title: 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.
authors.affiliation: Division of Applied Life Sciences (BK21), PMBBRC, Gyeongsang National University, Jinju, Gyeongnam, Republic of Korea.
authors.forename: Sunil Subhash
authors.initials: SS
authors.lastname: Ghatge
authors.forename: Amar Anandrao
authors.initials: AA
authors.lastname: Telke
authors.forename: Seo-Hee
authors.initials: SH
authors.lastname: Kang
authors.forename: Venkatesh
authors.initials: V
authors.lastname: Arulalapperumal
authors.forename: Keun-Woo
authors.initials: KW
authors.lastname: Lee
authors.forename: Sanjay Prabhu
authors.initials: SP
authors.lastname: Govindwar
authors.forename: Youngsoon
authors.initials: Y
authors.lastname: Um
authors.forename: Doo-Byoung
authors.initials: DB
authors.lastname: Oh
authors.forename: Hyun-Dong
authors.initials: HD
authors.lastname: Shin
authors.forename: Seon-Won
authors.initials: SW
authors.lastname: Kim
mesh_terms: D059001:Aquatic Organisms / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002475:Cellobiose / Q000378:metabolism; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D020563:Gammaproteobacteria / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005965:Glucuronates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009844:Oligosaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000378:metabolism; D013696:Temperature; D014990:Xylans / Q000378:metabolism
chemical_list: D005965:Glucuronates; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014990:Xylans; C570991:xylooligosaccharide; D002475:Cellobiose; D012965:Sodium Chloride; D002480:Cellulase; D002266:Carboxymethylcellulose Sodium; D002118:Calcium