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1.
Recombinant Candida rugosa lipase 5 (LIP5) has been functionally expressed along with other isoforms in our laboratory. However, the characterization and codon optimization of LIP5 have not been done. In this work, we characterized, codon-optimized and compared LIP5 with commercial lipase. LIP5 activity on hydrolysis of p-nitrophenyl (p-NP) butyrate was optimal at 55 °C as compared with 37 °C of the commercial lipase. Several assays were also performed to determine the substrate specificity of LIP5. p-NP butyrate (C(4)), butyryl-CoA (C(4)), cholesteryl laurate (C(12)), and N-carbobenzoxy-l-tyrosine-p-nitrophenyl ester (l-NBTNPE) were found as preferred substrates of LIP5. Interestingly, LIP5 specificity on hydrolysis of amino acid-derivative substrates was shown to be the highest among any lipase isoforms, but it had very weak preference on hydrolyzing triacylglycerol substrates. LIP5 also displays a pH-dependent maximum activity of a lipase but an esterase substrate preference in general. The characterization of LIP5 along with that of LIP1-LIP4 previously identified shows that each lipase isoform has a distinct substrate preference and catalytic activity.  相似文献   

2.
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3.
Candida rugosa contains several lipase (CRLs) genes, and CRLs show diverse enzyme activity despite being highly homologous across their entire protein family. Previous studies found that LIP4 has a high esterase activity and a low lipolytic activity and lacks interfacial activation. To investigate whether the C-terminal region of the CRLs mediates enzymatic activity, chimeras were generated in which the C-terminus of LIP4 from either residue 374, 396, 417, or 444 to residue 534 was swapped with the corresponding peptide from the isoform LIP1. A chimeric lipase containing the C-terminus from 396 to 534 of LIP1 on a LIP4 scaffold showed activity similar to that of commercial CRL on triolein, and lipolytic activity increased 2-6-fold over that of LIP4. Moreover, interfacial activation was also observed in the chimeric lipase. To improve its enzymatic properties, a novel glycosylation site was added at residue 314. The new glycosylated lipase showed improved thermostability and enhancement in enzymatic activity, indicating its potential for use in further application.  相似文献   

4.
Candida rugosa lipase (CRL), an important industrial enzyme, has been established, containing several different isoforms which were encoded by the high-identity lip gene family (lip1 to lip7). In this study, we compared the expression and biochemical characterization with three different engineered lip2 constructions in the yeast Pichia pastoris. Our results showed that lip2 (lip2) has an overall improvement of 50% higher production yield (1.446 U/mL) relative to that of nflip2 (0.964 U/mL) at 7 days of cultivation time. Codon-optimized lip2 (colip2) has a 2.3-fold higher production yield (2.182 U/mL) compared to that of lip2 (noncodon-optimized; 1.446 U/mL) and nflip2 (0.964 U/mL), with a cultivation time of 5 days. This finding demonstrated that the removal of the N-terminus and the regional codon optimization of the lip2 gene fragment at the 5' end can greatly increase the expression level of recombinant LIP2 in the P. pastoris system. The distinct biochemical properties of our purified recombinant nfLIP2 and LIP2 suggested that they are potentially useful for various industrial applications.  相似文献   

5.
Candida rugosa lipase (CRL), an important industrial enzyme, possesses several different isoforms encoded by the high-identity lip gene family (lip1 to lip7). In this study, an additional N-terminal peptide in front of the lip3 gene was removed by PCR, and the 18 nonuniversal serine codons (CTG) of the lip3 gene were converted into universal serine codons (TCT) by means of an overlap extension PCR-based multiple-site-directed mutagenesis to express an active recombinant LIP3 in the yeast Pichia pastoris. The regional synthetic DNA fragment (339 bp) is first recombined by primer assembly with 20 overlapping nucleotides, followed by specific overlap extension PCR with outside primers containing restriction enzyme sites for directional cloning into the pGAPZalphaC vector. The results show that the production yield (0.687 unit/mL) of N-fused lip3 (nflip3) has an overall improvement of 69-fold relative to that (0.01 unit/mL) of lip3 and of 52-fold (0.47 unit/mL) of codon-optimized lip3 (colip3) relative to that (0.01 unit/mL) of non-codon-optimized lip3 (lip3), with the cultivation time set at 5 days. This finding demonstrates that the reservation of the N terminus and the regional codon optimization of the lip3 gene fragment at the 5' end can greatly increase the expression level of recombinant LIP3 in the P. pastoris system. The purified recombinant LIP3 shows distinct biochemical properties compared with other isoforms.  相似文献   

