• Title/Summary/Keyword: Substrate specificity

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Identification and Characterization of a Novel Thermostable GDSL-Type Lipase from Geobacillus thermocatenulatus

  • Jo, Eunhye;Kim, Jihye;Lee, Areum;Moon, Keumok;Cha, Jaeho
    • Journal of Microbiology and Biotechnology
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    • v.31 no.3
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    • pp.483-491
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    • 2021
  • Two putative genes, lip29 and est29, encoding lipolytic enzymes from the thermophilic bacterium Geobacillus thermocatenulatus KCTC 3921 were cloned and overexpressed in Escherichia coli. The recombinant Lip29 and Est29 were purified 67.3-fold to homogeneity with specific activity of 2.27 U/mg and recovery of 5.8% and 14.4-fold with specific activity of 0.92 U/mg and recovery of 1.3%, respectively. The molecular mass of each purified enzyme was estimated to be 29 kDa by SDS-PAGE. The alignment analysis of amino acid sequences revealed that both enzymes belonged to GDSL lipase/esterase family including conserved blocks with SGNH catalytic residues which was mainly identified in plants before. While Est29 showed high specificity toward short-chain fatty acids (C4-C8), Lip29 showed strong lipolytic activity to long-chain fatty acids (C12-C16). The optimal activity of Lip29 toward p-nitrophenyl palmitate as a substrate was observed at 50℃ and pH 9.5, respectively, and its activity was maintained more than 24 h at optimal temperatures, indicating that Lip29 was thermostable. Lip29 exhibited high tolerance against detergents and metal ions. The homology modeling and substrate docking revealed that the long-chain substrates showed the greatest binding affinity toward enzyme. Based on the biochemical and insilico analyses, we present for the first time a GDSL-type lipase in the thermophilic bacteria group.

Roles of Carbohydrate-Binding Module (CBM) of an Endo-β-1,4-Glucanase (Cel5L) from Bacillus sp. KD1014 in Thermostability and Small-Substrate Hydrolyzing Activity

  • Lee, Jae Pil;Shin, Eun-Sun;Cho, Min Yeol;Lee, Kyung-Dong;Kim, Hoon
    • Journal of Microbiology and Biotechnology
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    • v.28 no.12
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    • pp.2036-2045
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    • 2018
  • An endo-${\beta}$-1,4-glucanase gene, cel5L, was cloned using the shot-gun method from Bacillus sp.. The gene, which contained a predicted signal peptide, encoded a protein of 496 amino acid residues, and the molecular mass of the mature Cel5L was estimated to be 51.8 kDa. Cel5L contained a catalytic domain of glycoside hydrolase (GH) family 5 and a carbohydrate-binding module family 3 (CBM_3). Chromatography using HiTrap Q and CHT-II resulted in the isolation of two truncated forms corresponding to 50 (Cel5L-p50) and 35 kDa (Cel5L-p35, CBM_3-deleted form). Both enzymes were optimally active at pH 4.5 and $55^{\circ}C$, but had different half-lives of 4.0 and 22.8 min, respectively, at $70^{\circ}C$. The relative activities of Cel5L-p50 and Cel5L-p35 for barley ${\beta}$-glucan were 377.0 and 246.7%, respectively, compared to those for carboxymethyl-cellulose. The affinity and hydrolysis rate of pNPC by Cel5L-p35 were 1.7 and 3.3 times higher, respectively, than those by Cel5L-p50. Additions of each to a commercial enzyme set increased saccharification of pretreated rice straw powder by 17.5 and 21.0%, respectively. These results suggest CBM_3 is significantly contributing to thermostability, and to affinity and substrate specificity for small substrates, and that these two enzymes could be used as additives to enhance enzymatic saccharification.

Purification, Characterization and Immobilization of Lipase from Proteus vulgaris OR34 for Synthesis of Methyl Oleate

  • Misbah, Asmae;Koraichi, Saad Ibnsouda;Jouti, Mohamed Ali Tahri
    • Microbiology and Biotechnology Letters
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    • v.48 no.4
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    • pp.491-505
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    • 2020
  • A newly isolated strain, Proteus vulgaris OR34, from olive mill waste was found to secrete an alkaline extracellular lipase at 11 U·ml-1 when cultivated on an optimized liquid medium. This lipase was purified 94.64-fold with a total yield of 9.11% and its maximal specific activity was shown to be 3232.58 and 1777.92 U·mg-1 when evaluated using the pH-stat technique at 55℃ and pH 9 and Tributyrin TC4 or olive oil as the substrate. The molecular mass of the pure OR34 lipase was estimated to be around 31 kDa, as revealed by SDS-PAGE and its substrate specificity was investigated using a variety of triglycerides. This assay revealed that OR34 lipase preferred short and medium chain fatty acids. In addition, this lipase was stable in the presence of high concentrations of bile salt (NaDC) and calcium ions appear not to be necessary for its activity. This lipase was inhibited by THL (Orlistat) which confirmed its identity as a serine enzyme. In addition, the immobilization of OR34 lipase by adsorption onto calcium carbonate increased its stability at higher temperatures and within a larger pH range. The immobilized lipase exhibited a high tolerance to organic solvents and retained 60% of its activity after 10 months of storage at 4℃. Finally, the OR34 lipase was applied in biodiesel synthesis via oleic acid mediated esterification of methanol when using hexane as solvent. The best conversion yield (67%) was obtained at 12 h and 40℃ using the immobilized enzyme and this enzyme could be reused for six cycles with the same efficiency.

