• 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.

• Journal of Microbiology and Biotechnology
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• 제10권6호
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• pp.764-769
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• 2000

The lipase gene(lip) from Bacillus stearothermophilus was recombined in vitro by utilizing the DNA shuffling technique. After four rounds of shuffling, transformation, and screening based on the initial rate of clear zone formation on a tricaprylin plate, a clone (M10) was isolated, the cell extract of which showed about 2.8-fold increased lipase activity. The DNA sequence of the mutant lipase gene (m10) showed 3 base changes, resulting in two cryptic mutations and one amino acid substitution: S113($AGC{\rightarrow}AGT$), L252 ($TTG{\rightarrow}TTA$), and G275E ($GGA{\rightarrow}GAA$). SDS-PAGE analysis revealed that the increased enzyme activity observed in M10 was partly caused by high expression of the m10 lipase gene. The amount of the expressed G275E lipase was estimated to comprise as much as 41% of the total soluble proteins of the cell. The maximum velocity ($V_{max}$) of the purified mutant enzyme for the hydrolysis of olive oil was measured to be 3,200 U/mg, which was 10% higher than that of the parental (WT) lipase (2,900 U/mg). Its optimum temperature for the hydrolysis of olive oil was $68^{\circ}C$ and it showed a typical $Ca^{2+}$-dependent thermostability, properties fo which were the same as those of the WT lipase. However, the mutant enzyme exhibited a high enantiospecificity towards (S)-naproxen compared with the WT lipase. In addition, it showed increased hydrolytic activity towards triolein, tricaprin, tricaprylin, and tricaproin.

• KSBB Journal
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• 제14권2호
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• pp.225-229
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• 1999

본 연구에서는 광학적으로 순수한 S-(+)-ketoprofen을 얻기 위한 효소적 분할공정을 개발하는 것을 그 내용으로 하고 있다. 결과에 의하면 Candida rugosa 유래의 lipase는 음이온 교환수지에 의해 두 가지 형태의 효소로 분리되었으며 특히 첫 번째 peak의 lipaserk (S)-enantiomer에 대해 선택성이 매우 높은 것으로 나타났다. 온도 pH, 첨가제의 영향에 대해서 조사한 결과 높은 활성을 유지하면서 광학적으로 순수한 (S)-ketoprofen을 얻는 조건을 찾지는 못하였으나, $37^{\circ}C$의 온도에서 선택성이 높았으며 ethylen glyco과 같은 polyalcohol 종류가 첨가될 경우 선택성이 증가한다는 결과를 얻을 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제22권1호
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• pp.84-91
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• 2012

Two staphylococcal lipases were obtained from Staphylococcus epidermidis S2 and Staphylococcus aureus S11 isolated from sebaceous areas on the skin of the human face. The molecular mass of both enzymes was estimated to be 45 kDa by SDS-PAGE. S2 lipase displayed its highest activity in the hydrolysis of olive oil at $32^{\circ}C$ and pH 8, whereas S11 lipase showed optimal activity at $31^{\circ}C$ and pH 8.5. The S2 lipase showed the property of cold-adaptation, with activation energy of 6.52 kcal/mol. In contrast, S11 lipase's activation energy, at 21 kcal/mol, was more characteristic of mesophilic lipases. S2 lipase was stable up to $45^{\circ}C$ and within the pH range from 5 to 9, whereas S11 lipase was stable up to $50^{\circ}C$ and from pH 6 to 10. Both enzymes had high activity against tributyrin, waste soybean oil, and fish oil. Sequence analysis of the S2 lipase gene showed an open reading frame of 2,067 bp encoding a signal peptide (35 aa), a pro-peptide (267 aa), and a mature enzyme (386 aa); the S11 lipase gene, at 2,076 bp, also encoded a signal peptide (37 aa), pro-peptide (255 aa), and mature enzyme (399 aa). The two enzymes maintained amino acid sequence identity of 98-99% with other similar staphylococcal lipases. Their microbial origins and biochemical properties may make these staphylococcal lipases isolated from facial sebaceous skin suitable for use as catalysts in the cosmetic, medicinal, food, or detergent industries.

• Journal of Microbiology and Biotechnology
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• 제32권5호
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• pp.672-679
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• 2022

Microbial lipases are used widely in the synthesis of various compounds due to their substrate specificity and position specificity. 4-Ethyl malate (4-EM) made from diethyl malate (DEM) is an important starting material used to make argon fluoride (ArF) photoresist. We tested several microbial lipases and found that Photobacterium lipolyticum M37 lipase position-specifically hydrolyzed DEM to produce 4-EM. We purified the reaction product through silica gel chromatography and confirmed that it was 4-EM through nuclear magnetic resonance analysis. To mass-produce 4-EM, DEM hydrolysis reaction was performed using an enzyme reactor system that could automatically control the temperature and pH. Effects of temperature and pH on the reaction process were investigated. As a result, 50℃ and pH 4.0 were confirmed as optimal reaction conditions, meaning that M37 was specifically an acid lipase. When the substrate concentration was increased to 6% corresponding to 0.32 M, the reaction yield reached almost 100%. When the substrate concentration was further increased to 12%, the reaction yield was 81%. This enzyme reactor system and position-specific M37 lipase can be used to mass-produce 4-EM, which is required to synthesize ArF photoresist.