6.
To obtain basic information about enzymatic deterioration of buckwheat flour, triacylglycerol lipase (LIP; EC 3.1.1.3) was purified from buckwheat seed. The LIP consisted of two isozymes, LIP I and LIP II, and they were purified with purification folds of 60 and 143 with final specific activities of 0.108 and 0.727 mumol of fatty acid released per minute per milligram of protein at 30 degrees C using triolein as a substrate. Molecular weights were estimated to be 150 (LIP I) and 28.4 kDa (LIP I) by gel filtration and 171 (LIP I) and 26.5 kDa (LIP II) by SDS-PAGE. Optimal pHs of LIP activities were 3.0 (LIP I) and 6.0 (Lip II) using triolein as a substrate. Both LIP I and II reacted in the acidic pH range. Optimal temperatures were 30 (LIP I) and 40 degrees C (LIP II), and both LIP I and II were stable below 30 degrees C when p-nitrophenyl-laurate was used as a substrate. However, they were inactivated above 60 degrees C. On the other hand, when triolein was used as a substrate, optimal temperatures were 30 degrees C for both LIP I and II, and they retained 40% of their activity after a 4 h incubation of enzymes at 70 degrees C. LIP I and II had higher activity against triolein than monoolein or tri/monopalmitin. Most of the LIP activity was distributed in the embryo.  相似文献   

7.
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8.
Structured lipids (SLs) containing palmitic and oleic acids were synthesized by transesterification of tripalmitin with either oleic acid or methyl oleate as acyl donor. This SL with palmitic acid at the sn-2 position and oleic acid at sn-1,3 positions is similar in structure to human milk fat triacylglycerol. LIP1, an isoform of Candida rugosa lipase (CRL), was used as biocatalyst. The effects of reaction temperature, substrate molar ratio, and time on incorporation of oleic acid were investigated. Reaction time and temperature were set at 6, 12, and 24 h, and 35, 45, and 55 degrees C, respectively. Substrate molar ratio was varied from 1:1 to 1:4. The highest incorporation of oleic acid (37.7%) was at 45 degrees C with methyl oleate as acyl donor. Oleic acid resulted in slightly lesser (26.3%) incorporation. Generally, higher percentage incorporation of oleic acid was observed with methyl oleate (transesterification) than with oleic acid (acidolysis). In both cases percentage incorporation increased with reaction time. Incorporation decreased with increase in temperature above 45 degrees C. Initially, oleic acid incorporation increased with increase in substrate molar ratio up to 1:3. LIP1 was also compared with Lipozyme RM IM as biocatalysts. The tested reaction parameters were selected on the basis of maximum incorporation of C18:1 obtained during optimization of LIP1 reaction conditions. Reaction temperature was maintained at 45, 55, and 65 degrees C. Lipozyme RM IM gave highest oleic acid incorporation (49.4%) at 65 degrees C with methyl oleate as acyl donor. Statistically significant (P < 0.05) differences were observed for both enzymes. SL prepared using Lipozyme RM IM may be more suitable for possible use in human milk fat substitutes.  相似文献   

9.
The esterification and hydrolytic activities of free and immobilized Candida rugosa lipase isoform 1 (LIP1) were investigated. Esterification activity was determined by reacting caprylic acid with glycerol in the presence of molecular sieves (30%, w/w), and the volume of 1.0 M NaOH consumed by the reaction products upon titration was used to calculate esterification activity. Caprylic acid was also reacted with cottonseed oil, and the amount of caprylic acid incorporated after 12 h of reaction was determined. Results indicated that LIP1 had little esterification activity, which was not significantly improved upon immobilization. Hydrolytic activity was determined by incubating tricaprylin emulsion (15%, w/w) with the respective lipases for 60 min, and the reaction products were titrated against 0.5 M NaOH. LIP1 showed hydrolytic activity comparable to Lipozyme RM IM. The hydrolytic activity improved significantly upon immobilization. Immobilization on Celite 545 produced the highest increase in hydrolytic activity.  相似文献   

10.
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11.
Rice bran has been reported to inhibit pancreatic lipase activity in vitro. This action shows that administration of rice bran may result in a decrease in plasma triglyceride levels and suppress accumulation of fat in vivo. We administered water extract of defatted rice bran (WED-rice bran) to rats to determine its effects. Single administration of WED-rice bran at a dose of 1 g/kg body weight caused a decrease in plasma triglyceride levels in fat emulsion induced hypertriglyceridemic rats. Four week administration of WED-rice bran suppressed accumulation of visceral fat and body weight gain without influencing food consumption, liver function, and renal function. These results indicate that a reduction of plasma triglycerides and suppression of visceral fat accumulation may be induced by pancreatic lipase inhibition caused by administration of WED-rice bran.  相似文献   

