Sung Kyum Kim

Molecular cloning and characterization of a novel family VIII alkaline esterase from a compost metagenomic library

A metagenomic library was constructed from completely fermented compost using a fosmid vector. From a total of 23,400 clones, 19 esterase-positive clones were selected on LB plates containing 1% glyceryl tributyrate as the substrate. The esterase gene of an esterase-positive clone, est2K, was on an ORF of 1299 bp and encoded a protein of 432 amino acids. Est2K had a SMTK motif and was a family VIII esterase. Unlike most family VIII esterases, Est2K had a signal peptide of 27 amino acids. The molecular mass and pI of the mature Est2K was calculated to be 44,668 Da and 4.48, respectively. The amino acid sequence of Est2K showed 72% identity with that of EstC, an esterase of an uncultured bacterium from leachate. The purified Est2K was optimally active at pH 10.0 and 50 degrees C. Est2K was stable in the presence of 30% methanol and exhibited a 2.4-fold higher activity in the presence of 5% methanol than in the presence of 1% isopropanol. Est2K preferred short to medium length p-nitrophenyl esters, especially p-nitrophenyl butyrate, as the substrate. Est2K did not hydrolyze beta-lactam antibiotics ampicillin and nitrocefin, even though Est2K showed the highest similarity to EstC.

The role of carbohydrate-binding module (CBM) repeat of a multimodular xylanase (XynX) from Clostridium thermocellum in cellulose and xylan binding

A non-cellulosomal xylanase from Clostridium thermocellum, XynX, consists of a family-22 carbohydratebinding module (CBM22), a family-10 glycoside hydrolase (GH10) catalytic module, two family-9 carbohydrate-binding modules (CBM9-I and CBM9-II), and an S-layer homology (SLH) module. E. coli BL21(DE3) (pKM29), a transformant carrying xynX′, produced several truncated forms of the enzyme. Among them, three major active species were purified by SDS-PAGE, activity staining, gel-slicing, and diffusion from the gel. The truncated xylanases were different from each other only in their C-terminal regions. In addition to the CBM22 and GH10 catalytic modules, XynX1 had the CBM9-I and most of the CBM9-II, XynX2 had the CBM9-I and about 40% of the CBM9-II, and XynX3 had about 75% of the CBM9-I. The truncated xylanases showed higher binding capacities toward Avicel than those toward insoluble xylan. XynX1 showed a higher affinity toward Avicel (70.5%) than XynX2 (46.0%) and XynX3 (42.1%); however, there were no significant differences in the affinities toward insoluble xylan. It is suggested that the CBM9 repeat, especially CBM9-II, of XynX plays a role in xylan degradation in nature by strengthening cellulose binding rather than by enhancing xylan binding.

Erratum to: Characterization of a GH family 8 β-1,3-1,4-glucanase with distinctive broad substrate specificity from Paenibacillus sp. X4

A β-1,3-1,4 glucanase gene of Paenibacillus sp. X4, bglc8H, was cloned and characterized. BGlc8H was predicted to be a protein of 409 amino acid residues, including a signal peptide of 31 amino acids. The mature enzyme was predicted to have 378 amino acid residues; ITS molecular mass and pI were estimated as 41,561 Da and 7.61, respectively. BGlc8H belongs to glycoside hydrolase family 8 (GH8). Site-directed mutants of Glu95 and Asp156 of BGlc8H showed a near-complete loss of activity, indicating that they are catalytically-active residues. Unlike other GH8 members, BGlc8H had broad substrate specificity and hydrolyzed barley-β-D-glucan > chitosan > carboxymethyl-cellulose > and lichenan. BGlc8H had a lower ratio of lichenase/barley-β-d-glucanase activities compared to GH16 enzymes. BGlc8H was optimally active at pH 5 and 50 °C, except for barley-β-d-glucanase (40 °C) and chitosanase (pH 7) activities. BGlc8H hydrolyzed cello-oligosaccharides (G3–G6) to G3 and G2 but not to G1. Ca2+ increased the activity and thermostability of BGlc8H for lichenan suggesting its use for the saccharification of cellulosic biomass.