Structural Insights into Porphyrin Recognition by the Human ATP-Binding Cassette Transporter ABCB6

  • Kim, Songwon;Lee, Sang Soo;Park, Jun Gyou;Kim, Ji Won;Ju, Seulgi;Choi, Seung Hun;Kim, Subin;Kim, Na Jin;Hong, Semi;Kang, Jin Young;Jin, Mi Sun
    • Molecules and Cells
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    • v.45 no.8
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    • pp.575-587
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    • 2022
  • Human ABCB6 is an ATP-binding cassette transporter that regulates heme biosynthesis by translocating various porphyrins from the cytoplasm into the mitochondria. Here we report the cryo-electron microscopy (cryo-EM) structures of human ABCB6 with its substrates, coproporphyrin III (CPIII) and hemin, at 3.5 and 3.7 Å resolution, respectively. Metal-free porphyrin CPIII binds to ABCB6 within the central cavity, where its propionic acids form hydrogen bonds with the highly conserved Y550. The resulting structure has an overall fold similar to the inward-facing apo structure, but the two nucleotide-binding domains (NBDs) are slightly closer to each other. In contrast, when ABCB6 binds a metal-centered porphyrin hemin in complex with two glutathione molecules (1 hemin: 2 glutathione), the two NBDs end up much closer together, aligning them to bind and hydrolyze ATP more efficiently. In our structures, a glycine-rich and highly flexible "bulge" loop on TM helix 7 undergoes significant conformational changes associated with substrate binding. Our findings suggest that ABCB6 utilizes at least two distinct mechanisms to fine-tune substrate specificity and transport efficiency.

Exploring the molecular characteristics, detoxification functions, and immune responses of two glutathione S-transferases in redlip mullet (Liza haematochelia)

  • Jeongeun Kim;Welivitiye Kankanamge Malithi Omeka;Qiang Wan;Jehee Lee
    • Fisheries and Aquatic Sciences
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    • v.27 no.5
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    • pp.314-328
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    • 2024
  • The mechanism for the elimination of xenobiotics undergoes three different phases of reactions in organisms. Among these, glutathione S-transferases (GSTs) are classified as phase II detoxification enzymes, catalyzing the conjugation of electrophilic substrates to glutathione or reduced hydroperoxides. This study aimed to investigate the molecular characteristics, detoxification functions, and immune responses of GST omega (LhGSTO1) and kappa (LhGSTK1) in redlip mullet. The open reading frames of LhGSTO1 (720 bp) and LhGSTK1 (687 bp) encoded proteins of 239 and 228 amino acids, respectively. Sequence analysis revealed that LhGSTO1 and LhGSTK1 possessed GSH-binding sites in their N-terminal domains. Substrate-binding sites in the C-terminal domain were exclusively identified in LhGSTO1. In the tissue-specific transcription profile analysis, both LhGSTO1 and LhGSTK1 were ubiquitously expressed in all tissues of healthy mullets. Temporal expression analysis of LhGSTO1 and LhGSTK1 in the blood showed that their expression was significantly modulated by polyinosinic:polycytidylic (poly I:C), lipopolysaccharide (LPS), and Lactococcus garvieae. Different chemical and cellular assays were performed to assess the detoxification and cellular protective abilities of the two proteins. A substrate specificity test using the recombinant proteins revealed that both proteins possessed specific activity toward 1-chloro-2,4-dinitrobenzene (CDNB). In the disk diffusion assay, the smallest clearance zones were observed for LhGSTO1 and LGSTK1 against CdCl2. In the cell protection assay, both LhGSTO1 and LhGSTK1 showed significant Cd detoxification ability compared to the control. Collectively, these results demonstrate that GST omega and kappa are involved in host defense against immune stimulants and xenobiotics in redlip mullet.

Characteristics of Catechol 2,3-Dioxygenase Produced by 4-Chlorobenzoate-degrading Pseudomonas sp. S-47

  • Kim, Ki-Pil;Seo, Dong-In;Min, Kyung-Hee;Ka, Jong-Ok;Park, Yong-Keun;Kim, Chi-Kyung
    • Journal of Microbiology
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    • v.35 no.4
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    • pp.295-299
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    • 1997
  • Pseudomonas sp. S-47 is capable of transforming 4-chlorobenzoate to 4-chlorocatechol which is subsequently oxidized bty meta-cleavage dioxygenase to prodyce 5-chloro-2-hydroxymuconic semialdehyde. Catechol 2,3-dioxygenase (C23O) produced by Pseudomonas sp. S-47 was purified and characterized in this study. The C23O enzyme was maximally produced in the late logarithmic growth phase, and the temperature and pH for maximunm enzyme activity were $30{\sim}35^{\circ}C$ and 7.0, respectively. The enzyme was purified and concentrated 5 fold from the crude cell extracts through Q Sepharose chromatography and Sephadex G-100 gel filtration after acetone precipitation. The enzyme was identified as consisting of 35 kDa subunits when analyzed by SDS-PAGE. The C23O produced by Pseudomonas sp. S-47 was similar to Xy1E of Pseudomonas putida with respect to substrate specificity for several catecholic compounds.