12.
A cDNA fragment of the Anman5A, a gene that encodes an acidophilic β-mannanase of Aspergillus niger LW-1 (abbreviated as AnMan5A), was cloned and functionally expressed in Pichia pastoris . Homology alignment of amino acid sequences verified that the AnMan5A belongs to the glycoside hydrolase (GH) family 5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay demonstrated that the recombinant AnMan5A (reAnMan5A), a N-glycosylated protein with an apparent molecular weight of 52.0 kDa, was secreted into the medium. The highest reAnMan5A activity expressed by one P. pastoris transformant, labeled as GSAnMan4-12, reached 29.0 units/mL. The purified reAnMan5A displayed the highest activity at pH 3.5 and 70 °C. It was stable at a pH range of 3.0-7.0 and at a temperature of 60 °C or below. Its activity was not significantly affected by an array of metal ions and ethylenediaminetetraacetic acid (EDTA). The K(m) and V(max) of the reAnMan5A, toward locust bean gum, were 1.10 mg/mL and 266.7 units/mg, respectively.  相似文献   

13.
A surfactant-coated lipase (SCL) prepared by mixing Candida rugosa lipase with emulsifier in ethanol was used to hydrolyze tuna oil in a two-phase aqueous-organic system. Both enzyme (SCL) and substrate (tuna oil) were soluble in the organic phase, and the hydrolysis could occur with water molecules from the aqueous phase. This hydrolysis could promptly proceed compared to that catalyzed by native lipases which only occurred at the interface between the two phases. Michaelis-Menten kinetics in the two-phase reactions showed that the K(m) value of the SCL was half that of the native lipase, while the maximum velocity (V(max)) was 11.5 times higher. The hydrolysis method resulted in enrichment of n-3 polyunsaturated fatty acid (n-3 PUFA) content in glyceride mixtures from 26.4% to 49.8% and DHA from 19.1% to 38.9%. The SCL acted as an efficient hydrolytic catalyst for tuna oil.  相似文献   

14.
A colorimetric method was established to determine the activity of recombinant lipase in extracts from transgenic corn seed. The system was an oil-in-water emulsion that was stabilized by a surfactant to accommodate the organic phase substrate and aqueous phase enzyme. The lipase activity was measured by monitoring the release of nitrophenol at 346 nm from the substrate, 4-nitrophenyl butyrate. Emulsions prepared with various surfactant types and concentrations were tested. For each surfactant, the measured activity was greatest when the surfactant concentration was close to the critical micelle concentration, consistent with the changing trend of oil droplet size as a function of surfactant concentration. The optimal system, with 0.01% (w/w) Tween 80, demonstrated good reproducibility, high sensitivity, robustness, and a linear response to lipase concentration.  相似文献   

15.
Experimental rats with hypertriglyceridemia were prepared by feeding a high-fructose diet. Dried Anka powder (2%), a rice product fermented with Monascus sp., was mixed with basic high-fructose (30%) or basal-diet feed. Serum and liver lipids were measured after 6 months. The concentrations of serum triglycerides, total cholesterol, VLDL-C, and LDL-C had significantly decreased, whereas that of HDL-C had slightly increased in 30% fructose-Anka-fed rats as compared with the 30% fructose-fed rats, but hepatic lipase activity had increased in the Anka-fed groups. The ratio of lipoprotein lipase/hepatic lipase was not significantly different between 30% fructose-Anka-fed rats and 30% fructose-fed rats. The dietary intake and weight of these two groups were approximately the same. Similar results were obtained in noninduced hypertriglyceridemic rats. The concentrations of triglycerides and cholesterol did not significantly differ in the liver. Interestingly, Anka can suppress serum triglycerides in rats with induced hypertriglyceridemia. The antioxidant enzyme SOD activity was also measured in serum, and no significant change was observed. On the basis of these findings, we suggest that Anka may be used to suppress hypertriglyceridemia and hyperlipidemia in rats and possibly in man.  相似文献   

16.
Aspergillus nidulans WG312 strain secreted lipase activity when cultured in liquid media with olive oil as carbon source. Highest lipase productivity was found when the mycelium was grown at 30 degrees C in a rich medium. The new enzyme was purified to homogeneity from the extracellular culture of A. nidulans by phenyl-Sepharose chromatography and affinity binding on linolenic acid-agarose. The lipase was monomeric with an apparent M(r) of 29 kDa and a pI of 4.85 and showed no glycosylation. Kinetic of enzyme activity versus substrate concentration showed a typical lipase behavior, with K(M) and K(cat) values of 0.28 mM and 494 s(-)(1) and 0.30 mM and 320 s(-)(1) for the isotropic solution and for the turbid emulsion, respectively. All glycerides assayed were hydrolyzed efficiently by the enzyme, but this showed preference toward esters of short- and middle-chain fatty acids. The optimum temperature and pH for the lipolytic activity were 40 degrees C and 6.5, with high activity in the range 0-20 degrees C and reduced thermal stability.  相似文献   

