• Journal of Microbiology and Biotechnology
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• v.25 no.10
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• pp.1660-1669
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• 2015

The present study describes the gene cloning, overexpression and characterization of a novel nitrilase from hyperthermophilic bacterium Thermotoga maritima MSB8. The nitrilase gene consisted of 804 base pairs, encoding a protein of 268 amino acid residues with a molecular mass of 30.07 kDa after SDS-PAGE analysis. The optimal temperature and pH of the purified enzyme were 45℃ and 7.5, respectively. The enzyme demonstrated good temperature tolerance, with 40% residual activity after 60 min of heat treatment at 75℃. The kinetic constants V_max and K_m of this nitrilase toward 3-cyanopyridine were 3.12 μmol/min/mg and 7.63 mM, respectively. Furthermore, this novel nitrilase exhibited a broad spectrum toward the hydrolysis of the aliphatic nitriles among the tested substrates, and particularly was specific to aliphatic dinitriles like succinonitrile, which was distinguished from most nitrilases ever reported. The catalytic efficiency k_cat/K_m was 0.44 /mM/s toward succinonitrile. This distinct characteristic might enable this nitrilase to be a potential candidate for industrial applications for biosynthesis of carboxylic acid.

• Proceedings of the Microbiological Society of Korea Conference
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• 2005.05a
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• pp.185.3-185
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• 2005

• Journal of Microbiology and Biotechnology
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• v.28 no.2
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• pp.255-261
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• 2018

Aglycon protopanaxatriol (APPT) has valuable pharmacological effects such as memory enhancement and tumor inhibition. ${\beta}$-Glycosidase from the hyperthermophilic bacterium Dictyoglomus turgidum (DT-bgl) hydrolyzes the glucose residues linked to APPT, but not other glycoside residues. ${\beta}$-Glycosidase from the hyperthermophilic bacterium Pyrococcus furiosus (PF-bgl) hydrolyzes the outer sugar at C-6 but not the inner glucose at C-6 or the glucose at C-20. Thus, the combined use of DT-bgl and PF-bgl is expected to increase the biotransformation of PPT-type ginsenosides to APPT. We optimized the ratio of PF-bgl to DT-bgl, the concentrations of substrate and enzyme, and the reaction time to increase the biotransformation of ginsenoside Re and PPT-type ginsenosides in Panax ginseng leaf extract to APPT. DT-bgl combined with PF-bgl converted 1.0 mg/ml PPT-type ginsenosides in ginseng leaf extract to 0.58 mg/ml APPT without other ginsenosides, with a molar conversion of 100%. We achieved the complete biotransformation of ginsenoside Re and PPT-type ginsenosides in ginseng leaf extract to APPT by the combined use of two ${\beta}$-glycosidases, suggesting that discarded ginseng leaves can be used as a source of the valuable ginsenoside APPT. To the best of our knowledge, this is the first quantitative production of APPT using ginsenoside Re, and we report the highest concentration and productivity of APPT from ginseng extract to date.

• Journal of Microbiology and Biotechnology
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• v.33 no.10
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• pp.1384-1389
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• 2023

This work aimed to evaluate the feasibility of biohydrogen production from Barley Straw and Miscanthus. The primary obstacle in plant biomass decomposition is the recalcitrance of the biomass itself. Plant cell walls consist of cellulose, hemicellulose, and lignin, which make the plant robust to decomposition. However, the hyperthermophilic bacterium, Caldicellulosiruptor bescii, can efficiently utilize lignocellulosic feedstocks (Barley Straw and Miscanthus) for energy production, and C. bescii can now be metabolically engineered or isolated to produce more hydrogen and other biochemicals. In the present study, two strains, C. bescii JWCB001 (wild-type) and JWCB018 (ΔpyrFA Δldh ΔcbeI), were tested for their ability to increase hydrogen production from Barley Straw and Miscanthus. The JWCB018 resulted in a redirection of carbon and electron (carried by NADH) flow from lactate production to acetate and hydrogen production. JWCB018 produced ~54% and 63% more acetate and hydrogen from Barley Straw, respectively than its wild-type counterpart, JWCB001. Also, 25% more hydrogen from Miscanthus was obtained by the JWCB018 strain with 33% more acetate relative to JWCB001. It was supported that the engineered C. bescii, such as the JWCB018, can be a parental strain to get more hydrogen and other biochemicals from various biomass.

• BMB Reports
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• v.33 no.1
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• pp.82-88
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• 2000

A homologous gene to alanine racemase was cloned from a hyperthermophilic bacterium, Aquifex pyrophilus. The cloned gene encodes a protein of 341 amino acids, which has a significant homology to alanine racemase of Bacillus stearothermophilus, Lactobacillus brevis, and E. coli. When the gene was expressed in Escherichia coli, it produced a 40 kDa protein. The purified protein contains one mole pyridoxal 5-phosphate per one mole of protein, which is essential for catalytic activity of alanine racemase. The purified protein catalyzed racemization of L-alanine to D-alanine, or vice versa, indicating that the cloned gene encoded alanine racemase. It also showed significant racemization activity against L-serine and ${\alpha}-aminobutylic$ acid. The A. pyrophilus alanine racemase showed strong thermostability, and it maintained catalytic activity in the presence of organic solvents.

