• 한국미생물·생명공학회지
• /
• 제50권1호
• /
• pp.71-80
• /
• 2022

Lipases (triacylglycerol acylhydrolases [EC 3.1.1.3]) are water-soluble enzymes. They catalyze the hydrolysis of fats and oils. A cold-active lipase from marine Bacillus cereus HSS, isolated from the Mediterranean Sea, Alexandria, Egypt, was purified and characterized. The total purification depending on lipase activity was 438.9 fold purification recording 632 U/mg protein. The molecular weight of the purified lipase was estimated to be 65 kDa using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The optimum substrate concentration, enzyme concentration, pH, and temperature were 1.5 mM, 100 µl, pH 6 and 10℃, respectively. The lipase was tolerant to NaCl concentrations ranging from 1.5 to 4.5%. The lipase was affected by the tested metal ions, and its activity was inhibited by 16% in the presence of 0.05 M SDS. The application of the cold-active lipase for the removal of an oil stain from a white cotton cloth showed that it is a promising biological agent for the treatment of oily wastes and other related applications. To the best of our knowledge, this is the first report of the purification and characterization of a lipase from marine B. cereus HSS isolated from the Mediterranean Sea.

• Journal of Microbiology
• /
• 제41권1호
• /
• pp.22-27
• /
• 2003

A lipase from Aeromonas sp. LPB 4, a psychrotophile isolated from a sea sediment was purified and characterized. The lipase was purified 53.5 fold to a homogeneous state by acetone precipitation and QAE sephadex column chromatography and its molecular weight was determined to be 50 kDa by SDS-PAGE. The enzyme exhibited maximum activity at 10$^{\circ}C$ and was stable at temperatures lower than 50$^{\circ}C$. This lipase favored substrates containing medium carbon chain of acyl group, while too low and high carbon chain decreased its activity. The lipolytic activity of purified lipase was slightly increased by the addition of 0.1% detergent, but decreased by 1% of detergent. Butanol severely decreased the lipase activity while methanol increased the activity about 15%.

• 한국생명과학회:학술대회논문집
• /
• 한국생명과학회 2007년도 제48회 학술심포지움 및 추계국제학술대회
• /
• pp.128.1-128.1
• /
• 2007

See Full Text

• Journal of Microbiology and Biotechnology
• /
• 제26권6호
• /
• pp.1067-1076
• /
• 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
• /
• 제17권4호
• /
• pp.604-610
• /
• 2007

A psychrotrophic strain 7195 showing extracellular lipolytic activity towards tributyrin was isolated from deep-sea sediment of Prydz Bay and identified as a Psychrobacter species. By screening a genomic DNA library of Psychrobacter sp. 7195, an open reading frame of 954 bp coding for a lipase gene, lipA1, was identified, cloned, and sequenced. The deduced LipA1 consisted of 317 amino acids with a molecular mass of 35,210 kDa. It had one consensus motif, G-N-S-M-G (GXSXG), containing the putative active-site serine, which was conserved in other cold-adapted lipolytic enzymes. The recombinant LipA1 was purified by column chromatography with DEAE Sepharose CL-4B, and Sephadex G-75, and preparative polyacrylamide gel electrophoresis, in sequence. The purified enzyme showed highest activity at $30^{\circ}C$, and was unstable at temperatures higher than $30^{\circ}C$, indicating that it was a typical cold-adapted enzyme. The optimal pH for activity was 9.0, and the enzyme was stable between pH 7.0-10.0 after 24h incubation at $4^{\circ}C$. The addition of $Ca^{2+}\;and\;Mg^{2+}$ enhanced the enzyme activity of LipA1, whereas the $Cd^{2+},\;Zn^{2+},\;CO^{2+},\;Fe^{3+},\;Hg^{2+},\;Fe^{2+},\;Rb^{2+}$, and EDTA strongly inhibited the activity. The LipA1 was activated by various detergents, such as Triton X-100, Tween 80, Tween 40, Span 60, Span 40, CHAPS, and SDS, and showed better resistance towards them. Substrate specificity analysis showed that there was a preference for trimyristin and p-nitrophenyl myristate $(C_{14}\;acyl\; groups)$.

