• KSBB Journal
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• 제7권3호
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• pp.172-178
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• 1992

담체로 사용된 polyurethane foam은 Rizopus chinensis의 균사가 부착하여 안정하게 증식할 수 있게 하였다. 고정화를 위해 사용된 네 종류의 polyurethane foam중 GP-160이 고정하ㅗ 매체로 우수한 성질을 보였고, 입자의 크기는 7-8mm가 적당하였다. Rizopus chinensis의 현탁 배양과 polyurethane foam에서의 고정화 배양을 비교할 때, 전체 리파아제의 활성도는 큰 변화가 없었지만, 고정화 배양의 경우 extracellualar lipase의 생성을 억제하여 intracellular lipase의 활성도를 현탁 배양의 경우보다 약 2배가량 높일 수가 있었다. 고정화 세포의 열안정성을 조사하기 위하여 35~$50^{\circ}C$사이에서 열에의한 비활성화 에너지값을 구해본 결과, 그 값이 28.7kcal/mol로서 본 연구에서 제조된 고정화 세포의 생촉매가 배교적 좋은 열안정성을 갖고 있다.

• Journal of Microbiology and Biotechnology
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• 제24권5호
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• pp.619-628
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• 2014

To identify lipase LipA (PFL_0617) from Pseudomonas protegens Pf-5, a lipA deletion mutant (Pf0617) and a complementary strain (Pf0617lipA) were constructed, and their effects on the lipase production were examined. Pf0617 remarkably decreased its whole-cell lipase activity, whereas Pf0617lipA made its whole-cell lipase activity not only restore to wild-type level but also get a further increment. However, the deletion and overexpression of lipA did not affect the extracellular lipase activity. In addition, the unbroken whole cells of these strains were able to catalyze the hydrolysis of membrane-permeable p-nitrophenyl esters, but could not hydrolyze the membrane-impermeable olive oil. These results confirmed that LipA was an intracellular lipase and Pf-5 could also be used as a natural whole-cell biocatalyst. To evaluate the potential of Pf-5 as a whole-cell biocatalyst and separately characterize the whole-cell LipA, the properties of the whole-cell lipases from Pf-5 and Top10lipA were characterized. The results demonstrated that both Pf-5 and Top10lipA exhibited high tolerance to alkaline condition, high temperature, heavy metal ions, surfactants, and organic solvents. Taken together, lipA can realize functional expression in E. coli Top10, and Pf-5 and Top10lipA as whole-cell biocatalysts may have enormous potential in applications.

• Journal of Microbiology and Biotechnology
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• 제28권5호
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• pp.739-747
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• 2018

The low solubility and high-cost recovery of Propionibacterium acnes polyunsaturated fatty acid isomerase (PAI) are key problems in the bioproduction of high value-added conjugated linoleic acid (CLA). To improve the solubility of recombinant PAI, six chaperone proteins were coexpressed with PAI. Introduction of GroELS proteins dramatically improved the PAI solubility from 29% to 97%, with increased activity by 57.8%. Combined expression of DnaKJ-GrpE and GroELS proteins increased the activity by 11.9%. In contrast, coexpression of DnaKJ-GrpE proteins significantly reduced the activity by 57.4%. Plasmids pTf16 harboring the tig gene and pG-Tf2 containing the tig and groEL-groES genes had no visible impact on PAI expression. The lytic protein E was then introduced into the recombinant Escherichia coli to develop a cell autolysis system. A 35% activity of total intracellular PAI was released from the cytoplasm by suspending the lysed cells in distilled water. The PAI recovery was further improved to 81% by optimizing the release conditions. The lipase from Rhizopus oryzae was also expressed in E. coli, with an extracellular activity of 110.9 U/ml. By using the free PAI and lipase as catalysts, a joint system was established for producing CLA from sunflower oil. Under the optimized conditions, the maximum titer of t-10,c-12-CLA reached 9.4 g/l. This work provides an effective and low-cost strategy to improve the solubility and recovery of the recombinant intracellular PAI for further large-scale production of CLA.

