• Journal of Microbiology and Biotechnology
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• v.28 no.8
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• pp.1339-1345
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• 2018

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, which catalyzes aldol cleavage and condensation reactions, has two distinct substrate-binding sites. The substrate-binding mode at the catalytic site and Schiff-base formation have been well studied. However, structural information on the phosphate-binding loop (P-loop) is limited. Zymomonas mobilis KDPG aldolase is one of the aldolases with a wide substrate spectrum. Its structure in complex with the substrate-mimicking 3-phosphoglycerate (3PG) shows that the phosphate moiety of 3PG interacts with the P-loop and a nearby conserved serine residue. 3PG-binding to the P-loop replaces water molecules aligned from the P-loop to the catalytic site, as observed in the apostructure. The extra electron density near the P-loop and comparison with other aldolases suggest the diversity and flexibility of the serine-containing loop among KDPG aldolases. These structural data may help to understand the substrate-binding mode and the broad substrate specificity of the Zymomonas KDPG aldolase.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.663-669
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• 2008

The solvent-tolerant bacterium Enterobacter sp. VKGH12 is capable of utilizing n-butanol and contains an $NAD^+$-dependent n-butanol dehydrogenase (BDH). The BDH from n-butanol-grown Enterobacter sp. was purified from a cell-free extract (soluble fraction) to near homogeneity using a 3-step procedure. The BDH was purified 15.37-fold with a recovery of only 10.51, and the molecular mass estimated to be 38 kDa. The apparent Michaelis-Menten constant ($K_m$) for the BDH was found to be 4 mM with respect to n-butanol. The BDH also had a broad range of substrate specificity, including primary alcohols, secondary alcohols, and aromatic alcohols, and exhibited an optimal activity at pH 9.0 and $40^{\circ}C$. Among the metal ions studied, $Mg^{2+}$ and $Mn^{2+}$ had no effect, whereas $Cu^{2+},\;Zn^{2+}$, and $Fe^{2+}$ at 1 mM completely inhibited the BDH activity. The BDH activity was not inhibited by PMSF, suggesting that serine is not involved in the catalytic site. The known metal ion chelator EDTA had no effect on the BDH activity. Thus, in addition to its physiological significance, some features of the enzyme, such as its activity at an alkaline pH and broad range of substrate specificity, including primary and secondary alcohols, are attractive for application to the enzymatic conversion of alcohols.

• Journal of Microbiology and Biotechnology
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• v.16 no.5
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• pp.778-785
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• 2006

Catechol 1,2-dioxygenase was purified from cells of R. rhodochrous N75 grown at the expense of benzoate and p-toluate as the sole sources of carbon. A single catechol 1,2-dioxygenase was found to be induced with either growth substrate. The enzyme has an estimated $M_r$ of 71,000 consisting of two identical subunits. Catechol 1,2-dioxygenase from R. rhodochrous N75 exhibits some unusual properties including: broad substrate specificity, extradiol cleavage activity with 4-methylcatechol and low $K_m$ values for halocatechols, suggesting that this enzyme is distinct from other known catechol and chlorocatechol 1,2-dioxygenases.

• Journal of Microbiology and Biotechnology
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• v.27 no.11
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• pp.1907-1915
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• 2017

Lipases are important enzymes with biotechnological applications in dairy, detergent, food, fine chemicals, and pharmaceutical industries. Specifically, hormone-sensitive lipase (HSL) is an intracellular lipase that can be stimulated by several hormones, such as catecholamine, glucagon, and adrenocorticotropic hormone. Bacterial hormone-sensitive lipases (bHSLs), which are homologous to the C-terminal domain of HSL, have ${\alpha}/{\beta}-hydrolase$ fold with a catalytic triad composed of His, Asp, and Ser. These bHSLs could be used for a wide variety of industrial applications because of their high activity, broad substrate specificity, and remarkable stability. In this review, the relationships among HSLs, the microbiological origins, the crystal structures, and the biotechnological properties of bHSLs are summarized.

• Journal of Microbiology and Biotechnology
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• v.17 no.12
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• pp.1991-1995
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• 2007

O-Methylation is a common substitution reaction found in microbes as well as in mammalians. Some of the O-methyltransferases (OMTs) have broad substrate specificity and could be used to methylate various compounds. DnrK from Streptomyces peucetius encodes an anthracycline 4-O-methyltransferase, which uses carminomycin as a substrate, and its crystal structure has been determined. Molecular docking experiments with DnrK using various flavonoids were successfully conducted, and some of the flavonoids such as apigenin and genistein were predicted to serve as substrates. Based on these results, O-methylations of various flavonoids with the DnrK were successfully carried out. The methylation position was determined to be at the hydroxyl group of C7. Important amino acid residues for the enzymatic reaction of DnrK with apigenin could be identified using site-directed mutagenesis. Molecular docking could be useful to predict the substrate specificity ranges of other OMTs.

