CHARACTERIZATION OF ENDOGLUCANASE AND GENOME ANALYSIS OF Lysobacter enzymogenes ISOLATED FROM TROPICAL SOIL

Abstract

A tropical Lysobacter sp. with lytic enzyme activities was isolated in Thailand, using chitin enrichment procedure. Among nine isolates capable of carboxymethylcellulose degradation, only the strain 521 was identified as Lysobacter enzymogenes based on morphological and physiological traits, together with 16S rRNA sequence comparison and phylogenetic analysis. Variation of phenotypic characters, growth and endoglucanase production of L. enzymogenes 521 with respect to its counterpart from the temperate, L. enzymogenes C3, were observed. Endoglucanase produced by L. enzymogenes 521 under the optimal condition was purified in order to study its biochemical properties. The purified endoglucanase, with a molecular mass of 41 kDa, exhibited bifunctional hydrolytic activities of carboxymethylcellulase (CMCase) and chitosanase as revealed by zymogram analysis. The optimal pHs and temperatures for CMCase and chitosanase activity were 5.0, 40ºC and 5.0, 50ºC, respectively. The purified enzyme was stable at 40°C, and between pH 4 and 11. Alpha-cellulose and colloidal chitosan were found to be its most specific substrates with about 49 and 45% relative activity, respectively, with respect to that of CMC (100%). The 5.8-Mbp genome of L. enzymogenes 521 consisted of 5,008 protein coding genes and 94 RNA genes. Comparative genomic analysis indicated that L. enzymogenes 521 genome exhibited highly conserved orthologs to bacteria belonging to Xanthomonadaceae. From analysis of 33 genes encoding proteins in the glycoside hydrolase class, seven genes encoded proteins involving in cellulolytic degradation. Phylogenetic analysis of the endoglucanase gene and multiple alignment of its deduced amino acid sequence suggested that the endoglucanase Y (Cel8A) from L. enzymogenes 521 belong to the Family 8 glycoside hydrolases (GHF-8), with high similarity to the bifunctional endoglucanase (Cel8A) from Lysobacter sp. IB-9374 (98.3% amino acid sequence similarity).