• Journal of Microbiology and Biotechnology
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• 제26권6호
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• pp.1087-1097
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• 2016

An intracellular lipase from Anoxybacillus flavithermus HBB 134 was purified to 7.4-fold. The molecular mass of the enzyme was found to be about 64 kDa. The maximum activity of the enzyme was at pH 9.0 and 50℃. The enzyme was stable between pH 6.0 and 11.0 at 25℃, 40℃, and 50℃ for 24 h. The K_m and V_max of the enzyme for pNPL substrate were determined as 0.084 mM and 500 U/mg, respectively. Glycerol, sorbitol, and mannitol enhanced the enzyme thermostability. The enzyme was found to be highly stable against acetone, ethyl acetate, and diethyl ether. The presence of PMSF, NBS, DTT and β-mercaptoethanol inhibited the enzyme activity. Hg²⁺, Fe³⁺, Pb²⁺, Al³⁺, and Zn²⁺ strongly inhibited the enzyme whereas Li⁺, Na⁺, K⁺, and NH₄⁺ slightly activated it. At least 60% of the enzyme activity and stability were retained against sodium deoxycholate, sodium taurocholate, n-octyl-β-D-glucopyranoside, and CHAPS. The presence of 1% Triton X-100 caused about 34% increase in the enzyme activity. The enzyme is thought to be a true lipase since it has preferred the long-chain triacylglycerols. The lipase of HBB 134 cleaved triolein at the 1- or 3-position.

• Journal of Microbiology and Biotechnology
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• 제18권12호
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• pp.1908-1914
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• 2008

An alkaliphilic bacterium, Bacillus clausii SKAL-16, was isolated from soil that had been contaminated with vegetable oil. The optimal pH and general pH range for bacterial growth was 8, and 7 to 10, respectively. The bacterium could grow on tributyrin and glycerol, but could not grow on acetate and butyrate. The SKAL-16 strain excreted butyric acid during growth on tributyrin, and selectively ingested glycerol during growth on a mixture of butyric acid and glycerol. The SKAL-16 generated intracellular lipase, but did not produce esterase and extracellular lipase. The DNA fragment amplified with the chromosomal DNA of SKAL-16 and primers designed on the basis of the esterase-coding gene of Bacillus clausii KSM-KI6 was not identical with the esterase-coding gene contained in the GenBank database. Pyruvate dehydrogenase, isocitrate dehydrogenase, and malate dehydrogenase activities were detected in the cell-free extract (crude enzyme).

• Journal of Microbiology and Biotechnology
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• 제27권11호
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• pp.1907-1915
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• 2017

Lipases are important enzymes with biotechnological applications in dairy, detergent, food, fine chemicals, and pharmaceutical industries. Specifically, hormone-sensitive lipase (HSL) is an intracellular lipase that can be stimulated by several hormones, such as catecholamine, glucagon, and adrenocorticotropic hormone. Bacterial hormone-sensitive lipases (bHSLs), which are homologous to the C-terminal domain of HSL, have ${\alpha}/{\beta}-hydrolase$ fold with a catalytic triad composed of His, Asp, and Ser. These bHSLs could be used for a wide variety of industrial applications because of their high activity, broad substrate specificity, and remarkable stability. In this review, the relationships among HSLs, the microbiological origins, the crystal structures, and the biotechnological properties of bHSLs are summarized.

• Food Science and Biotechnology
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• 제18권6호
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• pp.1500-1504
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• 2009

Effects of methanol extract from turmeric (Curcuma longa L.) (CME) on underlying mechanisms of lipolysis were investigated in 3T3-L1 adipocytes. Compared to the control, lipid accumulation with 72 hr treatment of CME at the concentration $20\;{\mu}g/mL$ was significantly decreased by 19.9% as quantified by Oil red O dye. Intracellular triglyceride (TG) content was also lowered by 19.3%. To determine the mechanism for TG content reduction, glycerol release level was measured. Incubation of 3T3-L1 adipocytes with 15 and $20\;{\mu}g/mL$ of CME significantly elevated the level of free glycerol released into the cultured medium by 20.4 and 28.6%, respectively. In subsequent measurements using quantitative real-time polymerase chain reaction (PCR), mRNA levels of hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) were significantly increased by 21.2 and 24.9%, respectively, at the concentration $20\;{\mu}g/mL$. Results indicated that CME stimulated lipolysis through induction of HSL and ATGL mRNA expressions, resulting in increased glycerol release.

