Improved activity and thermostability of Candida antarctica lipase B by DNA family shuffling

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.     

Preparation of regioisomers of structured TAG consisting of one mole of CLA and two moles of caprylic acid

TAG (MLM) with medium-chain FA (MCFA) at the 1,3-positions and long-chain FA (LCFA) at the 2-position, and TAG (LMM) with LCFA at the 1(3)-position and MCFA at 2,3(1)-positions are a pair of TAG regioisomers. Large-scale preparation of the two TAG regioisomers was attempted. A commercially available FFA mixture (FFA-CLA) containing 9-cis, 11-trans (9c, 11t)- and 10t,12c-CLA was selected as LCFA, and caprylic acid (C₈FA) was selected as MCFA. The MLM isomer was synthesized by acidolysis of acyglycerols (AG) containing two CLA isomers with C₈FA: A mixture of AG-CLA/C₈ FA (1∶10, mol/mol) and 4 wt% immobilized Rhizomucor miehei lipase was agitated at 30°C for 72 h. The ratio of MLM to total AG was 51.1 wt%. Meanwhile, LMM isomer was synthesized by acidolysis of tricaprylin with FFA-CLA: A mixture of tricaprylin/FFA-CLA (1∶2, mol/mol) and 4 wt% immobilized R. miehei lipase was agitated at 30°C for 24 h. The ratio of LMM to total AG was 51.8 wt%. MLM and LMM were purified from 1,968 and 813 g reaction mixtures by stepwise short-path distillation, respectively. Consequently, MLM was purified to 92.3% with 49.1% recovery, and LMM was purified to 93.2% with 52.3% recovery. Regiospecific analyses of MLM and LMM indicated that the 2-positions of MLM and LMM were 95.1 mol% LCFA and 98.3 mol% C₈ FA, respectively. The results showed that a process comprising lipase reaction and short-path distillation is effective for large-scale preparation of high-purity regiospecific TAG isomers.     

聚合物接枝脂肪酶的合成及其对酶活的影响

利用原子转移自由基聚合方法将含C₂～C₄烷基链的单体化合物分别接枝到皱褶假丝酵母脂肪酶（CRL）表面合成了HEMA-g-CRL、GMA-g-CRL、nPMA-g-CRL和BMA-g-CRL四种聚合物接枝CRL。CD和荧光光谱学分析显示,聚合物接枝导致HEMA-g-CRL、nPMA-g-CRL和BMA-g-CRL分子的α-螺旋和β-折叠含量增加。与此同时,聚合物接枝CRL分子荧光发射光谱也发生了蓝移,意味着其具有更加紧密的分子构象。酶活测定结果进一步显示,聚合物接枝增强CRL酶活227%～278%。随着单体化合物烷基链长增加,聚合物接枝CRL的K _m值由0.17 mmol·L^-1降至0.09 mmol·L^-1。与此同时,聚合物接枝CRL的k _cat值则提高至106～182 s^-1。BMA-g-CRL的催化效率达到了野生型CRL的3.28倍。这反映出聚合物接枝助力了CRL“盖子”结构开启和CRL活性中心暴露,促进底物的转化。稳定性测试表明,聚合物接枝提升了CRL的热稳定性并拓宽了其pH操作范围。     

A Novel Cold-Active Lipase from Candida albicans: Cloning, Expression and Characterization of the Recombinant Enzyme

A novel lipase gene lip5 from the yeast Candida albicans was cloned and sequenced. Alignment of amino acid sequences revealed that 86-34% identity exists with lipases from other Candida species. The lipase and its mutants were expressed in the yeast Pichia pastoris, where alternative codon usage caused the mistranslation of 154-Ser and 293-Ser as leucine. 154-Ser to leucine resulted in loss of expression of Lip5, and 293-Ser to leucine caused a marked reduction in the lipase activity. Lip5-DM, which has double mutations that revert 154 and 293 to serine residues, showed good lipase activity, and was overexpressed and purified by (NH(4))(2)SO(4) precipitation and ion-exchange chromatography. The pure Lip5-DM was stable at low temperatures ranging from 15-35 °C and pH 5-9, with the optimal conditions being 15-25 °C and pH 5-6. The activation energy of recombinant lipase was 8.5 Kcal/mol between 5 and 25 °C, suggesting that Lip5-DM was a cold-active lipase. Its activity was found to increase in the presence of Zn(2+), but it was strongly inhibited by Fe(2+), Fe(3+), Hg(2+) and some surfactants. In addition, the Lip5-DM could not tolerate water-miscible organic solvents. Lip5-DM exhibited a preference for the short-and medium-chain length p-nitrophenyl (C4 and C8 acyl group) esters rather than the long chain length p-nitrophenyl esters (C12, C16 and C18 acyl group) with highest activity observed with the C8 derivatives. The recombinant enzyme displayed activity toward triacylglycerols, such as olive oil and safflower oil.     

