Cloning, expression and characterization of a metagenome derived thermoactive/thermostable pectinase.
The gene encoding a thermostable pectinase was isolated from a soil metagenome sample. The gene sequence corresponded to an open reading frame of 1,311 bp encoding a translation product of 47.9 kDa. It showed maximum (93 %) identity to a Bacillus licheniformis glycoside hydrolase. Deduced amino acid analysis showed an absence of highly conserved cysteine residues in the N-terminal region at positions 24 and 42, and in the C-terminal region at positions 389, 394, 413 and 424. pQpecJKR01 (pQE30 expression vector containing the pectinase gene) was expressed in Escherichia coli strain M15 as a recombinant fusion protein containing an N-terminal 6× His tag. Biochemical properties of this pectinase were novel. The enzyme had temperature and pH optima of 70 °C and 7.0, respectively, but was active over a broad temperature and pH range. The enzyme was stable at 60 °C with a half-life of 5 h and the enzyme activity was inhibited by 0.1 % diethyl pyrocarbonate and 5 mM dicyclohexyl carbodiimide. The enzyme could be of great use in industrial processes due to its activity over a broad pH range and at high temperature.
Department of Biotechnology, Panjab University, Sector-14, Chandigarh 160014, India.
Rajvinder
R
Singh
Samriti
S
Dhawan
Kashmir
K
Singh
Jagdeep
J
Kaur
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D015870:Gene Expression; D006358:Hot Temperature; D054892:Metagenome; D008969:Molecular Sequence Data; D011096:Polygalacturonase / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D011096:Polygalacturonase