• 한국미생물·생명공학회지
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• 제14권6호
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• pp.473-478
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• 1986

미생물에 의한 천연유지의 분해자화과정을 극명함으로써 값싼 유지를 발효원으로서 착용하기 위하여 팜유자화성 유용균주인 Torulopsis candida Y-128과 Acinetobacter calcoaceticus KB-2가 생산하는 리파제의 특성과 이들 균주의 생리적인 특성을 검토하였다. T. candida Y-128은 팜유입자에 부착·자화하며 리파제의 작용에 의해 유리되는 분포화지방산을 포화지방산에 비해 쉽게 자화이용 함으로써 균체증식이 이루어지고 있었다. T. candida Y-128의 리파제는 대부분 균체내에 존재하는데 비해, A calcoaceticus KB-2는 배양시에 균체증식 대수기부터 균체외로 리파제가 생성됨을 알수 있었으며, 리파제에 의해 유리된 포화 지방산도 다른 균주에 비해 자화이용이 용이함을 알 수 있었다. 두 균주는 배양액중에 리파제를 축적하지 않고 균체생육에 필요한 정도를 생산하며 천연중지중 1(3-)-위치의 지방산에 작용하는 위치특이성을 나타내었다. 따라서 두 균주는 천연유지입자에 부착하여 1(3-)- 위치의 지방산을 분해하고, 분해생성물은 지방산대사경로를 거쳐 자화이용되는 것으로 보여진다.

• Journal of Genetic Medicine
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• 제2권2호
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• pp.65-70
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• 1998

Lysosomal acid lipase (LAL) plays a central role in the intracellular degradation of neutral lipids derived from plasma lipoproteins. In this study, we investigated the missense mutation within exon 2 of human LAL gene changing of codon -6 of prepeptide from threonine to proline. The Thr-6Pro mutation was detected by the HaeIII restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphism (SSCP). We analyzed the mutation in subjects with 221 unrelated randomly selected control samples and 86 patients with familial hypercholesterolemia (FH) in Korea. We observed that mutation is present with high frequency in Korea compared to other populations studied previously. The frequency of PP homozygote in the FH group was observed considerably higher than that of control. However, there was no significant difference of genotype frequency between two groups. These results, together with the fact that plasma lipids and lipoproteins levels between genotypes showed no statistical difference, suggest that the Thr-6Pro mutation in the LAL gene may have no association with the increased risk of FH development.

• 한국식품과학회지
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• 제47권2호
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• pp.246-253
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• 2015

본 연구에는 항비만소재로 연구되어진 11종의 소재를 대상으로 lipoprotein lipase (LPL)의 억제효능을 확인하고자 배양배지내 LPL의 함량과 LPL 효소활성을 측정하였다. 그 결과 3T3-L1 adipocyte에서 LPL의 분비를 억제하는 소재로 능이추출물(NE)을 선택할 수 있었다. 선택된 NE의 폴리페놀과 플라보노이드 함량을 측정한 결과 $16.61{\pm}0.44mg/g$과 $6.58{\pm}0.01mg/g$이 각각 확인되었다. NE의 LPL 분비억제기작을 확인하기위해 먼저 세포내 LPL단백질의 함량과 mRNA 발현을 확인하였다. 그 결과 함량이 감소했던 배양배지와는 다르게 NE를 처리한 3T3-L1 adipocyte의 세포내 LPL은 유의하게 증가한 것을 확인할 수 있었으며 mRNA의 발현에는 영향이 없음을 관찰할 수 있었다. 이를 바탕으로 생성된 LPL 단백질의 exocytosis에 문제가 발생했을 것으로 유추하고 다양한 단백질 이동 관련 유전자의 발현을 확인하였다. 그 결과 LPL의 이동과 분해에 관여하여 세포내 LPL의 활성을 조절하는 것으로 알려진 SorLA의 발현이 증가하는 것을 확인하고 이를 조절하는 transcription factor의 발현과 nuclear로의 이동에 NE가 미치는 영향을 검토하였다. 그 결과 NE를 처리함으로써 SorLA promoter에 작용하는 $C/EBP{\beta}$의 단백질 발현이 nuclear에서 증가하는 것을 확인할 수 있었다. 본 연구를 통해 NE가 SorLA 유전자의 transcription factor인 $C/EBP{\beta}$의 단백질 발현을 nuclear에서 증가시킴으로서 결과적으로 LPL의 분비억제가 가능함을 확인할 수 있었으며 이는 NE의 항비만 효과기전을 설명하는 기초자료를 제공하는 것이라 사료된다.