We found a novel lipase gene in the Paenibacillus pasadenensis CS0611 strain.The lipase gene sequence was cloned into the pET-28a expression vector to construct a recombinant lipase protein containing 6×His tags ...We found a novel lipase gene in the Paenibacillus pasadenensis CS0611 strain.The lipase gene sequence was cloned into the pET-28a expression vector to construct a recombinant lipase protein containing 6×His tags at the C-and N-termini,respectively.High-level expression of the lipase in E.coli BL21(DE3)was obtained upon induction with IPTG at 20°C.The recombinant lipase activity was approximately 1631-fold higher than the wild type.His-tagged recombinant lipase was purified rapidly and efficiently by using Ni-charged affinity chromatography with 63.5%recovery and a purification factor of 10.78.The purified lipase was stable in a broad range of temperatures and pH values,with the optimal temperature and pH being 50°C and 7.0,respectively.Its activity was stimulated to different degrees in the presence of metal ions such as Ca2+,Mg2+,and some non-ionic surfactants.In addition,the purified lipase was activated by a series of water-miscible organic solvents such as some short carbon chain alcohols and was highly tolerant to some water-immiscible organic solvents.展开更多
The stability of Candida rugosa lipase coated with glutamic acid didodecyl ester ribitol amide was investigated taking esterification of lauryl alcohol and lauric acid in isooctane as a model reaction. At 30C, the hal...The stability of Candida rugosa lipase coated with glutamic acid didodecyl ester ribitol amide was investigated taking esterification of lauryl alcohol and lauric acid in isooctane as a model reaction. At 30C, the half-life of the activity of the coated lipase was ca 10 h, the enzyme activity became less changed after 12 h and the residual activity was 39% of the initial value. The coated lipase obeyed a first-order deactivation model with a deactivation energy of 29.9J.mol-1.展开更多
The kinetics of Candida rugosa lipase catalyzed esterification of racemic ibuprofen with n-butanol in isooctane was studied. The kinetic study was carried out with the addition of 0.1% and 2% (by volume) of water for ...The kinetics of Candida rugosa lipase catalyzed esterification of racemic ibuprofen with n-butanol in isooctane was studied. The kinetic study was carried out with the addition of 0.1% and 2% (by volume) of water for enzyme activation respectively when celite was added into isooctane for enzyme dispersion. The specific initial rate for S-ibuprofen can be fitted with the Ping Pong Bi Bi mechanism with dead-end competitive inhibition by the alcohol. The time courses of the enantioselective esterification of the two ibuprofen enantiomers with different initial substrate concentrations and water contents were simulated with a model in which both effects of enzyme inactivation by long term reaction and reversed hydrolytic reaction under high water content were taken into consideration.展开更多
Optically active form of α-cyano-3-phenoxybenzyl (CPB) alcohol, building block of pyrethroid insecticides, was synthesized as its acetate by the combination of anion-exchange resin (D301)-catalyzed transcyanation bet...Optically active form of α-cyano-3-phenoxybenzyl (CPB) alcohol, building block of pyrethroid insecticides, was synthesized as its acetate by the combination of anion-exchange resin (D301)-catalyzed transcyanation between m-phenoxybenzaldehyde (m-PBA) and acetone cyanohydrin (AC), and lipase (from Alcaligenes sp.)-catalyzed enantioselective transesterification of the resulting cyanohydrin with vinyl acetate. Through optimizing technological conditions, the catalyzing efficiency was improved considerably compared to methods previously reported. Concentrations of CPB acetate were determined by gas chromatograph. The enantio excess (e.e.) values of CPB acetate were measured by NMR (nuclear magnetic resonance) method. Effects of solvents and temperatures on this reaction were studied. Cyclohexane was shown to be the best solvent among the three tested solvents. 55 °C was the optimal temperature for higher degree of conversion. External diffusion limitation was excluded by raising the rotational speed to 220 r/min. However, internal diffusion could not be ignored, since the catalyst (lipase) was an immobilized enzyme and its particle dimension was not made small enough. The reaction rate was substantially accelerated when the reactant (m-PBA) concentration was as high as 249 mmol/L, but decreased when the initial concentration of m-PBA reached to 277 mmol/L. It was also found that the catalyzing capability of recovered lipase was high enough to use several batches. Study of the mole ratio of AC to m-PBA showed that 2:1 was the best choice. The strategy of adding base catalyst D301 was found to be an important factor in improving the degree of conversion of the reaction from 20% to 80%. The highest degree of conversion of the reaction has reached up to 80%.展开更多
With recent development of enzyme engineering, the use of enzyme in organic solvents is rapidly increasing. The remarkable stereoselectivity and regioselectivity of reaction catalyzed by enzyme were used to resolve ma...With recent development of enzyme engineering, the use of enzyme in organic solvents is rapidly increasing. The remarkable stereoselectivity and regioselectivity of reaction catalyzed by enzyme were used to resolve many racemic alcohols and esters. A number of results have also been reported about the resolution of amino acids in organic solvents. For example, DL-phenylalanine methyl ester was resolved by α-chymotrypsin in toluene/water biphasic system; in fact, this reaction was carried, out in water;transesterification of DL-phenylalanine propyl ester and methanol was catalyzed by α-chymotrypsin in methanol, and the enzyme used was dissolved in water and the展开更多
分别采用水漂洗、有机溶剂浸提、脂肪酶酶解的方法对鲤鱼鱼肉进行脱脂。试验结果表明,水漂洗法中,清水漂洗效果最优;有机溶剂浸提法中,异丙醇脱脂效果最佳,并对其采用正交试验方案对异丙醇脱脂工艺进行优化,得到最佳试验条件是:脱水时间...分别采用水漂洗、有机溶剂浸提、脂肪酶酶解的方法对鲤鱼鱼肉进行脱脂。试验结果表明,水漂洗法中,清水漂洗效果最优;有机溶剂浸提法中,异丙醇脱脂效果最佳,并对其采用正交试验方案对异丙醇脱脂工艺进行优化,得到最佳试验条件是:脱水时间30 m in,脱脂时间110 m in,温度为75℃,溶剂添加量为50 g;脂肪酶脱脂试验得出最佳条件是加酶量40 U/m l,酶作用时间50 m in。3种方法中,脂肪酶脱脂效果最佳。展开更多
文摘We found a novel lipase gene in the Paenibacillus pasadenensis CS0611 strain.The lipase gene sequence was cloned into the pET-28a expression vector to construct a recombinant lipase protein containing 6×His tags at the C-and N-termini,respectively.High-level expression of the lipase in E.coli BL21(DE3)was obtained upon induction with IPTG at 20°C.The recombinant lipase activity was approximately 1631-fold higher than the wild type.His-tagged recombinant lipase was purified rapidly and efficiently by using Ni-charged affinity chromatography with 63.5%recovery and a purification factor of 10.78.The purified lipase was stable in a broad range of temperatures and pH values,with the optimal temperature and pH being 50°C and 7.0,respectively.Its activity was stimulated to different degrees in the presence of metal ions such as Ca2+,Mg2+,and some non-ionic surfactants.In addition,the purified lipase was activated by a series of water-miscible organic solvents such as some short carbon chain alcohols and was highly tolerant to some water-immiscible organic solvents.
