• 한국식품영양학회지
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• 제12권3호
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• pp.265-270
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• 1999

The stabilization of Aspergillus sp. $\alpha$-amylase was attained by modification with periodate-oxidized sol-uble starch. The pH stability of modified enzyme was increased at pH 3~4 and 9~11 in the presence of $\alpha$-cyclodextrin($\alpha$-CD) compared with that of native enzyme. Thermal stability of the modified enzyme was increased. After treatment at 6$0^{\circ}C$ for 30min the activity remained 20% for the enzyme modified at pH 9.7 in the presence of $\alpha$-CD and tested in the presence of $\alpha$-CD 10% for the enzyme modified at pH 9.7 in the presence of $\alpha$-CD 0% for the native enzyme. The native enzyme and modified enzyme showed one peak in HPLC. The substrate specificity of the modified enzyme was not changed in HPLC analysis of reaction product.

• Bulletin of the Korean Chemical Society
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• 제34권9호
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• pp.2741-2746
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• 2013

The immobilization of enzyme is one of the key issues both in the field of enzymatic research and industrialization. In this work, we reported a facile method to immobilize Candida Antarctica lipase B (CALB) in alginate carrier. In the presence of calcium cation, the enzyme-alginate suspension could be cross-linked to form beads with porous structure at room temperature, and the enzyme CALB was dispersed in the beads. Activity of the enzyme-alginate composite was verified by enzymatic hydrolysis reaction of p-nitrophenol butyrate in aqueous phase. The effects of reaction parameters such as temperature, pH, embedding and lyophilized time on the reactive behavior were discussed. Reuse cycle experiments for the hydrolysis of p-nitrophenol butyrate demonstrated that activity of the enzyme-alginate composite was maintained without marked deactivation up to 6 repeated cycles.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.445-449
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• 2003

As for the purpose, we first introduce an random mutation into wild-type gene to expand a mutation space, and then further recombine the mutant genes by staggered extension process PCR. As a result, we obtained the best clones 6-52 that showed a high activity and stability, from a round of error prone and staggered extension process PCR. The purified enzyme showed a similar pH stability to the wild-type enzyme and reveal a slightly high optimum pH at 12. In the optimum temperature, an identical dependency was also showed and a quite high stability in the thermal stability was obtained. Along with this, the enzyme was also stable at a reaction that supplement with a 15 % of ethanol as an additive. The addition of other solvents and surfactants did not improve the reaction and thus resulted in a similar profile to those of wild-type enzyme. The specific activity on the target compound rac-ketoprofen ethyl ester was calculated to be about 85, 000 unit, and the kinetic constants Km and Vmax were determined to be 0.2 mM and 90 mM/mg-protein/min respectively. The deduced amino acid alignment with the wild type enzyme revealed five mutations at L120P, I208V, T249A, D287H and T357A. Based on these observations, the site directed mutagenesis to delineate the mutagenic effect is under progress.

• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.513-517
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• 2010

A thermostable trehalose synthase (TtTSase) from Thermus thermophilus HJ6 was immobilized on chitosan activated with glutaraldehyde. The yield of immobilization was evaluated as 39.68%. The optimum pH of the immobilized enzyme was similar to that of the free enzyme. However, the optimal temperature ranges were shifted by about $4^{\circ}C$ owing to better thermal stability after immobilization. The half-life of heat inactivation for free and immobilized enzymes was 5.7 and 6.3 days at $70^{\circ}C$, respectively, thus showing a lager thermostability of the immobilized enzyme. When tested in batch reaction, the immobilized enzyme retained its relative activity of 53% after 30 reuses of reaction within 12 days, and still retained 82% of its initial activity even after 150 days at $4^{\circ}C$. A packed-bed bioreactor with immobilized enzyme showed a maximum yield of 56% trehalose from 100 mM maltose in a continuous recycling system (bed volume: 10 ml) under conditions of pH 7.0 and $70^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• 제26권8호
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• pp.1348-1357
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• 2016

An enzymatic reaction system was developed and optimized for bioconversion of resveratrol from glucose. Liquid enzyme extracts were prepared from Alternaria sp. MG1, an endophytic fungus from grape, and used directly or after immobilization with sodium alginate. When the enzyme solution was used, efficient production of resveratrol was found within 120 min in a manner that was pH-, reaction time-, enzyme amount-, substrate type-, and substrate concentration-dependent. After the optimization experiments using the response surface methodology, the highest value of resveratrol production (224.40 μg/l) was found under the conditions of pH 6.84, 0.35 g/l glucose, 0.02 mg/l coenzyme A, and 0.02 mg/l ATP. Immobilized enzyme extracts could keep high production of resveratrol during recycling use for two to five times. The developed system indicated a potential approach to resveratrol biosynthesis independent of plants and fungal cell growth, and provided a possible way to produce resveratrol within 2 h, the shortest period needed for biosynthesis of resveratrol so far.

