Hong-Ki Jun

Characterization of a gene encoding cellulase from uncultured soil bacteria.

To detect cellulases encoded by uncultured microorganisms, we constructed metagenomic libraries from Korean soil DNAs. Screenings of the libraries revealed a clone pCM2 that uses carboxymethyl cellulose (CMC) as a sole carbon source. Further analysis of the insert showed two consecutive ORFs (celM2 and xynM2) encoding proteins of 226 and 662 amino acids, respectively. A multiple sequence analysis with the deduced amino acid sequences of celM2 showed 36% sequence identity with cellulase from the Synechococcus sp., while xynM2 had 59% identity to endo-1,4-beta-xylanase A from Cellulomonas pachnodae. The highest enzymatic CMC hydrolysis was observable at pH 4.0 and 45 degrees C with recombinant CelM2 protein. Although the enzyme CelM2 additionally hydrolyzed avicel and xylan, no substrate hydrolysis was observed on oligosaccharides such as cellobiose, pNP-beta-cellobioside, pNP-beta-glucoside, and pNP-beta-xyloside. These results showed that CelM2 is a novel endo-type cellulase.

Isolation and sequence analysis of metalloprotease gene from Vibrio mimicus.

The vmc gene encoding a metalloprotease of Vibrio mimicus (ATCC 33653) was cloned in Escherichia coli and sequenced. The vmc gene contained 1884 nt sequence which codes a polypeptide of 628 amino acids with a predicted molecular mass of 71,275 Da. The deduced amino acid sequence had the similarity of 68.5% with V. parahaemolyticus metalloprotease. The consensus sequence of a zinc binding motif (HEXXH) was identified to be HEYTH. The zymography analysis showed a gelatinolytic protein band around molecular mass of 61 kDa, and this result suggested that the cloned metalloprotease may undergo processing during secretion.

Production of 2-O-α-D-glucopyranosyl L-ascorbic acid using cyclodextrin glucanotransferase from Paenibacillus sp.

Transglycosylation to produce a 2-O-α-d-glucopyranosyl l-ascorbic acid (AA-2G) was studied using cyclodextrin glucanotransferase (CGTase) from Paenibacillus sp. A series of maltooligosaccharides substituted 2-O-derivatives of l-ascorbic acid (AA) were analyzed by HPLC. The maltooligosaccharides were hydrolyzed by glucoamylase to give AA-2G. CGTase also produced AA-2G using dextrin as a glycosyl donor and AA as an acceptor. CGTase utilized α-, β-, and γ-CDs, amylose, soluble starch and corn starch as glycosyl donors but not glucose.

Crystal structure of engineered β‐glucosidase from a soil metagenome

Crystal structure of engineered b-glucosidase from a soil metagenome Ki Hyun Nam,1y Soo-Jin Kim,2y Min-Young Kim, Jae Hee Kim, Yun-Soo Yeo, Chang-Muk Lee, Hong-Ki Jun, and Kwang Yeon Hwang* 1Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, Korea 2Microbial Genetics Division, National Institute of Agricultural Biotechnology, Rural Development Administration, Suwon, Korea 3Department of Microbiology, Pusan National University, Pusan, Korea

Purification and characterization of extracellular adenosine deaminase from a Streptomyces sp.

Abstract Extracellular adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) was purified from the culture filtrate of Streptomyces sp. J-845S by ammonium sulfate precipitation and column chromatography on DEAE-cellulose, DEAE-Sephadex A-50, Sephadex G-100, and CM-cellulose. The purified enzyme preparation was homogeneous by the criterion of polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 90,000 by gel filtration with Sephadex G-100 and about 45,000 by SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme consisted of two subunits, and its isoelectric point was 7.8. The enzyme was stable at pH 3.5–5.5, the optimum pH for the reation was 5.0–6.0, and the optimum temperature was 55°C in the optimum pH range. Adenosine, 2′-deoxyadenosine, 2′,3′-isopropylidene adenosine, AMP, adenosine-3′-monophosphate, and cAMP were substrates of the enzyme, and the Km values for these compounds were 0.67, 0.22, 0.29, 0.17, 2.0, and 0.5 mM, respectively. The enzyme reaction was promoted by Fe3+, and inhibited by Hg2+, Ag+, and o-phenanthroline.

Purification and crystallization of adenosine deaminase in Pseudomonas iodinum IFO 3558

The purification and properties of adenosine deaminase (adenosine aminohydrolase: EC 3 5.4.4) have been described from calf intestinal mucosa [ 11, takadiastase and various animal sources [2-91. However, there are few studies of microbial intracellular adenosine deaminase. Recently, the attempt was made to purify the enzymes from the cytoplasm of Halobacterium cutirubrum and from the cytoplasm and the membrane of Micrococcus sodonensis, but these enzymes were unstable and could be purified only partially [lO,ll]. We describe here a purification and crystallization method for adenosine deaminase from Pseudomonas iodinum IF0 3 558. We found that although adenosine deaminase in Ps. iodinum cells was quite unstable, it could be stabilized by ethyl alcohol. The enzyme was purified by ammonium sulfate and ethyl alcohol precipitation and column chromatography on DEAEcellulose, Sephadex G-200, DEAE-Sephadex A-50, Sephacryl S-200 superfine and hydroxyapatite, in the presence of 15% ethyl alcohol. By these procedures, adenosine deaminase was purified 1800-2000-fold with a recovery of 25-30% activity originally present in the cell-free extract.

Purification and Properties of D-Xylose Isomerase from Lactococcus sp. JK-8

D-Xylose isomerase produced by Lactococcus sp. JK-8, isolated from kimchi, was purified 17-fold of homogeneity, and its physicochemical properties were determined. Although the N-terminal amino acid sequence of D-xylose isomerase was analysed to Ala-Tyr-Phe-Asn-Asp-Ile-Ala-Pro-Ile-Lys, it was not similar to that of Lactobacillus enzyme. The molecular weight of the purified enzyme was estimated to be 180 kDa by gel filtration, 45 kDa by SDS-PAGE and the enzyme was homotetramer. The optimum pH of the enzyme was around 7 and stable between pH 6 and 8. The optimum reaction temperature was 7 and stable up to 7 in the presence of 1 mM . Like other D-xylose isomerases, this enzyme required divalent cation, such as , , or for the activity and thermostability. was the best activator. Substrate specificity studies showed that this enzyme was highly active on D-xylose. The enzyme had an isoelectric point of 4.8, and fm values for D-xylose was 5.9 mM.