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1.
硫酸铵-丙酮协同沉淀法纯化南极假丝酵母产脂肪酶 总被引:2,自引:0,他引:2
采用硫酸铵-丙酮协同沉淀法,对南极假丝酵母产脂肪酶(CALB)进行了分离纯化研究.经正交实验确定纯化工艺为:向酶液中加入硫酸铵至饱和度45%的同时加入0.3倍酶液体积的丙酮;离心后再向上清液中加入硫酸铵至饱和度75%,分相后,相界面处沉淀即为脂肪酶粗品.经分离纯化后,酶活回收率为54%、纯化倍数为2.72. 相似文献
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双水相萃取技术分离纯化蛋白质的研究 总被引:6,自引:0,他引:6
阐述了双水相萃取原理,详细分析了影响双水相萃取分离纯化蛋白质的各种因素,探讨了双水相萃取技术在蛋白质分离纯化中的应用并对其前景进行了展望. 相似文献
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南极假丝酵母脂肪酶发酵条件优化及酶学性质 总被引:7,自引:0,他引:7
分别用摇瓶和15L发酵罐,对南极假丝酵母产胞外脂肪酶的培养基成分和操作条件进行了实验研究。得到最优的培养基组成为:豆粉40g/L,淀粉15g/L,豆油5mL/L,K2HPO4g/L,MgsO4·7H2O1g/L,Tween-800.1%,酵母膏5g/L;操作条件为:温度24℃,初始pH值为6.0,通气量为10.0L/min。在此培养条件下,发酵周期缩短至54h。由15L发酵罐生产的酶液酶活达到19.2U/mL。酶液在pH值为4.0~6.0和7.5~9.0范围内较稳定,其最适宜pH值范围为6~8.5;70℃时酶的催化活性最大.在40~70℃的温度范围内保持1h后残留酶活为60%。 相似文献
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固定化假丝酵母脂肪酶催化合成辛酸甘油酯 总被引:2,自引:0,他引:2
以自制固定化假丝酵母脂肪酶作为催化剂,研究了无溶剂体系中辛酸和甘油直接酯化合成辛酸甘油酯的反应条件。考察了酶的种类、底物的物质的量之比、温度、酶量、甘油的初始含水量和反应时间等因素对辛酸转化率和产物组成的影响。结果证明,以纺织物作为载体制备的固定化假丝酵母脂肪酶适宜催化辛酸甘油酯的合成。最优反应条件为:辛酸与甘油的物质的量之比为2∶1,固定化假丝酵母脂肪酶加量为0.5g /0.69g 甘油,温度为40 ℃ ,振荡培养箱转速为190 r/min。最优反应条件下辛酸转化率可以达到94%以上,经过简单处理的固定化酶可以重复使用4批。 相似文献
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双水相萃取技术研究进展 总被引:3,自引:1,他引:2
双水相萃取技术作为一项新的分离技术日益受到重视,它与传统的萃取方法相比有独特的优点.本文综述了双水相萃取技术基本原理、特点、应用及热力学模型,并对双水相萃取技术存在的问题和发展趋势作了论述. 相似文献
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双水相萃取技术研究进展 总被引:1,自引:0,他引:1
双水相萃取技术作为一项新的分离技术日益受到重视,它与传统的萃取方法相比有独特的优点,作者综述了又水相萃取技术基本原理、特点、应用及热力学模型,并对双水相萃取技术存在的问题和发展趋势作了论述. 相似文献
11.
N-乙基-N-丁基吗啉离子液体双水相体系萃取分离蛋白质 总被引:2,自引:0,他引:2
研究了新型离子液体N-乙基-N-丁基吗啉四氟硼酸盐([Nebm]BF4)和KH2PO4形成的双水相体系对牛血清白蛋白(BSA)的萃取行为,考察了盐的加入量、离子液体浓度、溶液pH值、蛋白质浓度等因素对萃取率的影响。结果表明:当KH2PO4的加入量为85 g/L、离子液体浓度在200~250 g/L、BSA的浓度60~120 mg/L、溶液酸度在pH4.5~7.0时,其萃取率达98.0%以上。该双水相体系对α-淀粉酶的萃取率也达98.5%。 相似文献
12.
Catalase from Amsonia orientalis was purified by ATPS, and its efficiency was compared against hydrophobic interaction chromatography. Activity recovery and purification fold of purified catalase by ATPS were examined under varying experimental conditions. The effects of various factors such as type of phase-forming salts, (PEG) mass, with their different concentrations, pH and temperature effects on partitioning were investigated. The highest activity recovery (156%) and purification fold (8.67) of catalase were obtained in the ATPS system containing 10% (g/g) PEG4000, 15% (g/g) Na2SO4 at pH 6.0 and room temperature. In hydrophobic interaction chromatography, the enzyme has been purified 12.54-fold with 57.5% recovery. The molecular weight of catalase was determined as 75 kDa by SDS-PAGE. 相似文献
13.