• 한국식품과학회지
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• 제17권2호
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• pp.75-80
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• 1985

Lipase를 유지분해에 이용하기 위한 기초연구로서 Candida cylindracea에서 추출된 lipase를 흡착법에 의해 고정화하고 그의 반응특이성을 본 결과는 다음과 같다. Lipase 흡착에 적합한 흡착제로는 silica gel이 선정되었으며, Silica gel 1.6g에 lipase 47.5 units를 $5^{\circ}C$, PH 7.0에서 100분간 흡착시키는 것이 좋았다. Silica gel에 고정화 시킨 lipase를 유지방과 olive oil의 분해에 적합한 최적온도 및 최적pH는 가용성효소와 비교시 $37^{\circ}C$, pH 7.0으로 변하지는 않았으나 활성의 범위는 넓어졌다. 또한 열안정성 및 pH안정성도 가용성 효소에 비하여 활성의 범위가 넓어졌다. 유지의 분해에 적합한 고정화 효소의 최적효소농도는 유지방의 경우 30g이었으며 올리브유의 경우 80g으로 선정하였다. 이 때 최적기질농도는 유지방 및 올리브유 모두 20%였다. 반응시간에 따른 반응률은 유지방을 이용하여 조사한 결과 가용성 효소는 반응 4시간까지는 급격한 분해를 나타냈으나 고정화 효소는 8시간 까지 급격한 증가를 나타내고 그 이상은 거의 일정하였다. 또한 유리되는 지방산의 profile은 가용성 효소와 비교시 capric acid의 생성은 모두 높았으며, myristic acid의 함량은 높고 butyic acid의 함량은 적었다.

• KSBB Journal
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• 제15권1호
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• pp.72-75
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• 2000

Candida cylindracea 유래의 효소 Lipase-OF 360,000을 사용하여 물고기 기름을 구성하고 있는 각 지방산의 가수분해 특성을 규명하여 보았다. 물고기 기름을 구성하고 있는 다양한 지방산중 $C_{14.0}$, $C_{16.0}$ 및 $C_{18.0}$의 포화지방산과 이중결합이 하나인 $C_{16.1}$, $C_{18.1}$(n-7), $C_{18.1}$(n-9), $C_{20.1}$ 및 $C_{22.1}$의 불포화 지방산은 $\omega$-3 다중불포화지방산에 비하여 쉽게 가수분해되었다. $\omega$-3다중불포화지방산중 탄소수는 동일하나 불포화도가 상이할 경우 불포화도가 낮은 지방산이 불포화도가 높은 지방산보다 쉽게 가수분해되는 특성을 나타내었으며 불포화도는 동일하나 탄소수가 다른 경우 탄소수가 적은 지방산이 탄소수가 많은 지방산보다 쉽게 가수분해되었다. $\omega$-3 다중불포화지방산중 가수분해 반응 후 모노-, 디- 및 트리-클리세라이드 혼합물에 가장 많이 농축되는 지방산은 DHA로 물고기 기름을 구성하는 총 $\omega$-3 지방산의 31.87%에서 가수분해반응 120시간 후에는 글리세라이드 혼합물을 구성하는 총 $\omega$-3 지방산의 51.89%까지 증가하였다.

• 한국미생물·생명공학회지
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• 제15권2호
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• pp.122-128
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• 1987

Candida rugosa와 Rhizopus arrhizus 리파제를 photocrosslinkable resin prepolymer에 고정화시켜서 이소옥탄을 유기용매로 사용한 이상계를 이용해서 유지분해 및 에스테르교환 반응을 보고자했다. Dioctylsulfosuccinate가 가장 좋은 surfactant였다. 소수성 젤인 ENTP-3000에 고정된 리파제가 좋은 활성을 나타냈고 친수성 젤인 ENT-4000에 고정된 리파제가 유기용매에 대해 안정했다. 고정화 matrix의 소수성이 증가될수록Vm(app)는 증가되었으나 Km(app)는 거의 일정했다. 리파제의 최적 pH는 소수성 젤인 ENTP-3000에 고정된 경우 C. rugosa와 R. arrhizus 리파제에 대해서 각각 6.0과 6.5였으나, 친수성 젤에 고정된 리파제는 짧은 시간 반응에는 pH에 크게 영향을 받지 않았으나 긴 시간 동안 반응시킬 때는 역시 pH6.0과 6.5에서 각각 C. rugosa와 R. arrhizus 리파제가 높은 양의 지방산을 분해시켰다. 리파제를 entrapment 시키면 열안정성이 증가됨을 알 수 있었다.

• 미생물학회지
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• 제30권3호
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• pp.171-186
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• 1992

Lipolytic characteristics of candia cylindracea lipase was studied by various triacylglycerols with different fatty acyl chains as substrate. The substrate was emulsified with gum arabic and the rate of hydrolysis was determined by pH stat method. The effects of gum concentration, pH, temperature, and $Ca^{2+}$ ion on the enzyme activities were examined. The results show that the effect of these factors are markedly depending on the structurla nature of substrates. The triolein was the best substrate among tested. Present study demonstrates that for characterization of lipolytic enzymes, it is critically important to select proper substrate and activator.r.

• 한국의류학회지
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• 제32권7호
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• pp.1075-1081
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• 2008

Eco-friendly processing using enzymes has been focused in textile industry in order to reduce environmental pollutions. This paper was suggested to hydrolyze non-cellulose, such as fats and waxes in cotton fabrics by lipase treatment. Enzymatic treatment conditions were controlled according to pH, temperature, enzyme concentration, and treatment time. The physical properties of the lipase-treated cotton fabrics were evaluated by measuring weight loss, moisture regain and dyeing properties. The surface morphology of lipase-treated cotton fabrics were observed by SEM. As a result, the optimum conditions for the lipase treatment were at pH 4.2, temperature 50$^{\circ}C$, concentration 50%, and treatment time 90 minutes. Calcium chloride and Triton X-100 were effective auxiliaries in lipase treatment.