Characterization of modular bifunctional processive endoglucanase Cel5 from Hahella chejuensis KCTC 2396

Abstract

Cel5 from marine Hahella chejuensis is composed of glycoside hydrolase family-5 (GH5) catalytic domain (CD) and two carbohydrate binding modules (CBM6-2). The enzyme was expressed in Escherichia coli and purified to homogeneity. The optimum endoglucanase and xylanase activities of recombinant Cel5 were observed at 65 °C, pH 6.5 and 55 °C, pH 5.5, respectively. It exhibited K m of 1.8 and 7.1 mg/ml for carboxymethyl cellulose and birchwood xylan, respectively. The addition of Ca(2+) greatly improved thermostability and endoglucanase activity of Cel5. The Cel5 retained 90 % of its endoglucanase activity after 24 h incubation in presence of 5 M concentration of NaCl. Recombinant Cel5 showed production of cellobiose after hydrolysis of cellulosic substrates (soluble/insoluble) and methylglucuronic acid substituted xylooligosaccharides after hydrolysis of glucuronoxylans by endo-wise cleavage. These results indicated that Cel5 as bifunctional enzyme having both processive endoglucanase and xylanase activities. The multidomain structure of Cel5 is clearly distinguished from the GH5 bifunctional glycoside hydrolases characterized to date, which are single domain enzymes. Sequence analysis and homology modeling suggested presence of two conserved binding sites with different substrate specificities in CBM6-2 and a single catalytic site in CD. Residues Glu132 and Glu219 were identified as key catalytic amino acids by sequence alignment and further verified by using site directed mutagenesis. CBM6-2 plays vital role in catalytic activity and thermostability of Cel5. The bifunctional activities and multiple substrate specificities of Cel5 can be utilized for efficient hydrolysis of cellulose and hemicellulose into soluble sugars.

Purification and characterizations of a novel recombinant Bacillus velezensis endoglucanase by aqueous two-phase system

“…The enzyme cannot digest pNPG, which indicates that Egls has no β-glucanase activity. This result was in agreement with the findings that some cellulases from Bacillus showed both endoglucanase activity and exoglucanase activity Ghatge et al 2014;Wei et al 2015). Egls is very useful for the pretreatment of lignocellulose of biomass to produce reducing sugar that is used in bio-products and bioenergy production.…”

“…The value K m /K cat of Egls was calculated as 1.74 × 10 −2 mg/ml S. The kinetic parameters (K m , V max , and K cat ) of an endoglucanase from Bacillus strain were found as 0.25 mg/ml, 20 μmol/ml/min, and 0.55/S respectively ). The K m of endoglucanase from the strain Hahella chejuensis KCTC 2396 was 1.8 mg/ml (Ghatge et al 2014). The higher K m value of our strain indicated that the enzyme Egls is less efficient in digesting CMC substrate than reported strains.…”

Extra carbohydrate binding module contributes to the processivity and catalytic activity of a non-modular hydrolase family 5 endoglucanase from Fomitiporia mediterranea MF3/22

“…As far as is known, our current knowledge of the roles of CBMs in processivity of cellulases is mainly based on the comparison of modular cellulases comprising a catalytic domain and different CBMs with their CBMs-deleted variants or catalytic domain mutants [5,6,[22][23][24]. However, it is known that more than 60% of cellulases in cellulose-degrading organisms are non-modular proteins without CBMs [25].…”

Multifunctional cellulolytic auxiliary activity protein HcAA10-2 from Hahella chejuensis enhances enzymatic hydrolysis of crystalline cellulose

“…Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out to analyze protein purity on a vertical slab gel with 5 % (w/v) staking gel and 12 % (w/v) separating gel under denaturing condition. The purification of recombinant Cel5 was carried out as described previously (Ghatge et al 2014). …”

“…Gel extraction and plasmid preparation kits were purchased from Qiagen (Valencia, CA, USA). The recombinant plasmid pECel5 (Ghatge et al 2014) was used for expression of endoglucanase Cel5 derived from H. chejuensis.…”