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Cloning, Expression And Characterization Of A Novel Cellulase Gene Cel28a With Xylanase Activity

Posted on:2013-04-26Degree:MasterType:Thesis
Country:ChinaCandidate:H Q JiangFull Text:PDF
GTID:2250330401985286Subject:Animal Nutrition and Feed Science
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In this study, based on important value of the cellulase and broad prospects ofdeveloping new cellulose in rumen, we screened a new cellulase from having been builtrumen microbial metagenomic DNA library by metagenomic technology, And thecharacterization of enzyme was studied. The main results are as follows:Using selective medium of sodium carboxymethyl cellulose (CMC), we screened sixpositive clones expressing cellulase activity from having been built Anhui white goatrumen microbial metagenomic library containing12,672clones. Then one positiveclone(28L14) with Higher activity was subcloned and sequenced. The result showed thatthe cellulase gene contained1596bp open reading frame (ORF) and encoded531aminoacids. Amino acids sequence of this gene shared51%identities and65%similarities withthe hypothetical protein derived from the Bacteroides cellulosilyticus DSM14838. Analysisof homology modeling by SwissModel showed that the cel28a shared30.4%similaritieswith2jepA. So we can conclude that cellulose gene is a novel gene, and named cel28a.The ORF of cel28a was cloned to vector pET-28a(+), and transferred into E.coliBL21(DE3). Protein of cel28a was overexpressed by IPTG induction. After Ni-NTAaffinity chromatography purification, its enzymatic properties were analyzed. The resultsshowed that the enzyme had cellulase activity and xylanase activity. The optimum pHand optimum temperature of the enzyme was5.0and50℃. Analysis of pH tolerance andthermal stability of the enzyme showed that it was more suitable for acidic environmentand medium temperature, and has no activity under the high temperature. As CMC wassubstrater, the Km was0.104mmol/L and Vmax was17μmol/min; As Oat xylan wassubstrater, the Km was1.98mmol/L and Vmax was2.92μmol/min. Metalions of Fe2+、Mn2+and Co2+on the enzyme activity were promoting, but Cu2+and Zn2+reduce itsactivity. As Oat xylan was substrater, promoting the enzyme activity was only Fe2+andNi2+. EDTA can reduce the enzyme activity to70%and SDS can result the enzyme inacti-vation.In the study, the cellulose and xylan bifunctional enzyme that we obtained waskeeping the same condition of the rumen environment. Sequence analysis showed that theenzyme was from uncultured microorganisms. So we can conclude that the enzyme mustbe from uncultured rumen microorganisms. Because of its bifunctionality, it has wideapplication prospects in the field of feed, food and other field.
Keywords/Search Tags:rumen microorganism, metagenome, cellulose, xylanase
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