• 미생물학회지
• /
• 제52권2호
• /
• pp.192-201
• /
• 2016

최근 북극해의 축치해(Chuckchi Sea)로부터 저온지질분해 효소활성을 보이는 Psychrobacter sp. ArcL13 균주가 분리되었다. 그러나 낮은 발현 양 때문에, 이 효소의 다양한 분야에서의 활용에 제약을 받아왔다. 따라서 유전자 재조합 기술을 이용하여, 이 효소를 대량생산하는 기술개발이 필요하였다. 재조합 지질분해효소를 만들기 위해서는 우선 해당 유전자의 동정이 필요하였기 때문에, Psychrobacter sp. ArcL13 균주로부터 PCR을 이용한 gene prospecting 방법으로 새로운 지질분해효소 유전자인 ArcL13-Lip을 분리하고 전체 염기 서열을 규명하였다. 염기 서열 분석결과 ArcL13-Lip은 일부 Psychrobacter 속 박테리아 유래의 지질분해효소들과 염기 서열의 유사성은 낮지만, 84-90%의 아미노산 서열 유사성을 보였다. ArcL13-Lip 전체 유전자를 대장균에서 발현시키고 전기영동으로 분석한 결과, 재조합 ArcL13-Lip은 약 35 kDa의 분자량을 보였으며 단백질 봉입체 형태로 발현되었다. Unfolding된 ArcL13-Lip을 다양한 첨가물이 포함된 완충용액에서 refolding 시킨 결과, glucose에 의해서 refolding 효율이 가장 크게 증가하였다. Refolding된 재조합 ArcL13-Lip은 다양한 p-nitrophenyl ester 중 p-nitrophenyl caprylate과 p-nitrophenyl decanoate에 대해 가장 높은 효소활성을 보였다. 온도에 따른 효소활성을 조사한 결과 ArcL13-Lip은 $40^{\circ}C$에서 최고의 활성을 나타내었고, $10^{\circ}C$와 $20^{\circ}C$에서 각각 최고 활성 대비 약 40%와 73%의 효소활성을 나타내었다. 이와 같이 ArcL13-Lip은 전형적인 저온활성 효소의 특징을 보여주었다.

• Journal of Microbiology and Biotechnology
• /
• 제24권11호
• /
• pp.1484-1489
• /
• 2014

A novel esterase gene, est01, was successfully unearthed from a biogas digester microbiota metagenomic library. The 1,194 bp est01 gene encodes a protein of 44,804 Da (designated Est01). The amino acid sequence of Est01 shows only moderate (33%) identity to a lipase/esterase. Phylogenetic analysis and biochemical characterization confirmed that Est01 is a new member of family VIII esterases. The purified Est01 from recombinant Escherichia coli BL21 (DE3) showed high hydrolytic activity against short-chain fatty acid esters, suggesting that it is a typical carboxylesterase rather than a lipase. Furthermore, the Est01 was even active at $10^{\circ}C$ (43% activity remained), with the optimal temperature at $20^{\circ}C$, and had a broad pH range from 5.0 to 10.0, with the optimal pH of 8.0. These properties suggest that Est01 is a cold-adaptive esterase and could have good potential for low-temperature hydrolysis application.

• 한국수산과학회지
• /
• 제28권2호
• /
• pp.123-130
• /
• 1995

우리나라에서 많이 양식하고 있는 주요 어종의 혈액 성분에 관한 기초 자료를 얻기 위하여, 저수온기(수온 $127^{\circ}C$)에 월동중인 조피볼락(Sebastes schlegeli), 농어 (Lateolarax japonicus), 넙치 (Paralichthys olivaceus), 돌돔(Oplegnathus fasciatus)과 함께 냉수성 어종인 은연어(Oncorhynchus kisutch)를 각 5-6마리씩 마취시키지 않은 상태로 꼬리 동맥에서 채혈하여 혈청 중의 총단백질(TP), 알부민(ALB),중성 지방(TG), 콜레스테롤(CHOL), 포도당(GLC), 리파제(LIPA), 나트륨(Na), 칼륨(K), 염소(Cl), 인(P), 아미노산전이효소(ALT, AST) 등을 혈액분석기로 분석하였다. TP 농도는 2.9-5.1g/dl의 수준이었으며, ALB 농도는 1.2-l.9g/dl의 수준이었다. A/G 비는 은연어가 1.1 정도로 가장 높았고 나머지는 0.5-0.6 정도로 비슷하였다. 지질 성분인 TG와 CHOL 농도도 어종에 따라 달라, 넙치는 TG 농도가 큰 대신에 CHOL 농도가 낮았으며, 농어는 반대로 TG농도가 낮은 대신에 CHOL 농도가 높았다. 두 지질 성분의 합은 넙치가 가장 많아 600mg/dl나 되었고, 농어나 조피볼락(약 400mg/dl), 돌돔이나 은연어(약 300mg/d1)의 순이었다. 혈당(GLC) 농도는 냉수성 어종인 은연어가 넙치에 비해 약 4배 가량 많아 61-76mg/dl이나 되었다. 한편 리파제(LIPA) 활성은 TG 농도와 정반대의 경향을 보여 은연어에서 가장 높았고, 넙치에서는 거의 확인되지 않았다. 무기질 중에서 Na, Cl과 K 농도는 160-204mmo1/l, 137-183mmol/l과 0.5-3.1mmol/l로 유사한 수준이었으나, P농도는 은연어나 조피볼락처럼 비교적 활동성이 강한 어종일수록 많았다. ALT 활성은 넙치가 가장 높았고, AST 활성은 은연어가 높았으며, AST 활성은 모든 어종에서 ALT 활성보다 높았다.