Molecular cloning and characterization of multidomain xylanase from manure library

Abstract

The gene (manf-x10) encoding xylanase from an environmental genomic DNA library was cloned and expressed in Escherichia coli. The manf-x10 encoded a predicted protein of 467 amino acids residues with a molecular mass of 50.3 kD. Sequence analysis of manf-x10 gene revealed that the N-terminus had high homology to the catalytic domain of other bacterial xylanase enzymes. The optimal pH and temperature for xylanase activity were 7.0 and 40°C, respectively. In the presence of 1 mM solution of Co 2? , Fe 2? , Mg 2? and Zn 2? , the relative xylanase activity was enhanced; however, it had almost no activity in the presence of 10 mM solution of Cu 2? . The apparent K m and V max values obtained for the hydrolysis of rye arabinoxylan were 2.8 mg/ml and 49.5 lmol/min/mg, respectively. The C-terminus of the enzyme had high homology to a domain of unknown function found in several mannanase enzymes. Biochemical characterization of the C-terminus of the enzyme revealed a previously unrecognized carbohydrate binding module.

Cloning, Expression and Characteristics of a Novel Alkalistable and Thermostable Xylanase Encoding Gene (Mxyl) Retrieved from Compost-Soil Metagenome

“…In addition, serine rich Q linker region (S 2 GS 2 DITVG 2 TS 2 G 2 TS 2 G 2 S 3 G 2 S 10 G 4 ) has also been detected from amino acid 213 to 248 just after catalytic domain. Such repeated amino acids make linker regions that usually discriminate catalytic domain from carbohydrate binding domain [22], [23], [24]. Moreover, linkers have also been reported as integral parts of various xylanases that connect thermo-stabilizing domains, surface layer homology domains and dockerin domains which play a role in stabilizing the protein [22], .…”

“…Such repeated amino acids make linker regions that usually discriminate catalytic domain from carbohydrate binding domain [22], [23], [24]. Moreover, linkers have also been reported as integral parts of various xylanases that connect thermo-stabilizing domains, surface layer homology domains and dockerin domains which play a role in stabilizing the protein [22], . Amino acid homology and hydrophobic cluster analysis categorized this high molecular weight xylanase into GH 11 family.…”

Isolation of α-Glucuronidase Enzyme from a Rumen Metagenomic Library

“…1% birchwood xylan was digested in 50 mM universal buffer, pH 7, at 40°C in reactions containing either a GH10 endoxylanase enzyme (0.9 nM) [16], a-glucuronidase (15 nM), or both. Hydrolysis of xylan substrate was determined by the detection of reducing sugar as measured by the 3,5-dinitrosalicyclic acid protocol [18].…”

“…Reactions were assembled that contained various combinations of endoxylanase enzyme (MANF-X10) [16] and a-glucuronidase enzyme (RUM630-AG).…”

Isolation and characterization of a novel GH67 α-glucuronidase from a mixed culture

“…Reactions using combinations of a GH10 endoxylanase enzyme (MANF-X10, 9.2 nM) [15] and DEG75-AG (0.35 M) were incubated with the xylan substrate at 45°C at pH 8 up to 24 h. The extent of enzymatic digestion was quantiWed by measuring the release of reducing sugars [16]. Reactions of 50 l were stopped by the addition of 75 l of DNSA reagent (1 % dinitrosalicylic acid and 30 % potassium sodium tartrate in 0.5 M sodium hydroxide), heated to 100°C for 5 min, and cooled to 23°C.…”