• Title/Summary/Keyword: Recombinant enzyme

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Construction of Bifunctional Fusion Enzyme between Maltooligosyltrehalose Synthase and Maltooligosyltrehalose Trehalohydrolase of Sulfolobus acidocaldarius and Overexpression in E. coli

  • Kim, Chung Ho
    • Journal of Applied Biological Chemistry
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    • v.43 no.4
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    • pp.240-245
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    • 2000
  • Two genes encoding maltooligosyltrehalose synthase (SaMTS) and maltooligosyltrehalose trehalohydrolase (SaMTH) were isolated from a hyperthermophilic microorganism, Sulfolobus acidocaldarius (ATCC 49462). ORFs of the SaMTS and SaMTH genes are 2,163 and 1,671 bp long and encode 720 and 556 amino acid residues, respectively. A bifunctional fusion enzyme (SaMTSH) was constructed through the gene fusion of SaMTS and SaMTH. Recombinant SaMTS, SaMTH, and SaMTSH fusion enzyme were overexpressed in E. coli BL21. SaMTS and SaMTH produced trehalose and maltotriose from maltopentaose in a sequential reaction. SaMTSH fusion enzyme catalyzed the sequential reaction in which the formation of maltotriosyltrehalose was followed by hydrolysis leading to the synthesis of trehalose and maltotriose. The SaMTSH fusion enzyme showed the highest activity at pH 5.0-5.5 and $70-75^{\circ}C$. SaMTS, SaMTH, and SaMTSH fusion enzyme were active in soluble starch, which resulted in the production of trehalose.

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Influence of Controlled- and Uncontrolled-pH Operations on Recombinant Phenylalanine Ammonia Lyase Production in Escherichia coli

  • Cui, Jian Dong;Zhao, Gui Xia;Zhang, Ya Nan;Jia, Shi Ru
    • Food Science and Biotechnology
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    • v.18 no.4
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    • pp.954-958
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    • 2009
  • Effects of controlled- and uncontrolled-pH operations on phenylalanine ammonia lyase (PAL) production by a recombinant Escherichia coli strain were investigated at uncontrolled-pH ($pH_{UC}$) and controlled-pH ($pH_C$) of 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 in bioreactor systems. The results showed that the recombinant PAL activity was improved significantly by controlled pH strategy. Among the $pH_C$ operations, the highest PAL activities were obtained under $pH_C$ 7.5 strategy where cell mass ($OD_{600\;nm}$) and PAL activity was 1.3 and 1.8 fold higher than those of $pH_{UC}$, respectively. The maximum PAL activity reached 123 U/g. The $pH_C$ 7.5 strategy made recombinant plasmid more stable and therefore allowed easier expression of PAL recombinant plasmid, which increased PAL production. It was indicated that the new approach (controlled-pH strategy) obtained in this work possessed a high potential for the industrial production of PAL, especially in the biosynthesis of L-phenylalanine.

Studies on the structure and expression of penicillin G acylase gene I (Penicillin G acylase 유전자의 구조와 발현기작에 관한 연구 I)

  • 김영창;구용범;오상진;강현삼
    • Korean Journal of Microbiology
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    • v.21 no.2
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    • pp.95-102
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    • 1983
  • The penicillin G acylase(pga) gene was cloned in the vector plasmid pKM $300(Ar^r,\;Tc^r,\;6.33kb)$ for the study of the structure and expression of the pga gene. This recombinant plasmid pPAKS-1 DNA(24.5 Kb) was cleaved into 2 fragments by restriction enzyme Eco R1.1fragment by BamH1, 4fragments by Hind III, and 2 fragments by Pst I. The pga gene was located on the Eco R1.Hind III-C fragement of pPAKS-1. The recombinant plasmids pPAKS-1 and pPAKS-2, in which the Hind III-B and Hind III-D fragments pPAKS-1 are deleted, are characterized. The results are summarized as follows : 1. Doubling times of bacterial strain bearing pPAKS-1 and pPAKS-2 are 90 and 60 minutes, respectively. 2. pPAKS-1 and pPAKS-2 are present at about 16-32 and 70 copies per cell, respectively, are 0.66 and 5.5 units, respectively, which represent 2-fold and 20-fold higher enzyme 4. pPAKS-1 is very unstable, but pPAKS-2 is stable.

