• Biotechnology and Bioprocess Engineering:BBE
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• 제9권6호
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• pp.465-470
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• 2004

Optimal conditions for the in situ immobilization of lipase in aldehyde-silica packed columns, via reductive amination, were investigated. A reactant mixture, containing lipase and sodium borohydride (NaCBH), was recirculated through an aldehyde-silica packed column, such that the covalent bonding of the lipase, via amination between the amine group of the enzyme and the aldehyde terminal of the silica, and the reduction of the resulting imine group by NaCBH, could occur inside the bed, in situ. Mobile phase conditions in the ranges of pH $7.0{\~}7.8$, temperatures between $22{\~}28^{circ}C$ and flow rates from $0.8{\~}1.5\;BV/min$ were found to be optimal for the in situ immobilization, which routinely resulted in an immobilization of more than 70 mg­lipase/g-silica. Also, the optimal ratio and concentration for feed reactants in the in situ immobilization: mass ratio [NaCBH]/[lipase] of 0.3, at NaCBH and lipase concentrations of 0.75 and 2.5 g/L, respectively, were found to display the best immobilization characteristics for concentrations of up to 80 mg-lipase/g-silica, which was more than a 2-fold increase in immobilization compared to that obtained by batch immobilization. For tributyrin hydrolysis, the in situ immobilized lipase displayed lower activity per unit mass of enzyme than the batch-immobilized or free lipase, while allowing more than a $45\%$ increase in lipase activity per unit mass of silica compared to batch immobilization, because the quantity of the immobilization on silica was aug­mented by the in situ immobilization methodology used in this study.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.951-956
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• 2001

A 28-kDa extracellular lipase (pI 8.7) was purified to homogeneity from the culture supernatant of Trichosporon sp. Y- 11 by mmonium sulfate precipitation, DEAE-Sephadex A-50, Bio-Gel P-30, CM- Sephadex C-50, and Bio-Gel P- 10 chromatographies. The purified enzyme exhibited a specific activity of $2,741{\;}{\mu}mol/min/mg$ based on the hydrolysis of triolein, and the optimal hydrolysis activity was dentified at pH 8.0 and $40^{\circ}C$. The enzyme activity was inhibited by $Ag^+$ and enhanced by $Fe^{2+}$, $Fe^{3+}$, $Mg^{2+}$, $Mn^{2+}$, and $Li^{+}$. The enzyme activity exhibited for the hydrolysis of both tributyrin and trilinolein. The ester synthesis of unsaturated fatty acids with various alcohols catalyzed by the purified lipase in a nonaqueous medium or microaqueous system was also investigated. The esterification activity of the lipase increased with an increase of the carbon chain length in the alcohol. The synthesis rate of linoleic acid and oleyl alcohol was the highest with an optimal temperature and pH of $40^{\circ}C$ and 8.0, respectively. The water content and agitation also affected the esterification activity of the lipase.

• BMB Reports
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• 제40권4호
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• pp.588-594
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• 2007

A novel hot spring thermophile, Anoxybacillus gonensis A4 (A. gonensis A4) was investigated in terms of capability of tributyrin degradation and characterization of its thermostable esterase activity by the hydrolysis of p-nitrophenyl butyrate (PNPB). It was observed that A. gonensis A4 has an esterase with a molecular weight of 62 kDa. The extracellular crude preparation was characterized in terms of substrate specificity, pH and temperature optima and stability, kinetic parameters and inhibition/activation behaviour towards some chemicals and metal ions. Tributyrin agar assay showed that A. gonensis A4 secreted an esterase and $V_{max}$ and $K_m$ values of its activity were found to be 800 U/L and 176.5 ${\mu}M$, respectively in the presence of PNPB substrate. The optimum temperature and pH, for A. gonensis A4 esterase was $60-80^{\circ}C$ and 5.5, respectively. Although the enzyme activity was not significantly changed by incubating crude extract solution at $30-70^{\circ}C$ for 1 h, the enzyme activity was fully lost at $80^{\circ}C$ for same incubation period. The pH-stability profile showed that original crude esterase activity increased nearly 2-fold at pH 6.0. The effect of some chemicals on crude esterase activity indicated that A. gonensis A4 produce an esterase having serine residue in active site and -SH groups were essential for its activity.

