tag containing 24 nucleotides (DYKDDDDK)and the reverse primer lacking in the native stop codon Theprimers were designed to be in frame with the C-terminal6x His tag located in the pPICZ120572C PCR was performed as

BioMed Research International 3

follows 1 cycle at 94∘C for 2min followed by 35 cycles ofthe sequence at 94∘C for 45 sec 62∘C for 45 sec and 72∘C for145min and final extension 72∘C for 5min After puri-fication the PCR product was digested with EcoR1 and Xba1and ligated into pPICZ120572C producing the P pastoris expres-sion plasmid pPICZ120572C GA The recombinant expressionplasmid containing glucoamylase cDNA (pPICZ120572C GA)wasthen transformed into Escherichia coli XL1-Blue The insertin pPICZ120572C GA was confirmed by PCR restriction enzymedigestion and sequencing analysis for correct orientation

25 Transformation and Selection of Glucoamylase Transfor-mant The pPICZ120572C GA carrying the insert was linearizedby SacI and transformed into P pastoris GS115 (Invitrogen)competent cells The competent cells were prepared byusing the Pichia EasyComp Transformation kit accordingto the instruction guidelines (Invitrogen) Linearized vectorpPICZ120572C without insert was also transformed into Ppastoris GS115 and used as a control After transformationPichia cells were plated onto yeast extract peptone dextrosesorbitol (YPDS) agar plate containing 150 120583gmLminus1 zeocinand incubated for 3 to 8 days at 30∘C Transformed colonieswere confirmed by colony PCR using 51015840AOX1 and 31015840AOX1primers and glucoamylase gene specific primers PutativeMut+ colony was selected based on PCR usingAXO1 primersSeveral Mut+ colonies from YPDS agar plate were selectedand independently blotted onto Buffered Methanol-complexMedium (BMMY) (containing 1 (wv) yeast extract 2(wv) peptone 134 (wv) YNB 4 times 10minus5 (wv) biotin05 (vv) methanol and 100mM potassium phosphatebuffer pH 60) agar plate containing 1 (wv) soluble starchGlucoamylase producing transformants were identified bythe formation of decolorization zone or clear halo around thePichia colonies after the addition of iodine solution

26 Expression of Recombinant Glucoamylase (rGA) TheMut+ GS115 single colony from YPDS agar plates wasinoculated into 25mL Buffered Glycerol-complex Medium((BMGY) containing 1 (wv) yeast extract 2 (wv)peptone 134 (wv) YNB 4 times 10minus5 (wv) biotin 1(vv) glycerol and 100mM potassium phosphate buffer pH60) in 100mL conical flask and incubated at 29∘C withshaking of 230 rpm until the culture reached an OD

600of

2ndash4 (approximately after 24 hours) After that cells wereharvested by centrifugation at 3000timesg for 5min at roomtemperature resuspended to an OD

600of 10 in Buffered

Methanol-complex Medium ((BMMY) containing 1 (wv)yeast extract 2 (wv) peptone 134 (wv) YNB 4 times 10minus5(wv) biotin 05 (vv) methanol and 100mM potassiumphosphate buffer pH 60) (40mL) in 250mL conical flaskand incubated for 10 days at 29∘C with shaking of 230 rpmApproximately 1mL of the culture was withdrawn at every24-hour intervals till 10-day period of incubation and addedwith absolute methanol into the culture to make the finalconcentration of 05 (vv) of methanol Based on the bestproduction of recombinant glucoamylase a Pichia cell wasscaled up by increasing the methanol concentration to upto 5 (vv) of final concentration Pichia pastoris GS115

containing vector without the insert was also expressed as theabove procedure and used as a control (Invitrogen 2010)

27 Electrophoresis of the Recombinant Glucoamylase Non-denaturing PAGE and complete SDS-PAGE were both usedto check on the glucoamylase enzyme activity and to estimatethe molecular weight of the rGA expressed Electrophoresiswas carried out by using a 12 (wv) acrylamide resolvinggel and 6 (wv) acrylamide stacking gel as described byLaemmli [25] using the Mini-Protean III electrophoresissystem (Bio-Rad Richmond CA USA) The proteins werestained with Coomassie Brilliant Blue R-250 As for amylaseactivity staining of the recombinant glucoamylase (sampleswithout heating and beta-mercaptoethanol on gel) was per-formed on a PAGE gel after washing the gel for 30minwith 01M sodium acetate buffer (pH 50) containing 02(vv) Triton X-100 to remove the SDS in gel After washingthe gel was washed again with 01M sodium acetate buffer(pH 50) containing 1 (wv) soluble starch for 30min Therecombinant glucoamylase activity appeared as clear band ondark blue background after the staining of iodine solution(015 (vv) of iodine and 05 (wv) of potassium iodide)Desired band in the complete SDS-PAGE analysis after thedestaining from Coomassie Brilliant Blue R-250 was cut outand subjected to peptide sequencing The analysis of theprotein was done by MALDI-TOFTOF mass spectrometryusing a 5800 Proteomics Analyzer The spectra generatedwere evaluated and analyzed to identify the protein ofinterest using Mascot sequence matching software (MatrixScience) with MSPnr100 Database This analysis was doneby Proteomics International Pty Ltd Broadway NedlandsWestern Australia

28 Recombinant Glucoamylase Enzyme Activity and ProteinAssay Glucoamylase activitywas determined by transferringa volume of 05mL of the dilute glucoamylase enzymefollowed by the addition of 05mL of 1 (wv) solublestarch solution in 01M sodium acetate buffer (pH 50)and incubated at 55∘C for 30min The released glucose wasmeasured using 35-dinitrosalicylic acid (DNS) reagent withsomemodification [26] andmeasured at 540 nmThe enzymeactivities were calculated using a calibration curve preparedusing D-glucose as standard One unit of glucoamylase activ-ity was defined as the amount of enzyme that released 1 120583molof glucose equivalent per minute from soluble starch underthe assay condition The protein content was determined bythe method of Bradford [27] with bovine serum albumin asstandard The specific activity of recombinant glucoamylasewas taken as unit of mgminus1 protein

29 Purification and Characterization of RecombinantGlucoamylase (rGA)

291 Purification Recombinant glucoamylase was purifiedby using an anti-FLAG M2 affinity gel according to themanufacturerrsquos instruction (Sigma Aldrich)


292 Effect of pH and Temperature The optimum pH foractivity was determined by measuring the glucoamylaseactivity at 55∘C for 30min using various types of buffersThe following 01M buffer systems were used sodium citrate(pH 30) sodium acetate (pH 40ndash50) potassium phosphate(pH 60ndash70) and Tris-HCI (pH 80ndash90) The optimumtemperature for activity was assayed by measuring enzymeactivity at optimum pH (01M sodium acetate buffer pH 50)over different temperature ranging from 20 to 90∘C

