• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.552-559
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• 2008

Ethyl (S)-4-chloro-3-hydroxybutyrate is an intermediate for the synthesis of Atorvastatin, a chiral drug used for hypercholesterolemia. A Rhodococcus erythropolisstrain (No.7) able to convert 4-chloro-3-hydroxybutyronitrile into 4-chloro-3-hydroxybutyric acid has recently been isolated from soil. This activity has been regarded as having been caused by the successive actions of the nitrile hydratase and amidase. In this instance, the corresponding amidase gene was cloned from the R. erythropolis strain and expressed in Escherichia coli cells. A soluble active form of amidase enzyme was obtained at $18^{\circ}C$. The Ni column-purified recombinant amidase was found to have a specific activity of 3.89 U/mg toward the substrate isobutyramide. The amidase was found to exhibit a higher degree of activity when used with mid-chain substrates than with short-chain ones. Put differently, amongst the various amides tested, isobutyramide and butyramide were found to be hydrolyzed the most rapidly. In addition to amidase activity, the enzyme was found to exhibit acyltransferase activity when hydroxyl amine was present. This dual activity has also been observed in other enzymes belonging to the same amidase group (E.C. 3.5.1.4). Moreover, the purified enzyme was proven to be able to enantioselectively hydrolyze 4-chloro-3-hydroxybutyramide into the corresponding acid. The e.e. value was measured to be 52% when the conversion yield was 57%. Although this e.e. value is low for direct commercial use, molecular evolution could eventually result in this amidase being used as a biocatalyst for the production of ethyl (S)-4-chloro-3-hydroxybutyrate.

• KSBB Journal
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• 제23권5호
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• pp.445-448
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• 2008

생명공학분야에서 매우 중요한 효소 중에 하나인 lipase는 여러 산업에 유용하게 사용되고 있다. lipase를 선별하기 위해서는 최적화된 발현 시스템이 필요하다. 많은 발현 시스템중에 E.coli 발현 시스템은 바람직한 특성을 갖는 효소를 스크리닝하거나, 선별된 변이체들의 특성을 확인하는 데에 소요되는 시간과 비용을 단축시켜 줄 것이다. 본 연구에서는 그 중에 BL21와 OrigamiB에서 CalB를 발현하였다. 그 결과 BL21 균주에서 발현된 CalB는 대부분이 불용성의 inclusion body를 형성하고, 전혀 활성을 나타내지 않았다. 이전의 타 연구와 더불어 이 결과에서 E.coli 균주에서 CalB의 기능적 발현이 상당히 어렵다는 것을 알 수 있다. 특히 불용성의 inclusion body형성과 lipase의 세포에 대한 유독성이 원인이 될 수 있다. 그러나 BL21와 비교해보면, OrigamiB에서 발현된 CalB 또한 많은 양의 inclusion body를 형성하지만, lipase의 주요 특성중의 하나인 가수분해 활성이 상당하게 나타나는 것을 알 수 있다. lipase의 구조 형성을 도와주는 변형된 OrigamiB와 저온유도시스템인 pCold 플라스미드를 사용했기 때문이다. 이처럼 균주나 플라스미드의 선택, 유도조건의 변경 등의 여러 연구를 통하여 유용한 효소를 선별할 수 있다.

• Journal of Microbiology and Biotechnology
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• 제25권7호
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• pp.1108-1113
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• 2015

Cadaverine (1,5-diaminopentane) is an important industrial chemical with a wide range of applications. Although there have been many efforts to produce cadaverine through fermentation, there are not many reports of the direct cadaverine production from lysine using biotransformation. Whole-cell reactions were examined using a recombinant Escherichia coli strain overexpressing the E. coli MG1655 cadA gene, and various parameters were investigated for the whole-cell bioconversion of lysine to cadaverine. A high concentration of lysine resulted in the synthesis of pyridoxal-5'-phosphate (PLP) and it was found to be a critical control factor for the biotransformation of lysine to cadaverine. When 0.025 mM PLP and 1.75 M lysine in 500 mM sodium acetate buffer (pH6) were used, consumption of 91% lysine and conversion of about 80% lysine to cadaverine were successfully achieved.

• Journal of Microbiology and Biotechnology
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• 제28권11호
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• pp.1850-1858
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• 2018

Glucosamine (GlcN) is widely used in the nutraceutical and pharmaceutical industries. Currently, GlcN is mainly produced by traditional multistep chemical synthesis and acid hydrolysis, which can cause severe environmental pollution, require a long prodution period but a lower yield. The aim of this work was to develop a whole-cell biocatalytic process for the environment-friendly synthesis of glucosamine (GlcN) from N-acetylglucosamine (GlcNAc). We constructed a recombinant Escherichia coli and Bacillus subtilis strains as efficient whole-cell biocatalysts via expression of diacetylchitobiose deacetylase ($Dac_{ph}$) from Pyrococcus furiosus. Although both strains were biocatalytically active, the performance of B. subtilis was better. To enhance GlcN production, optimal reaction conditions were found: B. subtilis whole-cell biocatalyst 18.6 g/l, temperature $40^{\circ}C$, pH 7.5, GlcNAc concentration 50 g/l and reaction time 3 h. Under the above conditions, the maximal titer of GlcN was 35.3 g/l, the molar conversion ratio was 86.8% in 3-L bioreactor. This paper shows an efficient biotransformation process for the biotechnological production of GlcN in B. subtilis that is more environmentally friendly than the traditional multistep chemical synthesis approach. The biocatalytic process described here has the advantage of less environmental pollution and thus has great potential for large-scale production of GlcN in an environment-friendly manner.