17.
The objective of the present study was to purify and characterize the lipoxygenase (LOX) from banana leaf (Giant Cavendishii, AAA), an unutilized bioresource. LOX was extracted, isolated, and purified 327-fold using 25-50% saturation of ammonium sulfate fractionation, hydroxyapatite column separation, and gel filtration on Superdex 200. The molecular mass of the purified LOX was 85 kDa, K(m) was 0.15 mM, and V(max) was 2.4 microM/min.mg using linoleic acid as substrate. Triton X-100 was required in the extraction medium; otherwise, no LOX activity was detected. LOX activity increased with the concentration of Triton X-100 with an optimum at 0.1%. The optimal pH of the purified LOX from banana leaf was 6.2, and optimal temperature was 40 degrees C. The LOX showed the highest reactivity toward 18:2 followed by 18:3 and 20:4. A very low reaction rate was observed toward 20:5 and 22:6. On the basis of retention time in normal phase HPLC, the products of 18:2 or 18:3 catalyzed by purified LOX were hydroperoxyoctadecadienoic acid or hydroperoxyoctadecatrienoic acid. It seems that 9-LOX is the predominant enzyme in banana leaf. Banada leaf dried at 110 degrees C for 2 h developed algal aroma. Banana leaf extract stored at 10 degrees C for 12 h formed an oolong tea-like flavor. Banana leaf extract reacted with 18:2 or soybean oil pretreated with bacterial lipase produced green and melon-like aroma, whereas the same reaction with 18:3 produced a sweet, fruity, cucumber-like flavor note.  相似文献   

18.
The enantioselectivity of the generation of 3-mercaptohexanal and 3-mercaptohexanol, two potent sulfur-containing aroma compounds, by lipase-catalyzed hydrolysis of the corresponding 3-acetylthioesters was investigated. The stereochemical course of the kinetic resolutions was followed by capillary gas chromatography using modified cyclodextrins as chiral stationary phases. The enzyme preparations tested varied significantly in terms of activity and enantioselectivity (E). The highest E value (E = 36) was observed for the hydrolysis of 3-acetylthiohexanal catalyzed by lipase B from Candida antarctica resulting in (S)-configured thiol products. Immobilization of the enzyme (E = 85) and the use of tert-butyl alcohol as cosolvent (E = 49) improved the enantioselectivity. Modification of the acyl moiety of the substrate (3-benzoylthiohexanal) had no significant impact. The sulfur-containing compounds investigated possess attractive odor properties, and only one of the enantiomers exhibits the pleasant citrus type note.  相似文献   

19.
The formation of chloropropanols was investigated using model systems comprised of lipase, vegetable oil or fat, water, and sodium chloride. The results showed that measurable levels of the foodborne carcinogen 3-chloro-1,2-propanediol (3-MCPD) are formed in the presence of commercially available lipases of mammalian, vegetable, and fungal origins, incubated at temperatures of 40 degrees C. The highest yield of 3-MCPD was obtained in reaction mixtures containing lipase from Rhizopus oryzae, and all the lipases studied exhibited a high hydrolytic activity toward triglycerides from palm and peanut oil. In contrast, hydrolysis over time and the yield of 3-MCPD in olive and sunflower oils were significantly lower (up to 10-fold), possibly linked to the relatively lower amount (<18%) of saturated fatty acids in these oils. We provide here for the first time evidence that lipases are able to induce the formation of chloropropanols under model system conditions. However, the key intermediates and precise mechanistic aspects governing the formation of 3-MCPD in the presence of lipase still need to be elucidated.  相似文献   

20.
An on-line supercritical fluid extraction (SFE)/enzymatic hydrolysis procedure using immobilized lipase has been developed for the determination of vitamin A in dairy and meat products. Several lipases were tried, of which Novozyme 435 (Candida antarctica type B) showed the highest activity toward retinyl palmitate. There was no observed activity with alpha-tocopheryl acetate. When pressure, temperature, modifiers, flow rate, extraction time, and water content were varied, high vitamin A recovery was obtained in milk powder. Collected extracts were analyzed by reversed-phase high-performance liquid chromatography with ultraviolet and fluorescence detection without additional sample cleanup. The procedure gave reliable values of vitamin A as well as of vitamin E in other food items such as infant formula, minced pork and beef meat, and low- and high-fat liver paste. The described method is faster and more automated than conventional methods based on liquid-liquid extraction, or SFE using off-line saponification, for vitamin A and E determination. Results obtained with the new method did not differ significantly from those obtained with the other two methods mentioned above.  相似文献   

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