293 pH Stability and Thermostability of rGA For pH sta-bility the recombinant glucoamylase was dispersed (1 1) into01M buffer solution pH 30 to 90 and incubated at 25∘C for24 h [28] and after that an aliquot was used to determine theremaining activity at optimum pH and temperatureThermalstability of the recombinant glucoamylase was determinedby incubating in 01M sodium acetate buffer pH 50 at60ndash100∘C for 90min and also enzyme was incubated inwater at 70∘C to observe the stability at neutral pH Timecourse aliquots were withdrawn cooled down in ice bath andassayed under standard conditions at optimum pH (pH 50)and temperature (70∘C)

294 Effect of Substrate Concentrations Enzyme activity wasassayed in the reaction mixture containing different amountof soluble starch (025ndash20 (wv)) at optimum pH 50 andtemperature at 70∘C with under standard assay conditionsThe data were plotted (according to Lineweaver-Burk) todetermine 119881max and119870m values

295 Effect of Metal Ions In order to investigate the effectof metal ions (Na+ K+ Cu2+ Fe2+ Pb2+ Ca2+ Mg2+ Cd2+and Zn2+) on the activity of recombinant glucoamylase anenzyme aliquot was incubated with a metal ion solution(the final concentration of 1mM and 5mM and the finalvolume after addition substrate) for 30 minutes at 37∘C Afterthat the remaining activity was measured under standardassay condition with optimum pH (pH 50) and temperature(70∘C) The relative glucoamylase enzyme activity which wasassayed in the absence of metal ions was taken as 100

296 Effect of Different Substrate Different substrates suchas soluble starch amylopectin glycogen and gelatinized rawsago starch were used to investigate the hydrolyzing capacityof the recombinant glucoamylase produced

297 Bioinformatics Nucleotide sequences of cDNA of glu-coamylase were used as the query sequences in the BLASTsearches using the BLAST X program Searches of proteinsequences were done in the NCBI (Protein BLAST)The phy-logenic tree of glucoamylase from Aspergillus flavus NSH9was constructed by using MEGA6 software [29] For thisthe alignment of amino acid sequences was performed usingthe CLUSTALW program and the evolutionary tree wascalculated from the manually adjusted alignment with theneighbor-joining clustering method and the bootstrappingprocedureThe number of bootstrap trails used was 1000 andimplemented in the MEGA6 program

3 Results

31 Identification and Analysis of Glucoamylase Gene Forisolation of glucoamylase from the cDNA and genomicDNA total RNA and DNA were extracted from Aspergillusflavus NSH9 and mRNA was converted to cDNA PCRamplified product by GAAsp F (51015840-CGCATGCGGAAC-AACCTTCTTT-31015840) and GAAsp R (51015840-CTACCACGACCC-AACAGTTGG-31015840) primer indicated that the full length ofglucoamylase was 1482 bp from cDNA and 1587 bp fromDNA A comparison of the genomic and cDNA sequencesof glucoamylase indicated the presence of two introns vary-ing in sizes of 59 bp and 46 bp The open reading frame(ORF) of A flavus NSH9 glucoamylase gene consisted of1482 nucleotides encoding 493 amino acid residues Thegenes were submitted to GeneBank and assigned with theiraccession number as KU095082 and KU095083 for glu-coamylase cDNA and genomic DNA respectively Based onthe sequence comparison with other fungal glycosyl hydro-lases the glucoamylase protein can be assigned to glycosylhydrolase family 15 and also in NCBI protein BLAST it wasobserved that this GA belonged to the glycoside hydrolasesrsquo15 families lacking in starch binding domain (SBD) Fivemotifs AEPKF WGRPQRDGP DLWEEV ALSNHK andAAELLYDA were found highly conserved among fungiglucoamylase The first 17 amino acids were presumed tobe the signal peptide Five putative asparagine-linked N-glycosylation sites (at 139 198 255 369 and 457 position)and another two possible glycosylation sites (at 120 and 384)which were deduced according to the rule of Asn-X-ThrSerwere present in the deduced amino acid sequence Searchesof protein sequences in the NCBI (Protein BLAST) servicerevealed that the sequence of A flavus NSH9 glucoamylaseshowed a high degree of identification with the glucoamylasesequences from other fungal glucoamylases

The phylogenic tree of glucoamylase from AspergillusflavusNSH9 was constructed by usingMEGA6 software [29](Figure 1) The results of BLAST between A flavus NSH9glucoamylase and other glucoamylases showed about 98-99identical of A flavus NRRL3357 (XP 0023750051 putativeprecursor) A oryzae 3042 (EIT733931) and A parasiticusSU1 (KJK647191) It was also found to be 70 identical withglucoamylase precursor of A terreus NH2624 (XP 0012151-581) About 57 to 60 identical were also observed fromAawamori (AEI589951) A niger (AIY230671) A ficuum(AAT580371) and Neurospora crassa OR74A (EAA277302)and A oryzae (BAA008411) and putative glucan 14 120572-gluco-sidase was observed from A flavus NRRL3357 (XP 002384-9461) In the phylogenic tree (Figure 1) and NCBI proteinBLAST it was found that glucoamylase from A flavus NSH9matched with the two types of glucoamylase from A oryzaeand A flavus NRRL3357 It is assumed that A flavus NSH9also produced two glucoamylases one that is lacking starchbinding domain and the other with the starch bindingdomain

The amino acid sequences of glucoamylase deducedfrom the nucleotide sequences were compared with thoseof other glucoamylases (gene accession numbers p69327and p69328) retrieved from the Universal Protein Resources


Glucoamylase Aspergillus oryzae 3042 (EIT733931)Glucoamylase Aspergillus oryzae (BAA252051)

AFNSH9 GA1Glucoamylase precursor putative Aspergillus flavus NRRL3357 (XP 0023750051)

Glycosyl hydrolases family 15 Aspergillus parasiticus SU-1 (KJK647191)Glycosyl hydrolase family 15 putative Aspergillus clavatus NRRL 1 (XP 0012726811)

Glucoamylase precursor Aspergillus terreus NIH2624 (XP 0012151581)Glucoamylase Neurospora crassa OR74A (XP 9569662)

Glucoamylase Aspergillus oryzae (BAA008411)Glucan 14-alpha-glucosidase putative Aspergillus flavus NRRL3357 (XP 0023849461)

Glucoamylase Aspergillus ficuum (AAT580371)Glucoamylase Aspergillus awamori (AEI589951)Glucoamylase Aspergillus niger (AIY230671)

100

57100

100

10063

100

74

10067

005

Figure 1 Homology tree between Aspergillus flavus NSH9 (AFNSH9 GA1) glucoamylase and other fungal glucoamylases based on aminoacids sequences by MEGA6 software Bar = 5 amino acid substitution per 100 amino acids