基金Supported by the National Natural Science Foundation of China(No.29876031)
文摘The stability of Candida rugosa lipase coated with glutamic acid didodecyl ester ribitol amide was investigated taking esterification of lauryl alcohol and lauric acid in isooctane as a model reaction. At 30C, the half-life of the activity of the coated lipase was ca 10 h, the enzyme activity became less changed after 12 h and the residual activity was 39% of the initial value. The coated lipase obeyed a first-order deactivation model with a deactivation energy of 29.9J.mol-1.
文摘The kinetics of Candida rugosa lipase catalyzed esterification of racemic ibuprofen with n-butanol in isooctane was studied. The kinetic study was carried out with the addition of 0.1% and 2% (by volume) of water for enzyme activation respectively when celite was added into isooctane for enzyme dispersion. The specific initial rate for S-ibuprofen can be fitted with the Ping Pong Bi Bi mechanism with dead-end competitive inhibition by the alcohol. The time courses of the enantioselective esterification of the two ibuprofen enantiomers with different initial substrate concentrations and water contents were simulated with a model in which both effects of enzyme inactivation by long term reaction and reversed hydrolytic reaction under high water content were taken into consideration.
基金Project supported by the National Natural Science Foundation of China (No. 20336010) and the National Basic Research Program (973)of China (No. 2003CB716008)
文摘Optically active form of α-cyano-3-phenoxybenzyl (CPB) alcohol, building block of pyrethroid insecticides, was synthesized as its acetate by the combination of anion-exchange resin (D301)-catalyzed transcyanation between m-phenoxybenzaldehyde (m-PBA) and acetone cyanohydrin (AC), and lipase (from Alcaligenes sp.)-catalyzed enantioselective transesterification of the resulting cyanohydrin with vinyl acetate. Through optimizing technological conditions, the catalyzing efficiency was improved considerably compared to methods previously reported. Concentrations of CPB acetate were determined by gas chromatograph. The enantio excess (e.e.) values of CPB acetate were measured by NMR (nuclear magnetic resonance) method. Effects of solvents and temperatures on this reaction were studied. Cyclohexane was shown to be the best solvent among the three tested solvents. 55 °C was the optimal temperature for higher degree of conversion. External diffusion limitation was excluded by raising the rotational speed to 220 r/min. However, internal diffusion could not be ignored, since the catalyst (lipase) was an immobilized enzyme and its particle dimension was not made small enough. The reaction rate was substantially accelerated when the reactant (m-PBA) concentration was as high as 249 mmol/L, but decreased when the initial concentration of m-PBA reached to 277 mmol/L. It was also found that the catalyzing capability of recovered lipase was high enough to use several batches. Study of the mole ratio of AC to m-PBA showed that 2:1 was the best choice. The strategy of adding base catalyst D301 was found to be an important factor in improving the degree of conversion of the reaction from 20% to 80%. The highest degree of conversion of the reaction has reached up to 80%.
文摘With recent development of enzyme engineering, the use of enzyme in organic solvents is rapidly increasing. The remarkable stereoselectivity and regioselectivity of reaction catalyzed by enzyme were used to resolve many racemic alcohols and esters. A number of results have also been reported about the resolution of amino acids in organic solvents. For example, DL-phenylalanine methyl ester was resolved by α-chymotrypsin in toluene/water biphasic system; in fact, this reaction was carried, out in water;transesterification of DL-phenylalanine propyl ester and methanol was catalyzed by α-chymotrypsin in methanol, and the enzyme used was dissolved in water and the
文摘分别采用水漂洗、有机溶剂浸提、脂肪酶酶解的方法对鲤鱼鱼肉进行脱脂。试验结果表明,水漂洗法中,清水漂洗效果最优;有机溶剂浸提法中,异丙醇脱脂效果最佳,并对其采用正交试验方案对异丙醇脱脂工艺进行优化,得到最佳试验条件是:脱水时间30 m in,脱脂时间110 m in,温度为75℃,溶剂添加量为50 g;脂肪酶脱脂试验得出最佳条件是加酶量40 U/m l,酶作用时间50 m in。3种方法中,脂肪酶脱脂效果最佳。