化学修饰Novozym-435酶催化合成维生素E琥珀酸酯的研究

Vitamin E succinate was synthesized in organic solvents using a modified Novozym-435 as catalyst.In order to improve the catalytic performance of Novozym-435,the enzyme was modified using acetic anhydride, propionic anhydride and succinic anhydride separately.We found that both the hydrolytic activity and the thermal stability of the modified Novozym-435 were enhanced compared with the unmodified enzyme.The modified Novozym-435 catalysts were used to synthesize the succinate derivative of vitamin E.Compared with the native Novozym-435,the catalytic activity of the modified novozym-435 in promoting the synthesis of vitamin E succinate was dramatically increased,with the novozym-435 modified with succinic anhydride(N435-S)as the most active catalyst.Conditions for the synthesis of vitamin E succinate were also optimized.A mixture of tert-butanol and DMSO(volume ratio of 2︰3)was the most suitable medium for the reaction,whereas the appropriate molar ratio of vitamin E to succinic anhydride and reaction temperature were 1︰5 and 40°C,respectively.Under these reaction conditions,the yield of vitamin E succinate reached 94.4%.N435-S could be reused for five batches.     

脂肪酶动力学拆分薄荷醇的研究进展

从脂肪酶拆分薄荷醇的转酯化、水解反应的工艺和影响因素等方面,分析了脂肪酶动力学拆分过程的转酯化和水解反应的催化路径与特点,指出了脂肪酶拆分薄荷醇的最佳路径和工艺选择. 综述了近10年来脂肪酶拆分制备工业重要的手性香料l-薄荷醇的研究进展,特别综述了具有工业化前景的脂肪酶催化拆分和单元操作工艺的研究进展. 同时展望了未来脂肪酶拆分薄荷醇的研究方向.     

A continuous ultrasound‐assisted packed‐bed bioreactor for the lipase‐catalyzed synthesis of caffeic acid phenethyl ester

BACKGROUND: The focus of this paper is the ultrasound‐assisted synthesis of caffeic acid phenethyl ester (CAPE) from caffeic acid and phenyl ethanol in a continuous packed‐bed bioreactor. Immobilized Novozym^® 435 (from Candida antarctica) is used as the catalyst. A three‐level–three‐factor Box–Behnken design and a response surface methodology (RSM) are employed to evaluate the effects of temperature, flow rate, and ultrasonic power on the percentage molar conversion of CAPE. RESULTS: Based on ridge max analysis, it is concluded that the optimum condition for synthesis is reaction temperature 72.66 °C, flow rate 0.046 mL min^?1, and ultrasonic power 1.64 W cm^?2. The expected molar conversion value is 97.84%. An experiment performed under these optimal conditions resulted in a molar conversion of 92.11 ± 0.75%. The enzyme in the bioreactor was found to be stable for at least 6 days. CONCLUSIONS: The lipase‐catalyzed synthesis of CAPE by an ultrasound‐assisted packed‐bed bioreactor uses mild reaction conditions. Enzymatic synthesis of CAPE is suitable for use in the nutraceutical and food production industries. Copyright © 2011 Society of Chemical Industry     

碱性脂肪酶生产菌假单孢菌4631菌株的筛选

从３２个土样中分离获得碱性脂肪酶产生菌１６６株，其中４６３１号菌株产酶能力最高，达到９．１ＩＵ／ｍｌ。初步鉴定为假单孢菌属。对研究结果统计分析表明，Ｄ／ｄ值与ＬＡ呈线性相关关系。     

脂肪酶水解L-亮氨酸异丁酯的工艺

以脂肪酶A为催化剂,水解L-亮氨酸异丁酯制备L-亮氨酸。研究了不同酶、缓冲液体系、物料比、反应温度、反应时间对L-亮氨酸收率的影响,通过正交试验确定了L-亮氨酸异丁酯最佳水解条件。以L-亮氨酸异丁酯为原料,在提取脂肪酶液为Gly-NaOH缓冲液体系、反应前添加L-亮氨酸达到L-亮氨酸溶解度的质量、物料比为1∶13、50℃、摇床转速为170r/min的条件下反应13h,L-亮氨酸的收率为95.23%。     

Synthesis and biodegradability evaluation of 2‐methylene‐1,3‐dioxepane and styrene copolymers

Biodegradable copolymers of 2‐methylene‐1,3‐dioxepane (MDO) and styrene (ST) were synthesized by free‐radical copolymerization using di‐t‐butyl peroxide (DTBP) as the initiator. The copolymers containing ester units were characterized by Fourier transform infrared (FTIR), ¹H‐NMR, and ¹³C‐NMR spectroscopy. Their molecular weight and polydispersity index were determined by gel permeation chromatography (GPC). In vitro enzymatic degradation of poly(MDO‐co‐ST) was performed at 37°C in phosphate buffer solution (PBS, pH = 7.4) in the presence of Pseudomonas lipase or crude enzyme extracted from earthworm. The experiment showed that incorporating ester units into C? C backbone chain of polystyrene would result in a biodegradable copolymer. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 1146–1151, 2007