Molecular cloning and nucleotide sequence of the gene encoding an endo-1,4- -glucanase from Bacillus sp. KSM-330

Abstract

The gene encoding an acid endo-1,4-P-glucanase from Bacillus sp. KSM-330 was cloned into the Hind111 site of pBR322 and expressed in Escherichia cofi HB101. The recombinant plasmid contained a 3.1 kb Hind111 insert, 1-8 kb of which was sufficient for the expression of endoglucanase activity in E cofi HB101. Nucleotide sequencing of this region (1816 bp) revealed an open reading frame of 1389 bp. The protein deduced from this sequence was composed of 463 amino acids with an M, of 51 882. The deduced amino acid sequence from amino acids 56 through 75 coincided with the amino-terminal sequence of the endoglucanase, Endo-K, purified from culture of Baciffus sp. KSM-330. The deduced amino acid sequence of Endo-K had 30% homology with that of the cefA enzyme from Cfostridum thermoceffum NCIB 10682 and 25 % homology with that of the enzyme from Ceffufomonas uda CB4.However, the Endo-K protein exhibited no homology with respect to either the nucleotide or the amino acid sequences of other endoglucanases from Buciffus that had been previously characterized. These results indicate that the gene for Endo-K in Bacillus sp. KSM-330 has evolved from an ancestral gene distinct from that of other Bacillus endoglucanases.

Purification and Characterization of Chitosanase from Bacillus sp. Strain KCTC 0377BP and Its Application for the Production of Chitosan Oligosaccharides

“…This sequence was 90% identical to the N-terminal sequence of an acidic endocellulase of Bacillus sp. strain KSM-330 (30). However, our chitosanase sequence had no similarity to the N-terminal sequences of other known chitosanases belonging to glycoside hydrolase family 46.…”

Atomic (0.94 Å) resolution structure of an inverting glycosidase in complex with substrate

“…Interestingly, the tyrosine residue is strictly conserved in all family 8 endoglucanases, but the aspartic acid residue is missing from two homologous enzymes from Bacillus, where it is replaced by an asparagine residue. 27,28 It follows that, at least for some family 8 glycosidases, the enhancement of water nucleophilicity by a carboxylate group might not be required for the reaction to occur. Alternatively, the conserved tyrosine residue could be acting as a base catalyst in these enzymes, although this would require a substantial modi®cation of the tyrosine pK a to account for the enzyme activity at slightly acidic pH values.…”

Application of the Upstream Region of aBacillusEndoglucanase Gene to High-level Expression of Foreign Genes inBacillus subtilis

“…Acid EG, a 1.8-kb HindIII-HpaI fragment that encoded the acid EG from Bacillus sp. KSM-330 was isolated from pKC3301 15 ) and was treated with T4 DNA polymerase to generate blunt ends. The fragment was then cloned into the SmaI sites of pHSP64 and pHY300PLK to give recombinant plasmids pHSP-KC331A and pHKC331, respectively.…”