• 생명과학회지
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• 제16권1호
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• pp.1-5
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• 2006

본 연구는 Penibacillus polymyxa 균주의 CFTase 유전자를 결손 변이시킨 고효율 효소 CFT108을 이용하여 cyclofructan (CF)의 대량생산 조건을 검토하고 생산된 CF의 순수 분리 정제 공정을 개발하였다. CF의 대량생산 조건은 $2\%$의 inulin 기질과 40 unit/g inulin의 기질에서 3시간 반응시켰을 때 최대 생산량 9.5 g/l의 수율을 달성할 수 있었으며, 이때의 in-ulin의 CF 전환율은 $47.5\%$였다. 생산된 CF를 순수 분리 정제하기 위해서 CFTase 반응액 을 exoinulinase 1 unit/ml로 6시간 처리하여 미 반응 inulin을 단당화시켰으며, 유리된 fructose는 $3\%$ CaO로 $CO_2$가스 하에서 $80^{\circ}C$, 10분간 3회 흡착제거 시킴으로써 순수 정제하였다. 정제된 CF를 HPLC로 확인한 결과 정제도는 $95\%$ 이상이였으며, $CF_6,\;CF_7,\;CF_8$ 비율이 72 : 27 : 1 이였다.

• 공업화학
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• 제23권4호
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• pp.394-398
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• 2012

아스트라갈린(AS)은 폴리페놀의 일종인 캠페롤(KR)에 글루코스가 하나 결합해있는 배당체로서, 천연에는 미량만이 존재하고 있다. 최근 tea seed extract (TSE)에 포함되어 있는 camelliaside A (CamA) 및 camelliaside B (CamB)로부터 복합효소인 Mash에 의한 부분가수분해에 의해 AS를 합성할 수 있다는 것이 알려졌다. 본 논문에서는, Mash에 의한 TSE의 선택적 가수분해에 의해 AS를 제조하는 공정에서 반응 온도, 효소량 및 반응물의 농도 등의 변수에 따른 반응성을 검토하였다. 반응 온도, 효소량 등이 증가할수록 AS의 생성 반응은 빨랐으나, 일단 생성된 AS가 급격하게 KR로 변환되는 문제점이 나타났다. 결론적으로, 반응 속도 및 AS의 선택성 측면을 고려하면, $50^{\circ}C$에서 TSE 대비 2배의 Mash를 사용하여 15%의 기질 농도에서 반응을 수행하는 것이 최적인 것으로 나타났다.

• 한국미생물·생명공학회지
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• 제27권6호
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• pp.471-476
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• 1999

Inulin fructotransferase(depolymerizing)(EC 2.4.1.93)(inulaseII) which converts inulin into di-D-fructofuranose-1,2':2,3'-dianhydride (DFAIII) was purified from Arthrobacter ureafaciens KCTC 3387 using column chromatography on DEAE-Toyopearl 650M and gel filtration of Sephadex G-200. The enzyme was purified 7-fold with a yield of 11% from a culture supernatant. The purified enzyme gave a single band on polyacrylamide gel electrophoresis, and the molecular weight of the enzyme was estimated to be 45,000 by SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature for the enzyme reaction were pH6.5~7.0 and $55{\circ}C$, respectively. The enzyme was stable within a pH range of 5.0 to 10.6 and up to $60^{\circ}C$. The Km of this enzyme for DFAIII production was 11.9mM. The enzyme was inactivated by $Hg^{2+}$ and after exhaustive digestion of inulin by this enzyme, 1-kestose and nystose were produced in addition of DFAIII.

• 한국미생물·생명공학회지
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• 제24권5호
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• pp.579-584
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• 1996

The molecular weight of the purified nucleoside oxidase estimated by gel filtration column chromatography was 480,000 and the enzyme protein was composed of four nonidentical subunits (81,000, 69,000, 32,000 and 16,000). On the basis of the visible absorption spectra and the enzymatic determination of the purified enzyme, the enzyme was supposed as a hemoprotein and also a flavoprotein containing 3 moles of FAD per I mole of enzyme. The isoelectric point of the enzyme was pH 5.1. Addition of metal salts such as 1 mM SnCl$_{2}$ and PbCl$_{2}$ into an enzyme reaction solution inhibited the enzyme activity by 94 and 90%, respectively. The enzyme activity was also lost significantly by hemoenzyme inhibitors such as NaCN and NaN$_{3}$ and flavoenzyme inhibitor, acriflavine and quinacrine. The maximal nucleoside oxidase activity was observed at pH 7.0 and 55$\circ$C. The nucleoside oxidase was relatively stable in the range of pH 5.5-9.0 and below 55$\circ$C.

• Journal of Microbiology and Biotechnology
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• 제28권8호
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• pp.1346-1351
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• 2018

Oxidoreductases are effective biocatalysts, but their practical use is limited by the need for large quantities of NAD(P)H. In this study, a whole-cell biocatalyst for NAD(P)H cofactor regeneration was developed using the economical substrate glycerol. This cofactor regeneration system employs permeabilized Escherichia coli cells in which the glpD and gldA genes were deleted and the gpsA gene, which encodes $NAD(P)^+-dependent$ glycerol-3-phosphate dehydrogenase, was overexpressed. These manipulations were applied to block a side reaction (i.e., the conversion of glycerol to dihydroxyacetone) and to switch the glpD-encoding enzyme reaction to a gpsA-encoding enzyme reaction that generates both NADH and NADPH. We demonstrated the performance of the cofactor regeneration system using a lactate dehydrogenase reaction as a coupling reaction model. The developed biocatalyst involves an economical substrate, bifunctional regeneration of NAD(P)H, and simple reaction conditions as well as a stable environment for enzymes, and is thus applicable to a variety of oxidoreductase reactions requiring NAD(P)H regeneration.