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Patel S.K.S.,Institute of SK KU Biomaterials | Mardina P.,Konkuk University | Kim S.-Y.,BioNgene Co. | Lee J.-K.,Konkuk University | Kim I.-W.,Institute of SK KU Biomaterials
Journal of Microbiology and Biotechnology | Year: 2016

Methane (CH4) is the most abundant component in natural gas. To reduce its harmful environmental effect as a greenhouse gas, CH4 can be utilized as a low-cost feed for the synthesis of methanol by methanotrophs. In this study, several methanotrophs were examined for their ability to produce methanol from CH4; including Methylocella silvestris, Methylocystis bryophila, Methyloferula stellata, and Methylomonas methanica. Among these methanotrophs, M. bryophila exhibited the highest methanol production. The optimum process parameters aided in significant enhancement of methanol production up to 4.63 mM. Maximum methanol production was observed at pH 6.8, 30°C, 175 rpm, 100 mM phosphate buffer, 50 mM MgCl2 as a methanol dehydrogenase inhibitor, 50% CH4 concentration, 24 h of incubation, and 9 mg of dry cell mass ml-1 inoculum load, respectively. Optimization of the process parameters, screening of methanol dehydrogenase inhibitors, and supplementation with formate resulted in significant improvements in methanol production using M. bryophila. This report suggests, for the first time, the potential of using M. bryophila for industrial methanol production from CH4. © 2016 by The Korean Society for Microbiology and Biotechnology. Source


Sigdel S.,Konkuk University | Singh R.,Konkuk University | Kim T.-S.,Konkuk University | Li J.,Konkuk University | And 6 more authors.
PLoS ONE | Year: 2015

The BaM6PI gene encoding a mannose-6-phosphate isomerase (M6PI, EC 5.3.1.8) was cloned from Bacillus amyloliquefaciens DSM7 and overexpressed in Escherichia coli. The enzyme activity of BaM6PI was optimal at pH and temperature of 7.5 and 70°C, respectively, with a kcat/Km of 13,900 s-1 mM-1 for mannose-6-phosphate (M6P). The purified BaM6PI demonstrated the highest catalytic efficiency of all characterized M6PIs. Although M6PIs have been characterized from several other sources, BaM6PI is distinguished from other M6PIs by its wide pH range and high catalytic efficiency for M6P. The binding orientation of the substrate M6P in the active site of BaM6PI shed light on the molecular basis of its unusually high activity. BaM6PI showed 97% substrate conversion from M6P to fructose-6- phosphate demonstrating the potential for using BaM6PI in industrial applications. Copyright: © 2015 Baikara et al. Source


Singh R.,Konkuk University | Singh R.K.,Konkuk University | Kim S.-Y.,BioNgene Co. | Sigdel S.,Konkuk University | And 4 more authors.
Biochemical Engineering Journal | Year: 2016

D-Ribulose, a potentially valuable rare sugar and an excellent building block in asymmetric synthesis, is usually produced by chemical synthesis, which unfortunately does not meet the increasing demand. In addition, ribitol oxidization for the production of d-ribulose has not yet become an industrial reality because of the lack of efficient biocatalysts, resulting in higher production costs as well as a poor yield. In this study, we have employed the Enterobacter aerogenes ribitol: NAD+ 2-oxidoreductase (EaRDH), which could efficiently and selectively convert ribitol to D-ribulose. The purified EaRDH enzyme and the recombinant Escherichia coli strain (as a whole-cell catalyst) were used to produce d-ribulose. Ribitol was efficiently converted to d-ribulose by EaRDH with a yield of ~85%, suggesting the usefulness of this enzyme for the in vivo and in vitro production of d-ribulose from ribitol. The oxidation of ribitol to D-ribulose by EaRDH was accomplished in the presence of stoichiometric amounts of NAD+; therefore, D-ribulose production was further enhanced by the incorporation of diaphorase for continuous NAD+ regeneration. The biocatalytic process presented should be a promising route for the biotechnological production of d-ribulose (and related branched pentoses) at an efficient and industrially relevant scale. © 2016 Elsevier B.V. Source


Kim J.-H.,BioNgene Co. | Prabhu P.,Konkuk University | Jeya M.,Konkuk University | Tiwari M.K.,Konkuk University | And 3 more authors.
Applied Microbiology and Biotechnology | Year: 2010

An isolated gene from Bacillus subtilis str. 168 encoding a putative isomerase was proposed as an L-arabinose isomerase (L-AI), cloned into Escherichia coli, and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1,491 bp, capable of encoding a polypeptide of 496 amino acid residues. The gene was overexpressed in E. coli and the protein was purified using nickel-nitrilotriacetic acid chromatography. The purified enzyme showed the highest catalytic efficiency ever reported, with a k cat of 14,504 min-1 and a k cat/K m of 121 min-1mM-1 for L-arabinose. A homology model of B. subtilis L-AI was constructed based on the X-ray crystal structure of E. coli L-AI. Molecular dynamics simulation studies of the enzyme with the natural substrate, L-arabinose, and an analogue, D-galactose, shed light on the unique substrate specificity displayed by B. subtilis L-AI only towards L-arabinose. Although L-AIs have been characterized from several other sources, B. subtilis L-AI is distinguished from other L-AIs by its high substrate specificity and catalytic efficiency for L-arabinose. © 2009 Springer-Verlag. Source


Kim T.-S.,Konkuk University | Yoo J.-H.,BioNgene Co. | Kim S.-Y.,BioNgene Co. | Pan C.-H.,Konkuk University | And 3 more authors.
Process Biochemistry | Year: 2015

A mutant of Agrobacterium tumefaciens (S02-13) producing high levels of coenzyme Q10 (CoQ10), was selected by high-throughput screening after repeated NTG mutagenesis. Mutant S02-13 was resistant to sodium azide and menadione and showed an eight-fold increase in CoQ10 production, as compared to that of the parent strain. The amount of CoQ10 produced by this mutant reached 350 mg l-1 in pH-stat fed-batch culture. Mutant S02-13 differed from wild-type in morphology, biochemical properties, and proteome profiles. Scanning electron microscopy results revealed a remarkable change in morphology: the wild-type strain had a rod shape, whereas the mutant S02-13 was coccoid. In contrast to wild-type, the mutant S02-13 strain showed a negative reaction to tryptophan deaminase and N2 reduction, and a positive reaction to nitrate production in API kit assays. The spots representing proteins that were increased in the proteome expression of the mutant S02-13 strain were identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glucosaminitol dehydrogenase (GlcNOH), and superoxide dismutase (SOD). In particular, the enzyme activities of GAPDH and GlcNOH increased eight-fold and three-fold, respectively, in the mutant, as compared to the wild-type. © 2014 Elsevier Ltd. All rights reserved. Source

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