• Proceedings of the Korean Biophysical Society Conference
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• 1996.07a
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• pp.30-30
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• 1996

A suproxide dismutase gene of Aquifex pyroprolus, a novel marine hypenhermophilic bacterium, was cloned, expressed, and characterized. The SOD of A pyrophilus (ApSOD) is an iron-containing homo-oligomeric protein with a monomeric molecular weight of 24.2 kDa. the amino acid sequence is similar to those of known Mn- and Fe-SODs from thermophilic archaea, and metal binding residues in all SOD sequences from different species are also conserved in A. pyrophilus SOD. (omitted)

• Journal of Microbiology and Biotechnology
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• v.31 no.4
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• pp.570-583
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• 2021

Pyrococcus furiosus α-amylase can hydrolyze α-1,4 linkages in starch and related carbohydrates under hyperthermophilic condition (~ 100℃), showing great potential in a wide range of industrial applications, while its relatively low productivity from heterologous hosts has limited the industrial applications. Bacillus subtilis, a gram-positive bacterium, has been widely used in industrial production for its non-pathogenic and powerful secretory characteristics. This study was conducted to increase production of P. furiosus α-amylase in B. subtilis through three strategies. Initial experiments showed that co-expression of P. furiosus molecular chaperone peptidyl-prolyl cis-trans isomerase through genomic integration mode, using a CRISPR/Cas9 system, increased soluble amylase production. Therefore, considering that native P. furiosus α-amylase is produced within a hyperthermophilic environment and is highly thermostable, heat treatment of intact culture at 90℃ for 15 min was performed, thereby greatly increasing soluble amylase production. After optimization of the culture conditions (nitrogen source, carbon source, metal ion, temperature and pH), experiments in a 3-L fermenter yielded a soluble activity of 3,806.7 U/ml, which was 3.3- and 28.2-fold those of a control without heat treatment (1,155.1 U/ml) and an empty expression vector control (135.1 U/ml), respectively. This represents the highest P. furiosus α-amylase production reported to date and should promote innovation in the starch liquefaction process and related industrial productions. Meanwhile, heat treatment, which may promote folding of aggregated P. furiosus α-amylase into a soluble, active form through the transfer of kinetic energy, may be of general benefit when producing proteins from thermophilic archaea.

• Journal of Microbiology and Biotechnology
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• v.14 no.5
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• pp.1022-1030
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• 2004

The gene encoding Aquifex pyrophilus (Apy) DNA polymerase was cloned and sequenced. The Apy DNA polymerase gene consists of 1,725 bp coding for a protein with 574 amino acid residues. The deduced amino acid sequence of Apy DNA. polymerase showed a high sequence homology to Escherichia coli DNA polymerase I-like DNA polymerases. It was deduced by amino acid sequence alignment that Apy DNA polymerase, like the Klenow fragment, has only the two domains, the $3'{\rightarrow}5'$ exonuclease domain and the $5'{\rightarrow}3'$ polymerase domain, containing the characteristic motifs. The Apy DNA polymerase gene was expressed under the control of T7lac promoter on the expression vector pET-22b(+) in E. coli. The expressed enzyme was purified by heat treatment, and Cibacron blue 3GA and $UNO^{TM}$ Q column chromatographies. The optimum pH of the purified enzyme was 7.5, and the optimal concentrations of KCl and $Mg^{2+}$ were 20 mM and 3 mM, respectively. Apy DNA polymerase contained a double strand-dependent $3'{\rightarrow}5'$ proofreading exonuclease activity, but lacked any detectable $5'{\rightarrow}3'$ exonuclease activity, which is consistent with its amino acid sequence. The somewhat lower thermostability of Apy DNA polymerase than the growth temperature of A. pyrophilus was analyzed by the comparison of amino acid composition and pressure effect.

• Microbiology and Biotechnology Letters
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• v.50 no.2
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• pp.218-227
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• 2022

Glycosylation of aesculetin was performed using amylosucrase from the hyperthermophilic bacterium Deinococcus geothermalis DSM 11300 to improve the solubility and biological activity of aesculetin. A newly synthesized aesculetin glycoside was identified as α-cichoriin (aesculetin 7-α-D-glucoside) by nuclear magnetic resonance analysis. The solubility of α-cichoriin was 11 times higher than that of aesculetin because of the attached glucose moiety. Aesculetin and α-cichoriin had no significant effect on the proliferation of normal cells, such as RAW 264.7, but they showed a cell proliferation inhibitory effect on B16F10 melanoma cells. Unlike treatment with aesculetin and α-cichoriin, aesculin (aesculetin 6-β-D-glucoside) showed no antiproliferative activity in B16F10 cells. Based on the molecular structures of aesculin and α-cichoriin, the position where glucose binds to aesculetin and the anomeric configuration between glucose and aesculetin are thought to be important for exerting an antiproliferative effect on the B16F10 cell line. Based on these results, we propose that α-cichoriin, the α-glycosylated form of aesculetin, may serve as a model for developing phytochemical analogs with therapeutic potential for the treatment of diseases associated with tumor cell proliferation without cytotoxicity to normal cells.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1471-1480
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• 2010

An extremely thermostable xylanase gene, xynB, from the hyperthermophilic bacterium Thermotoga maritima MSB8 was successful expressed in Kluyveromyces lactis. The response surface methodology (RSM) was also applied to optimize the medium components for the production of XynB secreted by the recombinant K. lactis. The secretion level (102 mg/l) and enzyme activity (49 U/ml) of XynB in the optimized medium (yeast extract, lactose, and urea; YLU) were much higher than those (56 mg/l, 16 U/ml) in the original medium (yeast extract, lactose, and peptone; YLP). The secretory efficiency of mature XynB was also improved when using the YLU medium. When the mRNA levels of 13 characterized secretion-related genes in the K. lactis cultured in YLP and YLU were detected using a semiquantitative RT-PCR method, the unfolded protein response (UPR)-related genes, including ero1, hac1, and kar2, were found to be up-regulated in the K. lactis cultured in YLU. Therefore, the nutrient ingredients, especially the nitrogen source, were shown to have a significant influence on the XynB secretory efficiency of the host K. lactis.