• Journal of Microbiology and Biotechnology
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• v.22 no.8
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• pp.1118-1126
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• 2012

Four putative GH12 genes were found in the Fusarium graminearum genome. The corresponding proteins were expressed in Escherichia coli, purified, and evaluated. FGSG_05851 and FGSG_11037 displayed high activities towards xyloglucan ($V_{max}$ of 4 and $11{\mu}mol/min$, respectively), whereas FGSG_07892 and FGSG_16349 were much less active with this substrate (0.081 and $0.004{\mu}mol/min$, respectively). However, all four of these enzymes had a similar binding affinity for xyloglucan. Xyloglucan was the substrate preferred by FGSG_05851, in contrast to the three other enzymes, which preferred ${\beta}$-glucan or lichenan. Therefore, FGSG_05851 is a xyloglucan-specific glucanase (E.C. 3.2.1.151) rather than an endoglucanase (E.C. 3.2.1.4) with broad substrate specificity. FGSG_11037 displayed a peculiar behavior in that the xyloglucan binding was highly cooperative, with a Hill coefficient of 2.5. Finally, FGSG_05851 essentially degraded xyloglucan into hepta-, octa-, and nonasaccharides, whereas the three other enzymes yielded hepta- and octa-saccharides as well as larger molecules.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.5
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• pp.710-719
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• 2001

Several amino acid transport systems in mammary gland have been characterized during the last few years. These systems may be divided into two broad categories based on whether they are sodium-dependent or $Na^{+}$-independent, and each of these categories is subdivided into 3 groups depending on whether the systems prefer zwitterionic, cationic or anionic substrates. The zwitterion preferring transport processes in mammary gland are $Na^{+}$-dependent system A and $Na^{+}$-independent systems L and T. System $y^{+}$ is a $Na^{+}$-independent transporter of cationic amino acids and $X_{AG^{-}}$ is a $Na^{+}$-dependent system for anionic amino acids. A ($Na^{+}+Cl^{-}$)-dependent system, selective for $\beta$-amino acids has been reported in rat mammary tissue. In addition, there is yet another class of transporters that have still broader specificity. The $Na^{+}$-dependent systems $BCl^{-}$-dependent and $BCl^{-}$-independent and $Na^{+}$-independent system $y^{+}L$ have been reported to mediate the transport of zwitterionic as well as cationic amino acids. Each system has been characterized with respect to its substrate specificity, affinity, kinetics and ion-dependence. Transport of amino acids by mammary tissue is regulated by i) the intracellular substrate concentration, ii) lactogenic hormones and iii) milk stasis. Four of the above transport systems (i.e. A, L, $y^{+}$ and $BCl^{-}$-independent) are up-regulated by lactogenic hormones (insulin, cortisol and prolactin) in mammary gland.

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.804-810
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• 1999

There is a possibility that carvone, a monoterpene from spearmint (Mentha spicata), could induce the bph degradative pathway and genes in Alcaligenes eutrophus H850, which is a known Gram-negative PCB degrader with a broad substrate specificity that was thoroughly investigated with Arthrobacter sp. BIB, a Gram-positive PCB degrader. The strains BIB and H850 were unable to utilize and grow on the plant terpene [(R)-(-)-carvone] (50ppm) to be recognized as a sole carbon source. Nevertheless, the carvone did induce 2,3-dihydroxybiphenyl 1,2-dioxygenase (encoded by bphC) in the strain B lB, as observed by a resting cell assay that monitors accumulation of a yellow meta ring fission product from 4,4'-dichlorobiphenyl (DCBp). The monoterpene, however, did not appear to induce the meta cleavage pathway in the strain H850. Instead, an assumption was made that the strain might be using an alternative pathway, probably the ortho-cleavage pathway. A reverse transcription (RT)-PCR system, utilizing primers designed from a conserved region of the bphC gene of Arthrobacter sp. M5, was employed to verify the occurrence of the alternative pathway. A successful amplification (182bp) of mRNA transcribed from the N-terminal region of the bphC gene was accomplished in H850 cells induced by carvone (50ppm) as well as in biphenyl-growth cells. It is, therefore, likely that H850 possesses a specific PCB degradation pathway and hence a different substrate specificity compared with B1B. This study will contribute to an elucidation of the dynamic aspects of PCB bioremediation in terms of roles played by PCB degraders and plant terpenes as natural inducer substrates that are ubiquitous and environmentally compatible.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.933-941
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• 2008

An extracellular $\beta$-glucosidase produced by Monascus purpureus NRRL1992 in submerged cultivation was purified by acetone precipitation, gel filtration, and hydrophobic interaction chromatography, resulting in a purification factor of 92-fold. A $2^2$ central-composite design (CCD) was performed to find the best temperature and pH conditions for enzyme activity. Maximum activity was observed in a wide range of temperature and pH values, with optimal conditions set at $50^{\circ}C$ and pH 5.5. The $\beta$-glucosidase showed moderate thermostability, was inhibited by $HgCl_2$, $K_2Cr_O_4$, and $K_2Cr_2O_7$, whereas other reagents including $\beta$-mercaptoethanol, SDS, and EDTA showed no effect. Activity was slightly stimulated by low concentrations of ethanol and methanol. Hydrolysis of p-nitrophenyl-$\beta$-D-glucopyranoside (pNPG), cellobiose, salicin, n-octyl-$\beta$-D-glucopyranoside, and maltose indicates that the $\beta$-glucosidase has broad substrate specificity. Apparently, glucosyl residues were removed from the nonreducing end of p-nitrophenyl-$\beta$-D-cellobiose. $\beta$-Glucosidase affinity and hydrolytic efficiency were higher for pNPG, followed by maltose and cellobiose. Glucose and cellobiose competitively inhibited pNPG hydrolysis.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.107-110
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• 2001

Among the tested ten enzymes, Optimase M-440 showed the highest activity in transesterification of N-t-Boc-L-Phe-OTFE with D-glucose. Monosaccharides and their derivatives acted as good acyl acceptors in the Optimase M -440 catalyzed transesterification of N-t-Boc-L-Phe-OTFE. Optimase M-440 showed a preferable catalytic activity on the primary hydroxyl group of saccharides and a good regioselectivity. Optimase M-440 showed the highest activity in pyricline among the tested solvents. As acyl donors, trifluoroethyl esters of amino acids showed a high reactivity in transesterification. Optimase M-440 showed a broad substrate specificity towards amin 。 acid esters and saccharides.