Improved activity and thermostability of Candida antarctica lipase B by DNA family shuffling 总被引:2,自引:0,他引:2
Suen WC Zhang N Xiao L Madison V Zaks A 《Protein engineering, design & selection : PEDS》2004,17(2):133-140
DNA family shuffling was used to create chimeric lipase B proteins with improved activity toward the hydrolysis of diethyl 3-(3',4'-dichlorophenyl)glutarate (DDG). Three homologous lipases from Candida antarctica ATCC 32657, Hyphozyma sp. CBS 648.91 and Crytococcus tsukubaensis ATCC 24555 were cloned and shuffled to generate a diverse gene library. A high-throughput screening assay was developed and used successfully to identify chimeric lipase B proteins having a 20-fold higher activity toward DDG than lipase B from C.antarctica ATCC 32657 and a 13-fold higher activity than the most active parent derived from C.tsukubaensis ATCC 24555. In addition, the stability characteristics of several highly active chimeric proteins were also improved as a result of family shuffling. For example, the half-life at 45 degrees C and melting point (T(m)) of one chimera exceeded those of lipase B from C.antarctica ATCC 32657 by 11-fold and 6.4 degrees C, respectively, which closely approached the stability characteristics of the most thermostable parent derived from Hyphozyma sp. CBS 648.91. 相似文献
14.
Conversion of vegetable oil to biodiesel using immobilized Candida antarctica lipase 总被引:11,自引:8,他引:3
Yuji Shimada Yomi Watanabe Taichi Samukawa Akio Sugihara Hideo Noda Hideki Fukuda Yoshio Tominaga 《Journal of the American Oil Chemists' Society》1999,76(7):789-793
Biodiesel derived from vegetable oils has drawn considerable attention with increasing environmental consciousness. We attempted
continuous methanolysis of vegetable oil by an enzymatic process. Immobilized Candida antarctica lipase was found to be the most effective for the methanolysis among lipases tested. The enzyme was inactivated by shaking
in a mixture containing more than 1.5 molar equivalents of methanol against the oil. To fully convert the oil to its corresponding
methyl esters, at least 3 molar equivalents of methanol are needed. Thus, the reaction was conducted by adding methanol stepwise
to avoid lipase inactivation. The first step of the reaction was conducted at 30°C for 10 h in a mixture of oil/methanol (1:1,
mol/mol) and 4% immobilized lipase with shaking at 130 oscillations/min. After more than 95% methanol was consumed in ester
formation, a second molar equivalent of methanol was added and the reaction continued for 14 h. The third molar equivalent
of methanol was finally added and the reaction continued for 24 h (total reaction time, 48 h). This three-step process converted
98.4% of the oil to its corresponding methyl esters. To investigate the stability of the lipase, the three-step methanolysis
process was repeated by transferring the immobilized lipase to a fresh substrate mixture. As a result, more than 95% of the
ester conversion was maintained even after 50 cycles of the reaction (100 d). 相似文献
15.
Ramesh N. Patel Amit Banerjee Venkata Nanduri Animesh Goswami F. T. Comezoglu 《Journal of the American Oil Chemists' Society》2000,77(10):1015-1019
Chiral intermediates S-(+)-2-pentanol and S-(+)-2-heptanol were prepared by a lipase-catalyzed enzymatic resolution proces. Among various lipases evaluated for the stereoselective
acylation of racemic alcohols, lipase B from Candida antarctica catalyzed the acylation of the undesired enantiomer of racemic alcohols leaving the desired S-(+)-alcohols unreacted. A reaction yield of 43–45% and an enantiomeric excess (e.e.) of >99% were obtained for S-(+)-2-pentanol or S-(+)-2-heptanol when the reaction was carried out using vinyl acetate or succnic anhydride as acylating agent. In an alternative
process, an enantioselective hydrolysis of 2-pentyl acetate was demonstrated using lipase B giving S-(+)-2-pentyl acetate and R-(−)-2-pentanol. A reaction yield of 45% and an e.e. of 98.6% were obtained for S-(+)-2-pentyl acetate.
This work was presented at the Biocatalysis Symposium in April 2000, held at the 91st Annual Meeting and Expo of the American
Oil Chemists' Society, San Diego, CA. 相似文献
16.
Kasrayan A Bocola M Sandström AG Lavén G Bäckvall JE 《Chembiochem : a European journal of chemical biology》2007,8(12):1409-1415
A number of model structures of the CalA suggested by comparative modeling were tested by site-directed mutagenesis. Enzyme variants were created where amino acids predicted to play key roles for the lipase activity in the different models were replaced by an inert amino acid (alanine). The results from activity measurements of the overproduced and purified mutant enzymes indicate a structure where the active site consists of amino acid residues Ser184, His366, and Asp334 and in which there is no lid. This model can be used for future targeted modifications of the enzyme to obtain new substrate acceptance, better thermostability, and higher enantioselectivity. 相似文献
17.