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Evaluation of a Chromogenic Medium Supplemented with Glucose for Detecting Enterobacter sakazakii

  • Song, Kwang-Young;Hyeon, Ji-Yeon;Shin, Ho-Chul;Park, Chan-Kyu;Choi, In-Soo;Seo, Kun-Ho
    • Journal of Microbiology and Biotechnology
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    • v.18 no.3
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    • pp.579-584
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    • 2008
  • A commercial chromogenic agar medium (DFI) was supplemented with glucose (mDFI) to enhance the specificity of Enterobacter sakazakii (E. sakazakit) detection. Escherichia vulneris (E. vulneris), a putative false-positive strain on the DFI medium, produces ${\alpha}$-glucosidase. The enzyme ${\alpha}$-glucosidase hydrolyzes a substrate, 5-bromo-4-chloro-3-indolyl-${\alpha}$, D-glucopyranoside $(X{\alpha}Glc)$, producing green colonies. E. sakazakii strains produced green colonies on both DFI and mDFI agar, whereas E. vulneris produced green colonies on DFI agar but small white colonies on mDFI agar. E. sakazakii and E. vulneris were also readily differentiated by colony color when the mixed culture of the two strains was plated on mDFI agar and incubated for 24 h at $37^{\circ}C$. The results indicate that the selectivity of the commercial chromogenic agar medium could be improved by a simple supplementation with glucose.

Histidylleucine Hydrolyzing Enzyme : A Dipeptidase From Hog Lung (Dipeptidase로서의 Histidylleucine 가수분해 효소)

  • Hyun-Jae Lee;Jung-Keun Park
    • Journal of the Korean Chemical Society
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    • v.19 no.5
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    • pp.375-380
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    • 1975
  • An enzyme capable of hydrolyzing histidylleucine was purified 50 fold from hog lung. The final preparation hydrolyzed $1.6{\mu}moles$ of histidylleucime per minute per mg of protein. The $K_m$ of the enzyme for the enzyme was found to be $2{\times}10^{-4}M$. The enzyme was required a number of free dipeptides for the substrate specificity, and was inhibited by EDTA and 1,10-phenan-throline. The molecular weight of the enzyme was estimated to be 80,000 daltons from sucrose density gradient sedimentation analysis. The corrected $s_{20,w}$ value was 5.3 S.

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Expression of Arabidiopsis CAX4 in tomato fruits increases calcium level with no accumulation of other metallic cations

  • Jeong, Se-Woon;Han, Jeung-Sul;Kim, Kyung-Min;Oh, Jung-Youl;Kim, Byung-Oh;Kim, Chang-Kil;Chung, Jae-Dong
    • Journal of Plant Biotechnology
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    • v.35 no.4
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    • pp.337-343
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    • 2008
  • We generated transgenic tomato plants with Arabidopsis thaliana $H^+$/cation exchanger gene (C4X4) by Agrobactrium-mediated transformation. We confirmed transgene copy number and transcription by Southern and Northern blot analyses. The intact CAX4-expressing tomato (Lycopersicon esculentum) fruits contained 63-71% more calcium ($Ca^{2+}$) than wild-type fruits. Moreover, ectopic expression of C4X4 in tomato fruits did not show any significant increase of the four kinds of metallic cations analyzed ($Mg^{2+}$, $Fe^{2+}$, $Mn^{2+}$, and $Cu^{2+})$. The C4X4-expressing tomato plants including their fruits did not show any morphological alternations during whole growth period. These results suggest the enhanced Ca-substrate specificity of CAX4 exchanger in tomato. Therefore, intact CAX4 exchanger can be a useful tool for $Ca^{2+}$ nutrient enrichment of tomato fruits with reduced accumulation of undesirable cations.

Cellulase 및 hemicellulase의 고생산균주 FJ1의 효소생산 특성 연구

  • Kim, Gyeong-Cheol;Yu, Seung-Su;O, Yeong-A;Jeong, Seon-Yong;Kim, Seong-Jun
    • 한국생물공학회:학술대회논문집
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    • 2001.11a
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    • pp.629-632
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    • 2001
  • The strain FJ1 isolated from a rotten wood showed high activity to hydrolysis of cellulosic materials. The strain produced largely enzymes related in hydrolysis of cellulose and hemicellulose, such as CMCase, xylanase, ${\beta}-glucosidase$ , and avicelase. The culture conditions(pH, temperature, inoculation concentration) and substrate specificity to various cellulosic materials were examined to elevate productivity of the enzymes. The enzyme activities of CMCase and xylanase were 13.5U/ml and 24.3U/ml in agitation culture using Mandel's medium, respectively. The high activity of the enzymes was earned when mixed cellulosic materials of rice straw, sawdust, and pulp as substrates, indicating that the strain FJ1 could use crystalline substrates.

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