Knowledgebase (UniPort) and GeneBank [30] databaseBased on the analysis of the data set it is observed that the twoamino acids aspartic acid at the 203 position and glutamicacids at 206 position were responsible for catalytic activityof glucoamylase of A flavus NSH9 and tryptophan at 147position was responsible for the binding

32 Transformation Expression of Glucoamylase cDNAand Purification of rGA Linearized recombinant plasmidpPICZ120572C GA was transformed into P pastoris GS115 byPichia EasyComp Transformation kit (Invitrogen) Thismethod produces low transformation efficiency with only 8colonies observed after 4 days of incubation on YPDS agarplate Five recombinant colonies were selected for colonyPCR using AXO1 primers The amplification results showedthat positive recombinants produced two close bands forMut+ in GS115 (21 kb and 22 kb fragments) using 51015840AXO1and 31015840AXO1 primers Meanwhile the control yeast trans-formed with pPICZ120572C produced a 600 bp band It wasobserved that recombinant GS115 (Mut+) produced recombi-nant glucoamylase on BMMY plates after 4 days of growthand also produced decolourization zoneclear halo aroundthe Pichia colonies with iodine solution compared to control(Figure 2)

Heterologous expression of glucoamylase was achievedunder the transcriptional control of the AOX1 promoterin P pastoris and the expression was induced by the sup-plement of methanol After eight days of induction GS115strain had the highest glucoamylase activity of 824UmLminus1at 1 (vv) methanol (Figure 3) and the recombinantglucoamylase expression level was 0145mgmLminus1 in theculture supernatant At higher concentration of methanolglucoamylase expression level was detected to decrease andno expression was observed when induced with 5 (vv) ofmethanol

Control 1

Control 2

Colony 1Colony 2

Colony 4

Colony 1

Colony 2Colony 4

Figure 2 Starch-substrate assay plates showing haloclear zone bythe recombinant Mut+ Pichia colonies on soluble starch Colonies1 2 and 4 were the recombinant GS115 and indicate the clear haloin plate controls 1 and 2 did not produce any glucoamylase on theplate

33 Characterization of Recombinant Glucoamylase

331 Molecular Weight Determination by SDS-PAGE Themolecular weight of the rGA was estimated to be approx-imately 78 kDa when analyzed on SDS-PAGE (Figure 4)This value was higher than the 551 kDa calculated fromthe deduced amino acid sequences of rGA The maturerGA had 514 amino acids which was calculated from theKex2 signal cleavage site to 6x his tag or stop codon of therecombinant plasmid pPICZ120572C GA Based on the results


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05 methanol 1 methanol2 methanol 3 methanol

Figure 3 Expression of recombinant glucoamylase (GA) in differ-ent concentration of methanol with time

6 54321M

Halo

(kDa)

10070

5040

30

25

14

Figure 4 SDS-PAGE and native PAGE analysis of recombinantglucoamylase (GA) Lane 1 and Lane 2 are the 2nd day and 5th day ofexpression respectively Lane 3 indicates the purified recombinantglucoamylase Lane 4 indicates control at 5th day Lane 6 alsoindicates control for activity in native PAGE in 5th day Lane5 indicates activity for expressed GA in native PAGE (purifiedsample)

of molecular weight in the SDS-PAGE and the calculatedweight of rGA the expressed recombinant glucoamylasein the culture was monomeric and might be glycosylatedThe rGA of Aspergillus flavus NSH9 expressed in Pichiapastoris was confirmed by four methods via its ability todecolorize or form halo around the Pichia colonies afterflooding with iodine solution on BMMY plate (Figure 2)zymogram analysis on SDS-PAGE (Figure 4) enzyme assayand MALDI-TOFTOF mass spectrometry analysis of pep-tide after digestion of protein by trypsin (data not shown)

332 Effects of pH Temperature and Kinetics The pH opti-mum of rGA for glucoamylase activity was pH 50 (Fig-ure 5(a)) and rGA was highly stable over a broad pHrange about 80 of the residual activity was retained afterbeing incubated at pH 30 to 90 and at 25∘C for 24 hours

Table 1 Thermal stability of recombinant glucoamylase (GA) indifferent temperature by incubation of diluted enzyme with 01Msodium acetate buffer pH 50 (1 1 250120583L diluted enzyme inwater 250 120583L 01M sodium acetate buffer pH 50)

TimeResidual enzyme activity in different

temperature60∘C 70∘C 80∘C 90∘C 100∘C

0min 100 100 100 100 1005min 832810min 9589 777315min 9825 9698 9153 8816 586720min 5149 496325min 3664 333830min 8082 7089 328 2502 201545min 503 3987 2532 1457 120260min 2415 1112 110 87 075min 1194 582 30 0 090min 845 0 0 0 0120min 0 0 0 0 0Assay condition was set at optimum pH (50) and temperature (70∘C) Eachvalue represents the mean of three independent observations

(Figure 5(c)) The optimum temperature of rGA was 70∘Cand the value was significantly higher from all values (Fig-ure 5(b)) The rGA was thermally more stable at 70∘C whenincubated at neutral pH (water) compared to pH 50 with01M sodium acetate buffer (Figure 5(d)) Approximately90 rGA residual activity was observed after 45min at 70∘Cat neutral pH and about 40 at pH 50 at the same conditionAbout 90 rGA residual activity was observed after 15minof incubation at 60∘C to 90∘C but at 100∘C the rGA residualactivity was about 59 after 15min (Table 1) About 50 rGAresidual activity was observed after incubation of 20min at90∘C and 100∘C (Table 1)

The values of Michaelis-Menten constant 119870m and max-imum velocity and 119881max of the recombinant glucoamylasewere determined using Lineweaver-Burk plot and recordedas 584mgmLminus1 and 15385Umgminus1 protein respectively forsoluble starch

333 Effects ofMetal Ions andDifferent Substrates Metal ionssuch as Na+ K+ Ca2+ and Cd2+ ions had no significanteffect on the recombinant glucoamylase activity at concen-tration of 1mM but higher concentration of 5mM Na+ hadnegative effect whilst Ca2+ had positive effect on enzymeactivity (Table 2) The recombinant glucoamylase enzymewas significantly stimulated by Cu2+ Fe2+ Pb2+ Zn2+and Mg2+ at 1mM concentration of ions but significantlyinhibited by Cu2+ Pb2+ and Zn2+ at 5mM concentration(Table 2) The results revealed that K+ and Cd2+ had noeffect on enzyme activity at different concentrations andrGA activity was significantly stimulated by only Mg2+ ionat different concentration In this work it was observed thatlow concentration of metal ions had a positive effect on


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Residual activity at 70∘C (incubation enzyme in water)Residual activity at 70∘C (incubation in to water buffer pH 50)

(d)