Peter Skagerlind Karin Larsson Martin Barfoed Karl Hult 《Journal of the American Oil Chemists' Society》1997,74(1):39-42
The surfactant, ethyl 6-O-decanoyl glucoside, was synthesized in microemulsion systems by lipase catalysis. The microemulsions were based on the two
substrates for the reaction, ethyl glucoside and fatty acid, and either the sodium salt of the fatty acid or the glucoside
ester was used as surfactant. The lipase used was component B from Candida antarctica. Reduced pressure was employed to eliminate the water of condensation. The reaction yield was good, with conversion of fatty
acid and ethyl glucoside reaching 77 and 96%, respectively. 相似文献
18.
Recovery of lipase from Penicillium cyclopium by aqueous two-phase extraction was studied with maximal possible crude enzyme loads. In polyethylene glycol/dextran and polyethylene glycol/salt systems the influences of molecular weight and concentration of polyethylene glycol, phase-forming salt and phase volume ratio were evaluated. Lipase partition coefficient 9 followed by the top phase yield 95.7% and purification factor 3.4 were achieved in 15% (w w?1) polyethylene glycol 4000/15% (w w?1) KH2PO4/70% (w w?1) crude enzyme. Efficient single-step recovery of lipase followed by partial enzyme purification indicated possible integration of production and primary bioseparation step by aqueous two-phase extraction. By varying phase volume ratio, the concentration of phosphate was reduced without decrease in lipase recovery. 相似文献
19.
Nyhlén J Martín-Matute B Sandström AG Bocola M Bäckvall JE 《Chembiochem : a European journal of chemical biology》2008,9(12):1968-1974
The selectivity of acetylation of delta-functionalized secondary alcohols catalyzed by Candida antarctica lipase B has been examined by molecular dynamics. The results from the simulation show that a delta-alcohol functionality forms a hydrogen bond with the carbonyl group of Thr 40. This interaction stabilizes the tetrahedral intermediate and thus leads to selective acetylation of the R enantiomer. A stabilizing interaction of the delta-(R)-acetoxy group with the peptide NH of alanine 282 was also observed. No stabilizing interaction could be found for the delta-keto functionality, and it is proposed that this is the reason for the experimentally observed decrease in enantioselectivity. From these results, it was hypothesized that the enantioselectivity could be restored by mutating the alanine in position 281 for serine. The mutation was made experimentally, and the results show that the E value increased from 9 to 120. 相似文献
20.
Yomi Watanabe Yuji Shimada Akio Sugihara Hideo Noda Hideki Fukuda Yoshio Tominaga 《Journal of the American Oil Chemists' Society》2000,77(4):355-360
Candida antarctica lipase is inactivated in a mixture of vegetable oil and more than 1∶2 molar equivalent of methanol against the total fatty
acids. We have revealed that the inactivation was eliminated by three successive additions of 1∶3 molar equivalent of methanol
and have developed a three-step methanolysis by which over 95% of the oil triacylglycerols (TAG) were converted to their corresponding
methyl esters (ME). In this study, the lipase was not inactivated even though 2∶3 molar equivalent of methanol was present
in a mixture of acylglycerols (AG) and 33% ME (AG/ME33). This finding led to a two-step methanolysis of the oil TAG: The first-step
was conducted at 30°C for 12 h with shaking in a mixture of the oil, 1∶3 molar equivalent of methanol, and 4% immobilized
lipase; the second-step reaction was done for 24 h after adding 2∶3 molar equivalent of methanol (36 h in total). The two-step
methanolysis achieved more than 95% of conversion. When two-step reaction was repeated by transferring the immobilized lipase
to a fresh substrate mixture, the enzyme could be used 70 cycles (105 d) without any decrease in the conversion. From the
viewpoint of the industrial production of biodiesel fuel production, the two-step reaction was conducted using a reactor with
impeller. However, the enzyme carrier was easily destroyed, and the lipase could be used only several times. Thus, we attempted
flow reaction using a column packed with immobilized Candida lipase. Because the lipase packed in the column was drastically inactivated by feeding a mixture of AG/ME33 and 2∶3 molar
equivalent of methanol, three-step flow reaction was performed using three columns packed with 3.0 g immobilized lipase. A
mixture of vegetable oil and 1∶3 molar equivalent of methanol was fed into the first column at a constant flow rate of 6.0
mL/h. The eluate and 1∶3 molar equivalent of methanol were mixed and then fed into the second column at the same flow rate.
The final step reaction was done by feeding a mixture of eluate from the second column and 1∶3 molar equivalent of methanol
at the same flow rate. The ME content in the final-step eluate reached 93%, and the lipase could be used for 100 d without
any decrease in the conversion. 相似文献