• KSBB Journal
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• 제16권6호
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• pp.562-567
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• 2001

Styrene oxide 계열의 라세믹 에폭사이드 기질에 대한 입체선택적 가수분해능이 우수한 Aspergillus nigerr계열의 생촉매를 선발하였고, A.niger LK 유래의 EHase의 기질 특이성을 분석하였다. A. niger LK의 EHase는 benzene ring에 oxirane ring이 직접 연결되어 있는 styrene oxide, p-nitrostyrene oxide 기질에 대해서는 (R)-이성질체, benzene ring과 oxirane ring사이에 ether 등의 연결 chain이 있는 기질에 대해서는 (S)-이 성질체에 대한 입체선택적 가수분해능이 우수하였다. A niger LK의 EHase 유전자를 RT-PCR 방법으로 클로닝하였고, sequencing을 통해 다른 미생물 유래의 EHase와의 sequence identity 분석 등을 통해 특성을 분석하였다. Yeast 유래의 EHase와는 32% 수준의 sequence identity를 보였으며, Agrobacterisum, Corynebacterium 등의 박테리아 유래 EHase와는 identity가 매우 낮은 특성을 보였다. E. coli 숙주에서 발현된 재조합 EHase의 활성은 라세믹 에폭사이드 기질에 대한 입체선택적 가수분해 반응을 통해 확인할 수 있었다. 클러닝된 EHase의 보다 효율적인 발현 연구가 필요하며, 이러한 재조합 EHase는 고부가가치 광학활성 에폭사이드 제조를 위한 생물전환공정 시스템의 생촉매로 응용될 수 있을 것으로 기대된다.

• Journal of Microbiology and Biotechnology
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• 제26권6호
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• pp.1067-1076
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• 2016

The gene encoding lipase (Lip98) from Aeromicrobium sp. SCSIO 25071 was cloned and functionally expressed in Escherichia coli. Lip98 amino acid sequence shares the highest (49%) identity to Rhodococcus jostii RHA1 lipase and contains a novel motif (GHSEG), which is different from other clusters in the lipase superfamily. The recombinant lipase was purified to homogeneity with Ni-NTA affinity chromatography. Lip98 showed an apparent molecular mass of 30 kDa on SDS gel. The optimal temperature and pH value for enzymatic activity were recorded at 30℃ and 7.5, respectively. Lip98 exhibited high activity at low temperatures with 35% maximum activity at 0℃ and good stability at temperatures below 35℃. Its calculated activation energy was 4.12 kcal/mol at the low temperature range of 15-30℃. Its activity was slightly affected by some metal ions such as K⁺, Ca²⁺, and Na⁺. The activity of Lip98 was increased by various organic solvents such as DMSO, ethanol, acetone, and hexane with the concentration of 30% (v/v) and retained more than 30% residual activity in neat organic solvent. The unique characteristics of Lip98 imply that it is a promising candidate for industrial application as a nonaqueous biocatalyst and food additive.

• Journal of Microbiology and Biotechnology
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• 제31권10호
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• pp.1455-1464
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• 2021

Trehalose is a non-reducing disaccharide in increasing demand for applications in food, nutraceutical, and pharmaceutical industries. Single-step trehalose production by trehalose synthase (TreS) using maltose as a starting material is a promising alternative process for industrial application due to its simplicity and cost advantage. Pseudomonas monteilii TBRC 1196 was identified using the developed screening method as a potent strain for TreS production. The TreS gene from P. monteilii TBRC 1196 was first cloned and expressed in Escherichia coli. Purified recombinant trehalose synthase (PmTreS) had a molecular weight of 76 kDa and showed optimal pH and temperature at 9.0 and 40℃, respectively. The enzyme exhibited >90% residual activity under mesophilic condition under a broad pH range of 7-10 for 6 h. Maximum trehalose yield by PmTreS was 68.1% with low yield of glucose (4%) as a byproduct under optimal conditions, equivalent to productivity of 4.5 g/l/h using enzyme loading of 2 mg/g substrate and high concentration maltose solution (100 g/l) in a lab-scale bioreactor. The enzyme represents a potent biocatalyst for energy-saving trehalose production with potential for inhibiting microbial contamination by alkaline condition.

• 한국해양바이오학회지
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• 제14권2호
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• pp.133-142
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• 2022

The increased production and consumption of polyethylene terephthalate (PET)-based products over the past several decades has resulted in the discharge of countless tons of PET waste into the marine environment. PET microparticles resulting from the physical erosion of general PET wastes end up in the ocean and pose a threat to the marine biosphere and human health, necessitating the development of new technologies for recycling and upcycling. Notably, enzyme-mediated PET degradation is an appealing option due to its eco-friendly and energy-saving characteristics. PETase, a PET-hydrolyzing enzyme originating from Ideonella sakaiensis, is one of the most thoroughly researched biological catalysts. However, the industrial application of PETase-mediated PET recycling is limited due to the low stability and poor reusability of the enzyme. Here we developed the whole-cell catalyst (WCC) in which functional PETase is attached to the outer membrane of Escherichia coli. Immunoassays are used to identify the surface-expressed PETase, and we demonstrated that the WCC degraded PET microparticles most efficiently at 30℃ and pH 9 without agitation. Furthermore, the WCC increased the PET-degrading activity in a concentration-dependent manner, surpassing the limited activity of soluble PETase above 100 nM. Finally, we demonstrated that the WCC could be recycled up to three times.