Cloning and molecular modelling of pectin degrading glycosyl hydrolase of family 28 from soil metagenomic library.
Western Ghats of India is recognized as one of the 12 mega diversity regions of the world and is the hot spot for unrevealed microbial diversity. To explore the diversity of polysaccharide degrading enzymes in that region, metagenomic library was constructed from forest soil of Southern Western Ghats region. Nine pectinolytic clones with the ability to degrade citrus pectin were isolated based on function based screening of the library. Sequence analysis of pg_4 clone containing revealed that it contained GH family 28 domain (pfam00295) belonging to polygalacturonase superfamily (PLN03003). Its amino acid sequence analysis showed 25-55 % identity to the other well-characterized polygalacturonases. Molecular modeling of pg_4 revealed that it comprised of three right handed-parallel β sheets, one anti-parallel β sheet and one α helix with three conserved catalytic residue D 2263, D 284-85 and H 312 at the C terminal end. The enzyme characterized was able to hydrolyze both apple and citrus pectin with K m values of 1.685 and 1.542 mg ml(-1) and retained more that 80 % of activity at pH 5-9 and temperature 20-60 °C.
Department of Food Microbiology, CSIR-Central Food Technological Research Institute, Mysore, 570020, India.
T A
TA
Sathya
Ani Methew
AM
Jacob
Mahejibin
M
Khan
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004789:Enzyme Activation; D015723:Gene Library; D023061:Gene Order; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006868:Hydrolysis; D056186:Metagenomics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D010368:Pectins / Q000378:metabolism*; D010802:Phylogeny; D011487:Protein Conformation; D016415:Sequence Alignment; D012988:Soil Microbiology; D013816:Thermodynamics
D010368:Pectins; D006026:Glycoside Hydrolases