• 한국미생물·생명공학회지
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• 제33권1호
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• pp.29-34
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• 2005

새만금 갯벌로부터 리파제를 생산하는 균주(S3)를 분리하였다. 생리적, 발효적 특성 및 계통분류학적 특성을 통해서 이 분리균이 Psychrobacter속에 속하는 것으로 판명되어서 Psychrobacter sp. S3로 명명하였다 이 균의 온도에 따른 배양특성을 구한 결과, $30^{\circ}C$에서 생장속도가 가장 빨랐으나, 리파제 효소의 활성은 $20^{\circ}C$에서 가장 높았다. S3리파제의 온도에 따른 p-nitrophenyl caproate 분해활성을 측정한 결과, 최적 온도가 $30^{\circ}C$로 판명되었으며 $10^{\circ}C$에서도 최고활성의 $80\%$ 이상의 활성을 유지하였다. 또한, $10-30^{\circ}C$범위에서의 효소활성에너지가 1.5 kcal/mol로 매우 낮게 계산되었다. 이것을 통해 S3 리파제가 전형적인 저온성 효소임이 확인되었다. 이 효소는 최적 pH가 $9.0\~9.5$인 알칼리성 효소로 확인되었다. 여러 길이의 트리글리세리드 기질을 분해할 수 있으며 그 중에서 $C_4,\;C_{14},\; C_{16}$기질을 가장 빠르게 분해하였다. S3리파제를 트리뷰티린-아가로스 젤에 가하여 온도별로 반응시킨 결과, $30^{\circ}C$와 $40^{\circ}C$에서 반응이 빠르게 진행되었으나, $4^{\circ}C$에서도 분해가 진행되었다.

• Journal of Microbiology and Biotechnology
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• 제17권10호
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• pp.1655-1660
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• 2007

A metagenome is a unique resource to search for novel microbial enzymes from the unculturable microorganisms in soil. A forest soil metagenomic library using a fosmid and soil microbial DNA from Gwangneung forest, Korea, was constructed in Escherichia coli and screened to select lipolytic genes. A total of seven unique lipolytic clones were selected by screening of the 31,000-member forest soil metagenome library based on tributyrin hydrolysis. The ORFs for lipolytic activity were subcloned in a high copy number plasmid by screening the secondary shortgun libraries from the seven clones. Since the lipolytic enzymes were well secreted in E. coli into the culture broth, the lipolytic activity of the subclones was confirmed by the hydrolysis of p-nitrophenyl butyrate using culture supernatant. Deduced amino acid sequence analysis of the identified ORFs for lipolytic activity revealed that 4 genes encode hormone-sensitive lipase (HSL) in lipase family IV. Phylogenetic analysis indicated that 4 proteins were clustered with HSL in the database and other metagenomic HSLs. The other 2 genes and 1 gene encode non-heme peroxidase-like enzymes of lipase family V and a GDSL family esterase/lipase in family II, respectively. The gene for the GDSL enzyme is the first description of the enzyme from metagenomic screening.

• 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.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권4호
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• pp.367-371
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• 2005

About 3,000 bacterial colonies with esterase activities were isolated from soil samples by enrichment culture and halo-size on Luria broth-tributyrin (LT) plates. The colonies were assayed for esterase activity in microtiter plates using enantiomerically pure (R)- and (S)-2-phenylbutyric acid resorufin ester (2PB-O-res) as substrates. Two enantioselective strains (JH2 and JH13) were selected by the ratio of initial rate of hydrolysis of enantiomerically pure (R)- and (S)-2-PB-O-res. When cell pellets were used, both strains showed high apparent enantioselectivity ($E_{app}>100$) for (R)-2PB-O-res and were identified as Exiguobacterium acetylicum. The JH13 strain showed high esterase activity on p-nitrophenyl acetate (pNPA), but showed low lipase activity on p-nitrophenyl palmitate (pNPP). The esterase was located in the soluble fraction of the cell extract. The crude intracellular enzyme preparation was stable at a pH range from 6.0 to 11.0.

• Journal of Microbiology and Biotechnology
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• 제15권5호
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• pp.1067-1072
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• 2005

A metagenomic library was constructed using a fosmid vector, and total genomic DNA was extracted directly from soil at Cisolok (hot spring area, Indonesia). This library was composed of 10,214 clones and screened for lipolytic enzyme on tributyrin agar plates. An esterase gene (estMa) was subcloned and sequenced from a positive lipolytic active clone. Esterase EstMa was encoded by a 954-bp open reading frame and showed low ($11-33\%$) amino acid similarity to known esterases. The amino acid sequence analysis demonstrated that the enzyme is a new member of lipolytic enzyme family VI. The estMa gene encodes a preprotein of 317 amino acids with a predicted molecular mass of 34,799 Da. The purified enzyme exhibited optimal activity at $50^{\circ}C$ and pH 6.5. The $K_m,\;and\;V_{max}$ values of EstMa for the hydrolysis of p-nitrophenyl valerate were $45.3\;{\mu}M$ and 4.45 U/mg, respectively.