Study On Endoglucanase Of Marine Bacterium Cellulophaga Sp. QY201

Cellulose is composed of D-glucose residues linked together to form linear chains viaβ-1,4-glycosidic linkages. It is the major polymeric component of plant cell wall and also the most abundant polysaccharide in nature. The value of cellulose as a renewable source of carbon and energy has made cellulose hydrolysis the subject of scientific research and industrial interest. Cellulase is a generic term of enzymes that can hydrolyze cellulose into glucose.Traditionally it has been classified into three categories, namely, endoglucanase, exoglucanase andβ-glucosidase. These enzymes can cooperate to degrade cellulose to cellooligosaccharide and cellobiose, and eventually to glucose. Degradation of cellulose by cellulase is more economical and effective than the traditionally physical and chemical methods, so it has a wide-range application in areas such as textile industry, commodity industry, food fermentation, laundry detergent and livestock feeding.An endoglucanase was purified 33.2-fold with a recovery yield of 3.2% from culture supernatants of Cellulophaga sp. QY201 to homogeneity using a combination of ammonium sulfate precipitation, DEAE-Sepharose FF anion-exchange chromatography and Superdex 75 gel filtration chromatography. The purified enzyme, which was named CelA, gave a specific activity of 59.8U/mg and a single band on SDS-PAGE with a molecular mass of 35.0 kDa.CelA was most active at 50℃and pH6.0, and stable below 40℃and over a range of pH4.0-6.6. The enzyme retained 50-55% of its maximum activity at 0℃. CelA could withstand 3 h at 40℃without irreversible loss of enzymatic activity and retain 88% of its activity at 40℃for 24h. The activity of the enzyme was enhanced in the presence of Na+,K+,Ni2+. Other compounds (Cu2+,Mg2+,Fe2+,Ba2+,Zn2+,Ca2+, Fe3+,SDS) inhibited the activity of the enzyme,but the inhibition is not significant. Analysis also showed that CelA could retain 95% of its activity after incubated with Proteinase K (0.1mg/mL) at 37℃for 30 min. Analysis of the substrate specificity showed that CelA exhibited high activity on CMCNa, but limited activity on whatman filter paper, xylan, microcrystalline cellulose and no activity on CMC, salicin and starch. The digestion products of various cellooligosaccharides by CelA were submitted to thin-layer chromatography (TLC), which indicated that CelA produced mainly cellobiose and cellotriose along with glucose as a minor product, and the minimal hydrolysis substrate of CelA was cellotetraose.To clone the celA gene, the purified CelA protein was sequenced, and ten N- terminal amino acids and 3 internal peptides were obtained. The gene encoding CelA was cloned using a strategy combining degenerate PCR and inverse PCR. Analysis showed that gene celA contained an open reading frame of 1080bp. The predicted product was a protein of 359 amino acids with a theoretical molecular mass of 40.799 kDa. And there was a signal peptide of 51 amino acids at the N-terminal of the precursor of CelA. The deduced molecular weight and isoelectric point of mature protein were 35.08 kDa and 5.0, respectively, which was consistent with SDS-PAGE result of the native enzyme. Amino acid sequence-based classification revealed that it belonged to glycosyl hydrolase family 5. In order to obtain sufficient amount of the enzyme, the celA gene was subcloned into pET24a (+)/E.coli BL21 (DE3) expression system. The recombinant enzyme was purified by Ni-affinity chromatography after ultrasonic cell crasher in 20 mmol Na2HPO4-citric acid buffer (pH4.6). SDS-PAGE analysis yielded a single band with an apparent molecular mass of 36 kDa, which was close to the predicted molecular mass. And the recombinant enzyme shared uniform enzymatic characteristics with the native enzyme.In conclusion, an neutral endoglucanase possessing activity at 0℃and high thermal stability, was purified from the culture supernatants of marine bacterium Cellulophaga sp. QY201. Its gene was cloned and expressed in pET24a (+)/E.coli BL21 (DE3) system. The discovery of CelA not only enriches the diversity of endoglucanases but also has great practical significance in industrial application areas such as textile industry and commodity industry.