• KSBB Journal
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• v.7 no.3
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• pp.172-178
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• 1992

A simple and effective method has been developed for the immobilization of lipase producing Rhizopus chinensis on polyurethane foam. In this method, the fungal cells with 1, 3 specific lipase in there inside are immobilized within the foam matrix. Four types of commercially available polyurethane foam were tested. The ultimate purpose of the process is to produce low-cost biocatalysts for lipase-catalyzed reactions, which are being increasingly used for industrial applications. Effects of several parameters were studied on the cell loading and the hydrolytic activity of intracellular lipase after acetone drying. These parameters were the type, size, and amount of polyurethane foam. In all the cases, the intracellular lipase activity obtained with the foam was approximately twice greater than that obtained in the absence of the foam.

• Journal of Microbiology and Biotechnology
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• v.24 no.5
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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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• v.28 no.5
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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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• v.26 no.6
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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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• v.18 no.12
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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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• v.27 no.11
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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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• v.18 no.6
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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.

• Microbiology and Biotechnology Letters
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• v.14 no.6
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• pp.473-478
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• 1986

In order to elucidate the patterns of natural oils and fats assimilation by microorganisms, lipases properties of yeast and bacterium strain, Torulopsis candia Y-128 and Acinetobacter calcoaceticus KB-2, which could assimilate palm oil efficiently, were investigated. T candida Y-128 attached palm oil droplets directly, and assimilated unsaturated fatty acid more easily than saturated acids liberated by the action of its lipase. Lipase of A. calcoaceticus KB-2 was extracellular and appeared quickly from the beginning of log phase of growth, whereas lipase of f candida Y-128 appealed intracellular. The lipases of two strains seem to be only enough to utilize the lipid materials for their own growth, without accumulation of lipases in the culture broth. Lipases of the strains have 1 (3-)-positional specificities on triglycerides. The patterns of palm oil assimilation showed that two strains attached droplets of lipid materials directly and split off fatty acids at 1 (3-)-position of triglycerides first, and assimilated the reaction products via fatty acids metabolic pathway.

• Journal of Genetic Medicine
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• v.2 no.2
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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.

• Korean Journal of Food Science and Technology
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• v.47 no.2
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• pp.246-253
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• 2015

In this study, we evaluated the lipoprotein lipase (LPL) inhibitory activity of 11 water extracts derived from Cinnamomum cassia Blume, Sarcodon aspratus, Cordyceps militaris, Crataegus pinnatifida Bunge, Corni fructus, Allium cepa, Coix lacryma-jobi, Plantago asiatica L., Lentinus edodes, Rosa rugosa, and Foeniculum fructus. The results of the LPL secretion and activity assay showed Sarcodon aspratus (NE) extract have an LPL secretion inhibitory acitivity. The cause of reduction in LPL secretion after NE treatment was investigated using molecular biology methods. NE treatment affected the LPL content in cells, but did not affect LPL mRNA expression. It also increased the mRNA expression level of sortilin-related receptor LDLR class A (SorLA), a receptor that induces endocytosis and intracellular trafficking of LPL. Finally, cell fractionation revealed that NE treatment induced the expression of CCAAT-enhancer-binding protein beta ($C/EBP{\beta}$), a SorLA transcription factor, in the nuclei of 3T3-L1 adipocytes. These results show that NE's anti-obesity effect involves inhibition of LPL secretion through $C/EBP{\beta}$-mediated induction of SorLA expression.