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Alginate Lyase Production of Halophilic Pseudomonas sp. by Recombinant Escherichia coli

  • Kong, In-Soo;Kim, Young-Ok;Kim, Jin-Man;Kim, Sung-Koo;Oh, Doo-Hwan;Yu, Ju-Hyun;Kong, Jal-Yul
    • Journal of Microbiology and Biotechnology
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    • v.5 no.2
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    • pp.92-95
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    • 1995
  • Halophilic Pseudomonas sp.W7 isolated from laver in the southem sea of Korea showed alginate lyase activity. Gene (aly) encoding alginate lyase was cloned in E.coli JM83 and the N-terminal amino acid sequence of the enzyme was determined after purificaion. The recombinant enzyme has been shown to have a molecular weight of about 40kDa after 12% SDS-polyacrylamide gel electrophoresis.

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Reaction Stability of the Recombinant Tyrosinase-CNK Originating from the Psychrophilic Marine Microorganism Candidatus Nitrosopumilus Koreensis (호냉성 균주 유래 재조합 티로시나아제 효소, tyrosinase-CNK의 반응 안정성 연구)

  • Choi, Yoo Rae;Do, Hyunsu;Jeong, Dawon;Park, Junetae;Choi, Yoo Seong
    • Clean Technology
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    • v.22 no.3
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    • pp.175-180
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    • 2016
  • Tyrosinases catalyze the hydroxylation of a monophenol (monophenolase activity) and the conversion of an o-diphenol to o-quinone (diphenolase activity), which are mainly involved in the modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (DOPA) and DOPA/DOPAquinone-derived intermolecular cross-linking. Previously, we obtained a slightly acidic and cold-active tyrosinase, tyrosinase-CNK, by our recombinant protein approach. The enzyme showed optimal activity at pH 6.0 and 20 ℃ with an abnormally high monophenolase/diphenolase activity ratio and still had approximately 50% activity compared with the highest activity even in ice water. Here, we investigated reaction stability of the recombinant tyrosinase-CNK as a psychrophilic enzyme. The enzyme showed remarkable thermal stability at 0 ℃ and the activity was well conserved in repeated freeze-thaw cycles. Although water-miscible organic solvent as reaction media caused the activity decrease of tyrosinase-CNK as expected, the enzyme activity was not additionally decreased with increased concentration in organic solvents such as ethanol and acetonitrile. Also, the enzyme showed high salt tolerance in chaotropic salts. It was remarkably considered that 2+ metal ions might inhibit the incorporation of Cu2+ into the active site. We expect that these results could be used to design tyrosinase-mediated enzymatic reaction at low temperature for the production of catechols through minimizing unwanted self-oxidation and enzyme inactivation.

Cloning and Expression of a Chitinase Gene from Thermoactinomyces vulgaris KFB-C100

  • Yooh, Ho-Geun;Kim, Hee-Yun;Lim, Young-Hee;Cho, Hong-Yon
    • Journal of Microbiology and Biotechnology
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    • v.8 no.6
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    • pp.560-567
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    • 1998
  • We have found that Thermoactinomyces vulgaris KFB-Cl00 produces a chitinase. The optimum temperature and pH of the enzyme activity were $55^{\circ}C$ and 6.5. The enzyme was stable after heat treatment at $80^{\circ}C$ for 30 min and stable in acidic and basic conditions (PH 6.0~11.0). The thermostable endo-chitinase from Thermoactinomyces vulgaris KFB-C100 was cloned into the plasmid pBR322 by using E. coli DH5$\alpha$ as a host strain. The positive clone carrying a recombinant plasmid (PKCHI23) with a 4.1-kb fragment containing the chitinase gene was found. The recombinant plasmid was analyzed to determine the essential region for chitinase activity and obtained a 2.3-kb fragment, which was sub cloned into pTrc99A using the PstI and SalI sites to construct pTrc99A/pKCHI23-3. The resulting plasmid exerted high chitinase activity upon transformation of E. coli XL1-Blue cells. Chitinase was overproduced 14 times more in the clone cells than in the wild-type cells and the enzyme was purified to homogeneity. The purified enzyme showed the similar properties as the native chitinase from T. vulgaris in terms of molecular weight and substrate specificity. The catalytic action of the cloned enzyme was an endo type, producing chitobiose as a major reaction product.

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Gentiobiose Synthesis from Glucose Using Recombinant $\beta$-Glucosidase from Thermus caldophilus GK24

  • Kim, Tae-Yeon;Lee, Dae-Sil;Shin, Hyun-Jae
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.8 no.3
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    • pp.210-212
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    • 2003
  • Recombinant $\beta$-glucosidase from Thermus caldophilus GK24 was easily purified partially by a heat treatment procedure, resulting in 8-fold and recovery yield of 80% from crude enzyme. When the $\beta$-glucosidase was incubated with a 80% glucose solution (w/w), gentiobiose ($\beta$1,6-glucobiose) was the major product in the reaction mixture. The optimal conditions for producing gentiobiose (11% yields of total sugar) were pH 8-9 and 7$0^{\circ}C$ for 72 h.