Figure 5 Characterization of the recombinant glucoamylase (a) Effect of pH on enzyme activity (b) Effect of temperature on enzyme activity(c) pH stability of enzyme after 24-hour incubation at 25∘C and the assay condition was at optimum pH (pH 50) and temperature (70∘C)(d) Thermostability of recombinant glucoamylase at 70∘C in two different conditions (diluted enzyme in 01M sodium acetate buffer at pH50 and diluted enzyme in water) assay condition was at optimum pH (pH 50) and temperature (70∘C) Each value in the panel representsthe mean plusmn SD (119899 = 3)

enzyme activity in all ions tested The relative activity ofthe recombinant glucoamylase was observed on 1 (wv)of different starches at optimum temperature (70∘C) andpH (50) The recombinant glucoamylases produced weremore active on the breakdown of gelatinized sago starchas compared to soluble starch amylopectin and glycogen(Table 3)

4 Discussion

The glucoamylase gene from Aspergillus flavus NSH9 con-sisted of two introns and the size of these introns (lt100 bp)is typical for most fungal introns [31]The splice sites of these

introns correspond to the fungal consensus sequences GT ndashAG boundaries and internal consensus for lariat formation[32] From protein BLAST (NCBI) it was determined thatthis glucoamylase cDNAbelonged to the glycoside hydrolases15 family excluding the starch binding domain (SBD) Thesingle domain structure of glucoamylase (glucoamylase with-out starch binding domain) was also observed in many fungisuch as in A niger [10] Rhizopus oryzae [11] and A oryzae[12] which was the similar to the glucoamylase fromA flavusNSH9 in this study Two different forms of glucoamylasewith and without starch binding domain are produced fromone gene or two different genes depending on mRNAsplicing [15] site of transcription initiation posttranslationalprocessing and limited proteolysis [4] For example A niger


Table 2 Effect of metal ion of recombinant glucoamylase (rGA)activity

Name of metalRelative activity of recombinant

glucoamylase (rGA)Metal salt (1mM) Metal salt (5mM)

Control (H2

O) 100 100Na+ 104 086lowast

K+ 104 099Ca++ 103 119lowast

Mg++ 106lowast 113lowast

Fe++ 150lowast 096Zn++ 115lowast 073lowast

Cu++ 163lowast 044lowast

Pb++ 139lowast 066lowast

Cd++ 102 104lowastHaving significant effect on activity of metal ion (119901 le 005 by independentsample 119905-test)

Table 3 Relative activity of recombinant glucoamylase on differentsubstrates

Substrates Relative activitySoluble starch 100Amylopectin 935Glycogen 331Sago starch 1047

and A awamori var kawachi produced two GA with andwithout SBD from the same gene due to proteolytic cleavageof SBD from large GA1 [13] but for A oryzae from twodifferent genes [14] According toHata et al [14] they purifiedtwo glucoamylases of A oryzae from solid-state culture (S-GA) and from submerged culture (L-GA) They concludedthat the two glucoamylases have different molecular weightand all of the enzymatic characteristics of the two glucoamy-lases were similar except the thermal stability and the rawstarch digestibility (L-GA could digest the raw starch) TheS-GA has a higher amount of carbohydrate than L-GA andthey assumed that the higher glycosylation of S-GA increasedits thermal stability and pH stability similar finding alsoobserved in this study Basically in the industry they usedthe glucoamylase having the starch binding domain likeL-GA due to digestibility of raw starch In this study theglucoamylase that discussed was similar to S-GA from Aoryzae having low raw starch digestibility [14] Glucoamylaseis different in the activity on raw and soluble starch based onthe structure and domain glucoamylases with SBD are moreactive on raw starch compared to single domain glucoamylaselike A flavus NSH9 glucoamylase without SBD in this study

The high degree of similarity of the glucoamylase in thisstudywas observedwith the single domain structure of othersglucoamylases such as theA flavusNRRL3357 (XP 0023750-051 putative precursor)A oryzae 3042 (EIT733931) andAparasiticus SU1 (KJK647191) Meanwhile the glucoamylasegene of A flavus NSH9 was comparatively less similar to

the glucoamylase having starch binding domain such as in Aawamori (AEI589951) A niger (AIY230671) and A oryzae(BAA008411)

In the expression study increasing the concentration ofmethanol above 1 had negative effect on expression may beat higher concentrations ofmethanol causes stress to the cellsdecreases cell viability and facilitates cell lysis and proteolyticdegradation [33] Heterologous expression of glucoamylasein this study was comparatively higher compared to glu-coamylases expressed in Clostridium thermosaccharolyticum[34] Sulfolobus solfataricus [35] and Thermomyces lanugi-nosus [36] Our finding was in parallel with previous reportedstudies with molecular weights of GA in fungi to be within arange of 25ndash112 kDa [9] Many researchers reported that themolecular weight of recombinant glucoamylase was higherthan the estimated molecular weight and it depended onthe glycosylation site within the sequence which supportedthis study [21 37ndash39] The study did not measure thedegree of glycosylation of the recombinant glucoamylaseand deglycosylation where this will give a clearer picture ofthe recombinant enzyme properties and become one of thelimitations of this study

It has been reported that fungal glucoamylases are activeat acidic pH [9] and the maximum catalytic activity at pH50 determined in our reinforces many other studies in Aniger (pH 48) [40]A tubingensis (45) [37] and F solani (pH45) [41] The optimum catalytic activity of the recombinantglucoamylase at pH 40 to 50 expressed in P pastoris wasobserved by many researchers [21 38] A higher optimumtemperature of recombinant glucoamylase activity reportedin this study was similar to the recombinant glucoamylasefrom A tubingensis expressed in S cerevisiae [37] and fromRhizomucor pusillus expressed in Pichia pastoris [38] TherGA fromA flavusNSH9had a higher optimum temperaturecompared to recombinant glucoamylase from Chaetomiumthermophilum [21] and A awamori [28] Meanwhile pHstability of rGA reported in this study was almost similar toa number of researchers [9 18 19 39 42] Glucoamylase thatshows high stability over a wide pH range for relatively longperiod time indicates its suitability for industrial applications[9 19]

