Arg235 is an essential catalytic residue of Bacillus pumilus DKS1 pectate lyase to degum ramie fibre

Abstract

After 24 h of incubation with only purified pectate lyase isolated from Bacillus pumilus DKS1 (EF467045), the weight loss of the ramie fibre was found to be 25%. To know the catalytic residue of pectate lyase the pel gene encoding a pectate lyase from the strain Bacillus pumilus DKS1 was cloned in E. coli XL1Blue and expressed in E. coli BL21 (DE3) pLysS. The pel gene was sequenced and showed 1032 bp length. After purification using CM-Sepharose the enzyme showed molecular weight of 35 kDa and maximal enzymatic activity was observed at 60°C and a pH range of 8.5-9.0. Both Ca²(+) and Mn²(+) ions were required for activity on Na-pectate salt substrates, while the enzyme was strongly inhibited by Zn²(+) and EDTA. The deduced nucleotide sequence of the DKS1 pectate lyase (EU652988) showed 90% homology to pectate lyases from Bacillus pumilus SAFR-032 (CP000813). The 3D structure as well as the catalytic residues was predicted using EasyPred software and Catalytic Site Atlas (CSA), respectively. Site directed mutagenesis confirmed that arginine is an essential catalytic residue of DKS1 pectate lyase.

Improving the thermoactivity and thermostability of pectate lyase from Bacillus pumilus for ramie degumming

“…Pel from Bacillus pumilus DKS1 was successfully cloned, expressed, and characterized in Escherichia coli, displaying maximal enzyme activity at 60°C and pH 8.5-9.0 (Basu et al 2011;Klug-Santner et al 2006). Previous studies have indicated that Ca 2+ ion is required for degrading Na-pectate salt substrates.…”

Bio-processing of bamboo fibres for textile applications: a mini review

“…Pectin is also regarded as the most structurally complex of polysaccharides (O ' Neill et al 1990), essentially consisting of long chains of (1,4)-α -D-polygalacturonate with a partial esterifi cation (Basu et al 2011). The pectin family is composed of α (1 -4) glycosidically linked to polygalacturonic acid (PGA) with small amounts of L-rhamnose (2 -4%) linked β (1 → 2) and various side-chains comprising L-arabinose, D-galactose and β -D-xylose (Mohnen 2008).…”

“…Pectin esterases de-esterify the methyl ester linkages of the pectin backbone releasing pectins and methanol (Jayani et al 2005). Pectin lyases are specifi c for methyl esterifi ed substrate (pectin) and act on glycosidic linkages next to methyl esteri fi ed carboxylic groups (Basu et al 2011;Gummadi & Kumar 2005). Pectate lyases show specifi city for unesterifi ed polygalacturonate (pectate) and catalyze the trans-eliminative cleavage of the galacturonic acid polymer, eliminating oligosaccharides of α (1→4) linked galacturonic acid residues (Basu et al 2011;Jayani et al 2005;Yadav et al 2009).…”

“…Pectin lyases are specifi c for methyl esterifi ed substrate (pectin) and act on glycosidic linkages next to methyl esteri fi ed carboxylic groups (Basu et al 2011;Gummadi & Kumar 2005). Pectate lyases show specifi city for unesterifi ed polygalacturonate (pectate) and catalyze the trans-eliminative cleavage of the galacturonic acid polymer, eliminating oligosaccharides of α (1→4) linked galacturonic acid residues (Basu et al 2011;Jayani et al 2005;Yadav et al 2009). Pectate lyases can catalyze the degradation of pectin located in the middle lamella and in the primary cell of higher plants and result in the maceration of plant tissues (Herron et al 2000).…”

“…Over the years, biotechnology has been employed in the textile fi bre processing such as fl ax and kenaf retting , ramie fi bre extraction (Zheng et al 2001;Basu et al 2011;Basu et al 2009), cotton bio-scouring (Agrawal et al 2008;Rajendran et al 2011), silk degumming (Arami et al 2007), wool functionalization (Queiroga et al 2007;Hossain et al 2009), etc.…”