Molecular Cloning, Gene Structure, Expression, and Enzyme Activity of a Serine Protease from Water Scorpion, Laccotrephes japonensis (Hemiptera: Nepidae)

  • Park, Kwan Ho;Choi, Young Cheol;Nam, Seong Hee;Hwang, Jae Sam;Nho, Si Kab
    • International Journal of Industrial Entomology and Biomaterials
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    • v.25 no.2
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    • pp.187-193
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    • 2012
  • Serine proteases are major insect enzymes involved in the digestion of dietary proteins and in the process of blood meal digestion. In this study, cDNA was constructed using the whole body of Laccotrephes japonensis. The flanking sequences of the 5- and 3- end of this gene were characterized by RACE-PCR. Sequence analysis showed that this gene contained a 963-bp ORF encoding 320 amino acids. The deduced amino acid sequence showed 62% identity with the Creontiades dilutus serine protease, 58% with the Lygus lineolaris trypsin precursor, and 54% with the Triatoma infestans salivary trypsin. To assess the expression of the L. japonensis serine protease (JGsp), the JGsp gene was cloned into a baculovirus transfer vector, pBac-1, and expressed in Sf9 cells (Spodoptera frugiperda). SDS-PAGE and western blot analysis have shown that the JGsp recombinant protein was a monomer with a molecular weight of about 32 kDa. Recombinant JGsp has shown activity in the protease enzyme assay using gelatin as a substrate.

Cloning of the Bacillus subtilis AMX-4 Xylanase Gene and Characterization of the Gene Product

  • Yoon, Ki-Hong
    • Journal of Microbiology and Biotechnology
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    • v.19 no.12
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    • pp.1514-1519
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    • 2009
  • A gene encoding the xylanase of Bacillus subtilis AMX-4 isolated from soil was cloned into Escherichia coli and the gene product was purified from the cell-free extract of the recombinant strain. The gene, designated xylA, consisted of 639 nucleotides encoding a polypeptide of 213 residues. The deduced amino acid sequence was highly homologous to those of xylanases belonging to glycosyl hydrolase family 11. The molecular mass of the purified xylanase was 23 kDa as estimated by SDS-PAGE. The enzyme had a pH optimum of 6.0-7.0 and a temperature optimum of $50-55^{\circ}C$. Xylanase activity was significantly inhibited by 5 mM $Cu^{2+}$ and 5 mM $Mn^{2+}$, and noticeably enhanced by 5 mM $Fe^{2+}$. The enzyme was active on xylans including arabinoxylan, birchwood xylan, and oat spelt xylan, but it did not exhibit activity toward carboxymethylcellulose or p-nitrophenyl-$\beta$-xylopyranoside. The predominant products resulting from xylan and xylooligosaccharide hydrolysis were xylobiose and xylotriose. The enzyme could hydrolyze xylooligosaccharides larger than xylotriose.

Surface Immobilizntion on Silica of Endoxylanase Produced from Recombinant Bacillus subtilis

  • Kang, Su-Cheol;Kim, Hye-Jeong;Nam, Soo-Wan;Oh, Deok-Kun
    • Journal of Microbiology and Biotechnology
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    • v.12 no.5
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    • pp.766-772
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    • 2002
  • The plasmid, pJHKJ4, containing the endoxylanase gene, was introduced into Bacillus subtilis DB 104. The recombinant cells produced 587 unit/ml of endoxylanase at 33 h. The endoxylanase was immobilized covalently on the surface of silica fur effective xylan hydrolysis. The activities of the immobilized and free endoxylanases were optimal at pH 6.5 and 10 mM $MnSO_4$. The optimal temperature of the immobilized endoxylanase was $60^{\circ}C$, whereas that of the free endoxylanase was $65^{\circ}C$. Under these optimal conditions, the activity of the immobilized endoxylanase was 1.7 times higher than that of the fee endoxylanase. From microscope photographs, the immobilized endoxylanase was found to be bounded and evenly distributed on the surface of silica, a nonporous solid support. The enzyme kinetics between the immobilized and free endoxylanases was estimated to be uncompetitive, when plotting double-reciprocal plots against xylan concentrations and endoxylanase activities. These results suggest that the higher activity of the immobilized endoxylanase may be due to increased formation of enzyme-substrate complex, because of the easy accessibility of the immobilized enzyme to the polysaccharide-xylan as a high molecular weight substrate.