As for the production of glucose in the starch processingindustry it involves two stage processing steps the liquefac-tion step at a high temperature (95ndash105∘C) by thermostablealpha amylase and saccharification step by glucoamylasewith additional debranching enzymes [43] are normallyperformed in the industry Currently glucoamylases derivedfromAspergillus sp are commonly used in starch industry forthe saccharification process [39 43] But the glucoamylasefrom fungal sources are active at acidic condition and alsoless thermostable and the industrial saccharification takesa long time to produce the desired yield [4] for this it alsorequired pH and temperature adjustments from liquefactionto saccharification steps [39] The reported recombinantglucoamylase (rGA) fromA flavusNSH9 canbe used directlyafter liquefaction process for a complete hydrolysis of thedextrin into 120573-D-glucose due to its thermostability Manyresearchers reported that rapid denaturation of glucoamylaseoccurred at temperatures above 60∘C from F solani [41]


and glucoamylase derived from A citrinus [44] was veryunstable at temperatures above 55∘C The recombinant glu-coamylase from A flavus NSH9 has lower 119870m value com-pared toAcremonium sp [45] andR oryzae [46] respectivelybut higher than F solani [41] and A niger NCIM [47] forsoluble starch Lower 119870m value for starch may be due to ahigher number of interactions between the active site of theenzyme and the substrate molecule resulting in an increasedaffinity of the enzyme towards the substrate [47] Our resultsalso observed that low concentration of metal ions exertspositive effect on recombinant enzyme activity Our resultssupports the findings reported by Bhatti et al [41] for somemetal such as Mg2+ and Ca2+ and inhibitory effect of Cu2+and Zn2+ at higher concentration as reported by Koc andMetin [19] There are many methods which will improvethe stability of rGA from Aspergillus flavus NSH9 suchas immobilization modification and protein engineering[48] Immobilization of enzyme enhances their properties forefficient utilization in industrial process [48] Magnetically(magnetic nanoparticles MNPs) immobilized enzymes haveadvantages for commercial application due to their ease ofseparation and reusability [48]

5 Conclusion

In this work a novel thermostable glucoamylase cDNAwithout the substrate binding domain (SBD) of Aspergillusflavus NSH9 has been successfully isolated characterizedand overexpressed in Pichia pastorisGS115 producing biolog-ically active recombinant glucoamylase (rGA) These findingopens up for the possibility of upscaling andmass productionof the recombinant thermostable glucoamylase in an easiermanner Subsequently this rGA has the potential for useand application in the saccharification steps due to itsthermostability in the starch processing industries

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

Acknowledgments

This research project was financially supported by the Min-istry of Higher Education Malaysia (MoHE) under theResearch Acculturation Grant Scheme (RAGS) RAGSb(9)9312012(32)

References

[1] J Sauer B W Sigurskjold U Christensen et al ldquoGlucoamy-lase structurefunction relationships and protein engineeringrdquoBiochimica et Biophysica Acta (BBA)mdashProtein Structure andMolecular Enzymology vol 1543 no 2 pp 275ndash293 2000

[2] Q D Nguyen J M Rezessy-Szabo M Claeyssens I Stals andA Hoschke ldquoPurification and characterisation of amylolyticenzymes from thermophilic fungus Thermomyces lanuginosusstrain ATCC 34626rdquo Enzyme and Microbial Technology vol 31no 3 pp 345ndash352 2002

[3] A Pandey P Nigam V T Soccol D Singh and R MohanldquoAdvances in microbial enzymesrdquo Biotechnology and AppliedBiochemistry vol 31 pp 135ndash152 2000

[4] P Kumar and T Satyanarayana ldquoMicrobial glucoamylasescharacteristics and applicationsrdquo Critical Reviews in Biotechnol-ogy vol 29 no 3 pp 225ndash255 2009

[5] B I Cantarel P M Coutinho C Rancurel T Bernard V Lom-bard and B Henrissat ldquoThe Carbohydrate-Active EnZymesdatabase (CAZy) an expert resource for glycogenomicsrdquoNucleic Acids Research vol 37 no 1 pp D233ndashD238 2009

[6] X-J Wang J-G Bai and Y-X Liang ldquoOptimization of mul-tienzyme production by two mixed strains in solid-state fer-mentationrdquoAppliedMicrobiology and Biotechnology vol 73 no3 pp 533ndash540 2006

[7] R Te Biesebeke E Record N Van Biezen et al ldquoBranchingmutants of Aspergillus oryzae with improved amylase andprotease production on solid substratesrdquo Applied Microbiologyand Biotechnology vol 69 no 1 pp 44ndash50 2005

[8] B Jensen and J Olsen ldquoAmylases and their inudstrial poten-tialrdquo in Thermophilic Moulds in Biotechnology B N Johri TSatyanarayana and J Olsen Eds pp 115ndash137 Kluwer AcademicPublishers Amsterdam The Netherlands 1999

[9] D Norouzian A Akbarzadeh J M Scharer and M M YoungldquoFungal glucoamylasesrdquo Biotechnology Advances vol 24 no 1pp 80ndash85 2006

[10] A D Joslashrgensen J Noslashhr J S Kastrup et al ldquoSmall angle X-raystudies reveal that Aspergillus niger glucoamylase has a definedextended conformation and can form dimers in solutionrdquo TheJournal of Biological Chemistry vol 283 no 21 pp 14772ndash147802008

[11] J A Mertens and C D Skory ldquoIsolation and characterizationof two genes that encode active glucoamylase without a starchbinding domain from Rhizopus oryzaerdquo Current Microbiologyvol 54 no 6 pp 462ndash466 2007

[12] Y Hata H Ishida E Ichikawa A Kawato K Suginami and SImayasu ldquoNucleotide sequence of an alternative glucoamylase-encoding gene (glaB) expressed in solid-state culture ofAspergillus oryzaerdquo Gene vol 207 no 2 pp 127ndash134 1998

[13] S Hayashida K Nakahara W Kanlayakrit T Kara and Y Ter-amoto ldquoCharacteristics and function of raw-starch-affinity siteon Aspergillus awamori var kawachiglucoamylase I moleculerdquoAgricultural and Biological Chemistry vol 53 no 1 pp 143ndash1491989

[14] Y Hata H Ishida Y Kojima et al ldquoComparison of two glu-coamylases produced byAspergillus oryzae in solid-state culture(koji) and in submerged culturerdquo Journal of Fermentation andBioengineering vol 84 no 6 pp 532ndash537 1997

[15] E Boel I Hjort B Svensson F Norris K E Norris and NP Fiil ldquoGlucoamylases G1 and G2 from Aspergillus niger aresynthesized from two different but closely related mRNAsrdquoTheEMBO Journal vol 3 no 5 pp 1097ndash1102 1984

[16] S Hayashida K Nakahara K Kuroda T Miyata and SIwanaga ldquoStructure of the raw-starch-affinity site on theAspergillus awamori var kawachi glucoamylase I moleculerdquoAgricultural and Biological Chemistry vol 53 no 1 pp 135ndash1411989

[17] M S El-Abyad F A El-Sayed and M Hafez ldquoEffects ofculture conditions on amylase production by some soil fungirdquoZentralblatt fur Mikrobiologie vol 147 no 1-2 pp 23ndash34 1992

[18] E Gomes S R De Souza R P Grandi and RD Silva ldquoProduc-tion of thermostable glucoamylase by newly isolatedAspergillus


flavus a 11 and Thermomyces lanuginosus A 1337rdquo BrazilianJournal of Microbiology vol 36 no 1 pp 75ndash82 2005

[19] O Koc and K Metin ldquoPurification and characterization ofa thermostable glucoamylase produced by Aspergillus flavusHBF34rdquo African Journal of Biotechnology vol 9 no 23 pp3414ndash3424 2010

