Functional cloning and expression of a novel Endo-alpha-1,5-L-arabinanase from a metagenomic library.
A novel endo-alpha-L-arabinanase gene (arn2) was isolated, and expressed in E. coli in active form. The recombinant enzyme (ARN2) had optimum activity at pH 6.0 and 45-50( degrees )C with stability between pH 5.0-8.0 and at temperatures up to 40( degrees )C. The recombinant ARN2 catalyzed internal cleavage of alpha-1,5 glycosidic bonds of CM-arabinan, debranched arabinan, linear arabinan, and sugar beet (native) arabinan at rates of decreasing order, and was inactive on wheat arabinoxylan and p-nitrophenyl-alpha-L-arabinofuranoside. Kinetic analysis showed that branching in the arabinan did not significantly affect the apparent K(m) values, and the difference in the reaction rates was likely due to the chemical step after substrate binding. The enzyme hydrolyzed arabino-oligosaccharides of DP> or =6 to smaller oligomers and mostly arabinotriose. Natural and modified arabinans were cleaved to oligomers of various chain lengths, which were progressively hydrolyzed to yield arabinotriose. The pattern of degradation revealed an endo-acting mechanism with arabinotriose as the end product.
Wong
Dominic W S
DW
Western Regional Research Center, Agricultural Research Service, US Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA. dwsw@pw.usda.gov
Chan
Victor J
VJ
McCormack
Amanda A
AA
D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D056186:Metagenomics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012417:Rumen / Q000382:microbiology*; D016415:Sequence Alignment; D013379:Substrate Specificity
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C434938:exo-alpha-L-arabinanase