Structural And Functional Studies Of The Endoglucanase Cel10 From Klebsiella Pneumoniae

Cellulase is a set of enzyme for the degradation of cellulose.Microbial cellulase has an important significance in transforming insoluble cellulose into glucose the utilization of these cellulase in industrial processes is fast becoming a hot topic.Here,a Cel10 was identified in a strain of Klebsiella pneumoniae,isolated from Chinese bamboo rat gut.The objective of this study was to identify prospective cellulase-producing gene and characterize the cellulolytic activity and structural function of Cel10,and finally make adjustment on the enzyme to get higher cellulase activity based on the structure of Cel10.1.Analysis of substrate specificity showed that Cel10 is able to hydrolyze amorphous carboxymethyl cellulose(CMC)and crystalline forms of cellulose(Avicel and xylan)but is unable to hydrolyze p-nitrophenol-d-glucopyranoside(p-NPG),proving that Cel10 is an endoglucanase.A phylogenetic tree analysis indicates that Cel10 belongs to the glycoside hydrolase 8(GH8)subfamily.The optimum temperature and pH for this Cel10 activity was 50? and pH 5.0.And was stable between the range of pH 4.0-8.0,and 20-60?,respectively.The enzyme was activated by Mn²⁺and Co²⁺,but inhibited by SDS,EDTA,DTT,Hg⁺,K⁺,Na⁺,Cd²⁺,Fe²⁺ and Pb²⁺.2.In order to further understanding of its substrate specificity,the structure of Cel10 was solved by molecular replacement and refined to 1.76 A resolution.The overall fold is distinct from those of most other enzymes belonging to the GH8 subfamily.Although it forms the typical(?/?)6 barrel motif fold,like Acetobacterxylinum CMCax,one helix is missing.Structural comparisons with Clostridium thermocellum CelA(CtCelA),the best characterized GH8 endoglucanase,revealed that sugar-recognition subsite-3 is completely missing in Cel10.The absence of this subsite correlates to a more open substrate-binding cleft on the cellooligosaccharide reducing-end side.3.In order to make effective in the cellulose-production process from dynamic lignocellulosic material,we require potentially acting and stable cellulolytic enzymes.In our investigation,the endoglucanase Cel10 was subjected to site-directed mutagenesis and carbohydrate-binding module(CBM)engineering.For this purpose,amino acids around the active-site region were targeted.Results indicated that two single mutants showed no significant difference as compared to the wild-type.Conclusion:The present investigation provides a basis for of the cellulose biosynthesis from family 8 cellulases.The architecture of the active-site cleft,presenting at least five glucosyl-binding subsites,explains why family 8 cellulases cleave cellooligosaccharide polymers that are at least five D-glucosyl subunits long.However,structure comparison at the active site between Cel10 and CtCelA revealed notable differences;two out of five aromatic residues that,with stacking interactions,are critical for substrate recognition by CtCelA,corresponding to Trp205 and Tyr369 of CtCelA are not conserved in Cel10.And it is believed that the nature of these the amino acids play a significant role in the different catalytic activities observed in the enzymes.It is hoped that,future researchers would shed more light on the efficiency and effectiveness of cellulosic substrates.