[20] G Potvin A Ahmad and Z Zhang ldquoBioprocess engineeringaspects of heterologous protein production in Pichia pastoris areviewrdquo Biochemical Engineering Journal vol 64 no 1 pp 91ndash105 2012

[21] J Chen Y-Q Zhang C-Q Zhao A-N Li Q-X Zhou and D-C Li ldquoCloning of a gene encoding thermostable glucoamylasefrom Chaetomium thermophilum and its expression in Pichiapastorisrdquo Journal of Applied Microbiology vol 103 no 6 pp2277ndash2284 2007

[22] U SchumannNA Smith andM-BWang ldquoA fast and efficientmethod for preparation of high-quality RNA from fungalmyceliardquo BMC Research Notes vol 6 no 1 article 71 2013

[23] O F Cubero A Crespo J Fatehi and P D Bridge ldquoDNAextraction and PCR amplification method suitable for freshherbarium-stored lichenized and other fungirdquo Plant Systemat-ics and Evolution vol 216 no 3-4 pp 243ndash249 1999

[24] H-B Shen and K-C Chou ldquoSignal-3L a 3-layer approachfor predicting signal peptidesrdquo Biochemical and BiophysicalResearch Communications vol 363 no 2 pp 297ndash303 2007

[25] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[26] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959

[27] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein-dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[28] F C Pavezzi E Gomes and R da Silva ldquoProduction and char-acterization of glucoamylase from fungus Aspergillus awamoriexpressed in yeast Saccharomyces cerevisiae using differentcarbon sourcesrdquo Brazilian Journal of Microbiology vol 39 no1 pp 108ndash114 2008

[29] K Tamura G Stecher D Peterson A Filipski and S KumarldquoMEGA6molecular evolutionary genetics analysis version 60rdquoMolecular Biology and Evolution vol 30 no 12 pp 2725ndash27292013

[30] D A Benson I Karsch-Mizrachi D J Lipman J Ostell and EW Sayers ldquoGenBankrdquo Nucleic Acids Research vol 39 no 1 ppD32ndashD37 2011

[31] S J Gurr S E Unkles and J R Kinghorn ldquoThe structure andorganization of nuclear genes of filamentous fungirdquo in GeneStructure in Eukaryotic Microbes J R Kinghorn Ed pp 93ndash139 IRL Oxford UK 1987

[32] R A Padgett M M Konarska P J Grabowski S F Hardy andP A Sharp ldquoLariat RNArsquos as intermediates and products in thesplicing of messenger RNA precursorsrdquo Science vol 225 no4665 pp 898ndash903 1984

[33] H Hohenblum K Vorauer-Uhl H Katinger and DMattanovich ldquoBacterial expression and refolding of humantrypsinogenrdquo Journal of Biotechnology vol 109 no 1-2 pp 3ndash112004

[34] U Specka F Mayer and G Antranikian ldquoPurification andproperties of a thermoactive glucoamylase from Clostridium

thermosaccharolyticumrdquo Applied and Environmental Microbiol-ogy vol 57 no 8 pp 2317ndash2323 1991

[35] M-S Kim J-T Park Y-W Kim et al ldquoProperties of anovel thermostable glucoamylase from the hyperthermophilicarchaeon sulfolobus solfataricus in relation to starch process-ingrdquo Applied and Environmental Microbiology vol 70 no 7 pp3933ndash3940 2004

[36] T S Thorsen A H Johnsen K Josefsen and B Jensen ldquoIden-tification and characterization of glucoamylase from the fun-gus Thermomyces lanuginosusrdquo Biochimica et Biophysica Acta(BBA)mdashProteins and Proteomics vol 1764 no 4 pp 671ndash6762006

[37] M J Viktor S H Rose W H Van Zyl and M Viljoen-BloomldquoRaw starch conversion by Saccharomyces cerevisiae expressingAspergillus tubingensis amylasesrdquo Biotechnology for Biofuels vol6 no 1 article 167 pp 1ndash11 2013

[38] Z He L Zhang Y Mao et al ldquoCloning of a novel thermostableglucoamylase from thermophilic fungus Rhizomucor pusillusand high-level co-expression with 120572-amylase in Pichia pastorisrdquoBMC Biotechnology vol 14 article 114 2014

[39] HHua H Luo Y Bai et al ldquoA thermostable glucoamylase fromBispora sp MEY-1 with stability over a broad pH range andsignificant starch hydrolysis capacityrdquo PLoS ONE vol 9 no 11Article ID e113581 2014

[40] J Jafari-Aghdam K Khajeh B Ranjbar and M Nemat-Gorgani ldquoDeglycosylation of glucoamylase from Aspergillusniger effects on structure activity and stabilityrdquo Biochimica etBiophysica ActamdashProteins and Proteomics vol 1750 no 1 pp61ndash68 2005

[41] H N Bhatti M H Rashid R Nawaz M Asgher R Perveenand A Jabbar ldquoPurification and characterization of a novelglucoamylase from Fusarium solanirdquo Food Chemistry vol 103no 2 pp 338ndash343 2007

[42] FGMoreira V Lenartovicz andRM Peralta ldquoA thermostablemaltose-tolerant120572-amylase fromAspergillus tamariirdquo Journal ofBasic Microbiology vol 44 no 1 pp 29ndash35 2004

[43] N Goyal J K Gupta and S K Soni ldquoA novel raw starchdigesting thermostable 120572-amylase from Bacillus sp I-3 and itsuse in the direct hydrolysis of raw potato starchrdquo Enzyme andMicrobial Technology vol 37 no 7 pp 723ndash734 2005

[44] M Niaz M Y Ghafoor A Jabbar E Rasul A Wahid andR Ahmed ldquoIsolation and purification of glucoamylases fromArachniotus citrinus under solid phase growth conditionsrdquoInternational Journal of Biology and Biotechnology vol 1 no 1pp 15ndash23 2004

[45] Y Marlida N Saari Z Hassan S Radu and J Bakar ldquoPurifica-tion and characterization of sago starch-degrading glucoamy-lase from Acremonium sp endophytic fungusrdquo Food Chemistryvol 71 no 2 pp 221ndash227 2000

[46] W Suntornsuk andW D Hang ldquoPurification and characterisa-tion of glucoamylase from Rhizopus oryzae mutantrdquo Journal ofthe Science Society of Thailand vol 23 no 3 pp 199ndash208 1997

[47] P Selvakumar L Ashakumary A Helen and A PandeyldquoPurification and characterization of glucoamylase produced byAspergillus niger in solid state fermentationrdquo Letters in AppliedMicrobiology vol 23 no 6 pp 403ndash406 1996

[48] H Vaghari H Jafarizadeh-Malmiri M Mohammadlou etal ldquoApplication of magnetic nanoparticles in smart enzymeimmobilizationrdquo Biotechnology Letters vol 38 no 2 pp 223ndash233 2016