Chitinibacter suncheonensis sp. nov., a chitinolytic bacterium from a mud flat in Suncheon Bay

A chitinolytic bacterium, designated strain SK16T, was isolated from a mud flat in Suncheon Bay, Republic of Korea. Strain SK16T is Gram-negative, strictly aerobic, motile by a polar flagellum, and short rod-shaped. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain belonged to the genus Chitinibacter and was most closely related to Chitinibacter tainanensis S1T (98.2% similarity). DNA-DNA hybridization analyses showed a low association value of 20.45±4.08% between them. The major cellular fatty acids, the G+C content of the genomic DNA, and the predominant quinone of the strain were summed feature 3 (iso-C15:0 2-OH and/or C16:1ω7c; 50.5%) and C12:0 (12.5%), 52.26 mol%, and Q-8, respectively. Based on the phylogenetic, chemotaxonomic, and phenotypic properties, strain SK16T represents a novel species of the genus Chitinibacter, for which the name Chitinibacter suncheonensis sp. nov. is proposed. The type strain is SK16T (=KCTC 23839T =DSM 25421T).

Characterization of truncated endo -β-1,4-glucanases from a compost metagenomic library and their saccharification potentials

A gene encoding an endo-β-1,4-glucanase (Cel6H-f481) was cloned from a compost metagenomic library. The gene, cel6H-f481, was composed of 1446 bp to encode a fused protein of 481 amino acid residues (50,429 Da), i.e., 445 residues (Cel6H-445) from the metagenome, and 36 residues from the pUC19 vector at N-terminus. Cel6H-445 belonged to glycosyl hydrolase (GH) family 6 and showed 71% identity with Actinotalea fermentans endoglucanase with low coverage. Several active bands of truncated forms were observed by activity staining of the crude extract. Major truncated enzymes of 35 (Cel6H-p35) and 23 kDa (Cel6H-p23) were separated by HiTrap Q chromatography. The two enzymes had the same optimum temperature (50 °C) and pH (5.5), but Cel6H-p35 was more thermostable than Cel6H-p23 and other GH6 endoglucanases reported. Both enzymes efficiently hydrolyzed carboxymethyl-cellulose (CMC) and barley β-glucan, but hardly hydrolyzed other substrates tested. The Vmax of Cel6H-p35 for CMC was 1.4 times greater than that of Cel6H-p23. The addition of the crude enzymes to a commercial enzyme set increased the saccharification of pretreated rice straw powder by up to 30.9%. These results suggest the N-terminal region of Cel6H-p35 contributes to thermostability and specific activity, and that the enzymes might be a useful additive for saccharification.

Disruption of rsmA gene of Pectobacterium carotovorum subsp. carotovorum LY34 and effect on pathogenicity

The rsmA gene was cloned from soft-rot bacterium Pectobacterium carotovorum subsp. carotovorum LY34 (Pcc LY34), and its role in pathogenicity was investigated by marker exchange mutagenesis. From a cosmid library of Pcc LY34 genomic DNA, a positive clone carrying the rsmA gene was selected, and the gene was cloned by polymerase chain reaction (PCR) amplification. The gene is 186 bp in size and encodes a protein of 62 amino acids with a predicted molecular mass of 6,839 Da. The calculated pI of the RsmA is 8.16. The phylogenetic tree showed that the RsmA of Pcc LY34 appeared genetically identical to the CsrA of Pectobacterium atrosepticum SCRI1043 (100% identity) and similar to the CsrA of Yersinia pestis KIM10+ (98.3%). The gene was disrupted by the Kmr gene, and the cells became mutated (i.e., RsmA− mutant). The pathogenicity test revealed that the disease rating of the RsmA− mutant only differed slightly from that of the wild type on a slice of potato tuber and a Chinese cabbage stalk. These results suggest that RsmA is not an essential factor for the pathogenicity of Pcc LY34 and that the rsmA gene of Pcc LY34 is not completely derepressed in the RsmA− mutant for virulence-related genes, contrary to the results of Erwinia carotovora subsp. carotovora RsmA− mutant, which proved hypervirulent for celery petioles. These results showed that the microenvironmental conditions of the host and/or strain of pathogen are important for the coordination of virulence gene expression.