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


productions of glucoamylase in the enzyme industry areachieved by Aspergillus niger [6] Aspergillus oryzae andRhizopus oryzae [7]

Fungi often produce more than one form of enzyme thatmay have different molecular weights amino acid composi-tion glycosylation ability to digest soluble and raw starchesand stability [8] The majority of fungal glucoamylase is mul-tidomain consisting of a catalytic domain at the N-terminusand a starch binding domain (SBD) at the C-terminus [9]which is separated by an O-glycosylated linker region rich inserine and threonine residues But with exception glucoamy-lases with a single domain structure are also available in Aniger [10]Rhizopus oryzae [11] andA oryzae [12] Two differ-ent forms of glucoamylase with and without starch bindingdomain are produced from one gene or two different genesFor example A niger and A awamori var kawachi producetwo forms of glucoamylase (GA) from the same gene [13]while A oryzae produces two GA from two different genes[14]

Due to industrial importance glucoamylase encodinggene has been cloned characterized and heterologouslyexpressed which derived the Aspergillus species A niger[15] A awamori var kawachi [16] and A oryzae [12]El-Abyad and his coworker [17] screened 21 species foramylolytic activity and found that A flavus was among themost active amylase producer It is also reported that Aflavus NSH9 was the best amylase producer among fourteenisolates of Aspergillus sp [18] Although glucoamylase fromA flavus has been characterized [18 19] little is knownabout its molecular information The genetic sequencemolecular information substrate specificity and biochemicalcharacteristic of glucoamylase from A flavus are not yetwell known On the other hand Pichia pastoris is one ofthe most used eukaryotic systems for the production ofrecombinant proteins It is easy to culture and can reachhigh cell densities and has strong and regulated promotersand ability to secrete proteins as well as introducing post-translational modifications [20 21] Here we described theisolation cloning and sequence analysis of glucoamylasegene (GA) derived from Aspergillus flavus and its expressionin Pichia pastoris

2 Materials and Methods

21 Maintenance and Culturing of Aspergillus Strain Anewly fungal strain of Aspergillus flavus NSH9 with starchdegrading properties was previously isolated from the sagohumus and kept as collection at the Department ofMolecularBiology Faculty of Resource Science and Technology Uni-versiti Malaysia Sarawak Malaysia For enzyme inductionthe actively growing fungal mycelium was transferred frompotato dextrose agar (PDA) plate to a culture mediumcontaining (in g Lminus1) 20 g raw sago starch 3 g KH

2PO4 1 g

(NH4)2SO4 05 g MgSO

4sdot7H2O and 4 g of yeast extract The

mycelia were collected after 4 days of growth in the culturemedia at room temperature of 28∘C with shaking at 150 rpmand then subsequently used to extract the total RNA

22 Extraction of Total RNA and DNA and First-Strand cDNASynthesis Total RNA was extracted by using TRIzol Reagentaccording to the protocol as described by Schumann et al[22] and the genomic DNA was extracted according to themethod of Cubero et al [23] Total RNAwas purified by beingtreated with DNAse and reverse transcriptions were carriedout in 20120583L reaction mixture containing the following 01ndash5 120583g DNA free RNA as a template 05 120583g oligo (dt)

18 4 120583L

of 5x reaction buffer 2 120583L 10mM dNTP Mix 20U RiblockRNase Inhibitor and 40U Moloney Murine Leukemia Virus(M-MuLV) reverse transcriptase (Thermo Scientific) Thereaction was performed at 37∘C for 60min and at 70∘Cfor 5min The product was either used immediately in thesubsequent PCR analysis or stored at minus80∘C

23 Isolation and Subcloning of Glucoamylase cDNA Glu-coamylase gene fromAspergillus flavusNSH9 was isolated byusing GAAsp-F and GAAsp-R primers that were designedbased on sequences of A flavus NRRL3357 (glucoamy-lase precursor putative mRNA XM 0023749641) obtainedfrom the National Center for Biotechnology Information(NCBI) The PCR was carried out in 50120583L reaction mixturecontaining the following 5 120583L 10x Pol Buffer A (1x finalconcentration) 5mMMgCl

2 02mMdNTP 01ndash05 120583Meach

of the forward and reverse gene specific primers 05 120583gcDNA template and 125U Taq DNA polymerase (EURxGdansk Poland)The full length ofA flavusNSH9 glucoamy-lase cDNA and glucoamylase gene were amplified by PCRusing the following oligonucleotide primers GAAsp F (51015840-CGCATGCGGAACAACCTTCTTT-31015840) and GAAsp R (51015840-CTACCACGACCCAACAGTTGG-31015840) The PCR reactionwas carried out using the following conditions denaturationat 94∘C for 2min for one cycle followed by denaturation at94∘C for 45 sec annealing at 55∘C for 45 sec and elongationat 72∘C for 145min for 35 cycles and a final elongationwas at 72∘C for 5min The purified PCR product was thencloned into pGEMT-Easy Vector (Promega) as described bythe manufacturer protocol and transformed into E coli XL1-Blue Purified plasmid was run on agarose gel and sequencedusing T7 and SP6 primers

24 Cloning of Glucoamylase cDNA into Yeast ExpressionVector ThepPICZ120572C (Invitrogen)was selected as expressionvector and used for the cloning of glucoamylase cDNAwithout signal peptide sequences A putative signalsequence was predicted by the Signal-3L program [24]and glucoamylase cDNA without the signal peptide wasamplified using the following list of primers forward primer(51015840-AGCGAATTCAGATTACAAG GACGACGACGAT-AAGCCGTCCTTCCCTATCCAT-31015840) and reverse primer(51015840-ACGACTCTAGAGCCCACGACCCAACAGTTGG-31015840)The designed primers contained an EcoR1 site and Xbal sitein the forward and reverse primers (bold and underline)respectively In addition the forward primer was designed toinclude FLAG tag containing 24 nucleotides (DYKDDDDK)and the reverse primer lacking in the native stop codon Theprimers were designed to be in frame with the C-terminal6x His tag located in the pPICZ120572C PCR was performed as





600of
















3 Results











100

57100

100

10063

100

74

10067

005





Control 1

Control 2

Colony 1Colony 2

Colony 4

Colony 1

Colony 2Colony 4





0123456789

10

0 2 4 6 8 10 12

Enzy

me a

ctiv

ity (U

mL

min

)

(Days)



6 54321M

Halo

(kDa)

10070

5040

30

25

14












0

1

2

3

4

5

6

7

2 3 4 5 6 7 8 9 10Different pH

Activ

ity (120583

mol

mL

min

)

(a)

0

1

2

3

4

5

6

7

8

9

10

10 20 30 40 50 60 70 80 90 100

Activ

ity (120583

mol

mL

min

)

Temperature (∘C)

(b)

0

20

40

60

80

100

120

0

1

2

3

4

5

6

7

8

9

3 4 5 6 7 8 9

Resid

ual a

ctiv

ity (

)

pH

Residual activity

Activ

ity (120583

mol

mL

min

)

After 24hAt 0min

(c)

0

20

40

60

80

100

120

0 15 30 45 60 75 90 105 120

Resid

ual a

ctiv

ity

Time (min)


(d)



4 Discussion







Control (H2

O) 100 100Na+ 104 086lowast

K+ 104 099Ca++ 103 119lowast
















5 Conclusion


Competing Interests


Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





600of
















3 Results











100

57100

100

10063

100

74

10067

005





Control 1

Control 2

Colony 1Colony 2

Colony 4

Colony 1

Colony 2Colony 4





0123456789

10

0 2 4 6 8 10 12

Enzy

me a

ctiv

ity (U

mL

min

)

(Days)



6 54321M

Halo

(kDa)

10070

5040

30

25

14












0

1

2

3

4

5

6

7

2 3 4 5 6 7 8 9 10Different pH

Activ

ity (120583

mol

mL

min

)

(a)

0

1

2

3

4

5

6

7

8

9

10

10 20 30 40 50 60 70 80 90 100

Activ

ity (120583

mol

mL

min

)

Temperature (∘C)

(b)

0

20

40

60

80

100

120

0

1

2

3

4

5

6

7

8

9

3 4 5 6 7 8 9

Resid

ual a

ctiv

ity (

)

pH

Residual activity

Activ

ity (120583

mol

mL

min

)

After 24hAt 0min

(c)

0

20

40

60

80

100

120

0 15 30 45 60 75 90 105 120

Resid

ual a

ctiv

ity

Time (min)


(d)



4 Discussion







Control (H2

O) 100 100Na+ 104 086lowast

K+ 104 099Ca++ 103 119lowast
















5 Conclusion


Competing Interests


Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








3 Results











100

57100

100

10063

100

74

10067

005





Control 1

Control 2

Colony 1Colony 2

Colony 4

Colony 1

Colony 2Colony 4





0123456789

10

0 2 4 6 8 10 12

Enzy

me a

ctiv

ity (U

mL

min

)

(Days)



6 54321M

Halo

(kDa)

10070

5040

30

25

14












0

1

2

3

4

5

6

7

2 3 4 5 6 7 8 9 10Different pH

Activ

ity (120583

mol

mL

min

)

(a)

0

1

2

3

4

5

6

7

8

9

10

10 20 30 40 50 60 70 80 90 100

Activ

ity (120583

mol

mL

min

)

Temperature (∘C)

(b)

0

20

40

60

80

100

120

0

1

2

3

4

5

6

7

8

9

3 4 5 6 7 8 9

Resid

ual a

ctiv

ity (

)

pH

Residual activity

Activ

ity (120583

mol

mL

min

)

After 24hAt 0min

(c)

0

20

40

60

80

100

120

0 15 30 45 60 75 90 105 120

Resid

ual a

ctiv

ity

Time (min)


(d)



4 Discussion







Control (H2

O) 100 100Na+ 104 086lowast

K+ 104 099Ca++ 103 119lowast
















5 Conclusion


Competing Interests


Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








100

57100

100

10063

100

74

10067

005





Control 1

Control 2

Colony 1Colony 2

Colony 4

Colony 1

Colony 2Colony 4





0123456789

10

0 2 4 6 8 10 12

Enzy

me a

ctiv

ity (U

mL

min

)

(Days)



6 54321M

Halo

(kDa)

10070

5040

30

25

14












0

1

2

3

4

5

6

7

2 3 4 5 6 7 8 9 10Different pH

Activ

ity (120583

mol

mL

min

)

(a)

0

1

2

3

4

5

6

7

8

9

10

10 20 30 40 50 60 70 80 90 100

Activ

ity (120583

mol

mL

min

)

Temperature (∘C)

(b)

0

20

40

60

80

100

120

0

1

2

3

4

5

6

7

8

9

3 4 5 6 7 8 9

Resid

ual a

ctiv

ity (

)

pH

Residual activity

Activ

ity (120583

mol

mL

min

)

After 24hAt 0min

(c)

0

20

40

60

80

100

120

0 15 30 45 60 75 90 105 120

Resid

ual a

ctiv

ity

Time (min)


(d)



4 Discussion







Control (H2

O) 100 100Na+ 104 086lowast

K+ 104 099Ca++ 103 119lowast
















5 Conclusion


Competing Interests


Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0123456789

10

0 2 4 6 8 10 12

Enzy

me a

ctiv

ity (U

mL

min

)

(Days)



6 54321M

Halo

(kDa)

10070

5040

30

25

14












0

1

2

3

4

5

6

7

2 3 4 5 6 7 8 9 10Different pH

Activ

ity (120583

mol

mL

min

)

(a)

0

1

2

3

4

5

6

7

8

9

10

10 20 30 40 50 60 70 80 90 100

Activ

ity (120583

mol

mL

min

)

Temperature (∘C)

(b)

0

20

40

60

80

100

120

0

1

2

3

4

5

6

7

8

9

3 4 5 6 7 8 9

Resid

ual a

ctiv

ity (

)

pH

Residual activity

Activ

ity (120583

mol

mL

min

)

After 24hAt 0min

(c)

0

20

40

60

80

100

120

0 15 30 45 60 75 90 105 120

Resid

ual a

ctiv

ity

Time (min)


(d)



4 Discussion







Control (H2

O) 100 100Na+ 104 086lowast

K+ 104 099Ca++ 103 119lowast
















5 Conclusion


Competing Interests


Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

1

2

3

4

5

6

7

2 3 4 5 6 7 8 9 10Different pH

Activ

ity (120583

mol

mL

min

)

(a)

0

1

2

3

4

5

6

7

8

9

10

10 20 30 40 50 60 70 80 90 100

Activ

ity (120583

mol

mL

min

)

Temperature (∘C)

(b)

0

20

40

60

80

100

120

0

1

2

3

4

5

6

7

8

9

3 4 5 6 7 8 9

Resid

ual a

ctiv

ity (

)

pH

Residual activity

Activ

ity (120583

mol

mL

min

)

After 24hAt 0min

(c)

0

20

40

60

80

100

120

0 15 30 45 60 75 90 105 120

Resid

ual a

ctiv

ity

Time (min)


(d)



4 Discussion







Control (H2

O) 100 100Na+ 104 086lowast

K+ 104 099Ca++ 103 119lowast
















5 Conclusion


Competing Interests


Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Control (H2

O) 100 100Na+ 104 086lowast

K+ 104 099Ca++ 103 119lowast
















5 Conclusion


Competing Interests


Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



5 Conclusion


Competing Interests


Acknowledgments


References



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article heterologous, expression, and...

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