微环境对脂肪酶催化乙酸甲基苯甲酯立体选择性氨解的影响

系统研究了反应介质、水活度、温度、p H等因素对脂肪酶 N ovozym 435催化乙酸甲基苯甲酯立体选择性氨解反应的影响。以正己烷为反应介质 ,酶表现出较高的催化活性和对映体选择性 ;适宜的反应温度为 2 5～ 35℃ ;最适反应体系初始水活度为 0 .33;较适宜的 p H范围为 6～ 7。     

枯草芽孢杆菌ATCC6633催化7-苯乙酰胺基-3-Z-丙烯基-3-头孢菌素-4-对甲氧基苄酯的水解

以枯草芽孢杆菌ATCC6633菌体为催化剂,催化头孢丙烯中间体顺式7-苯乙酰胺基-3-2-丙烯基-3-头孢菌素-4对甲氧基苄酯(顺式GPRE)水解生成顺式7-苯乙酰胺基-3-2-丙烯基-3-头孢菌素4-羧酸(顺式GPRA),经过优化反应产率达63%.而脂肪酶Novozyme435和Amino PS IM均无此活性.     

酶法改良大豆油制备质构脂质的研究

研究了固定化脂肪酶催化大豆油与辛酸酸解制备质构脂质的工艺.脂肪酶筛选实验表明,在所选用的三种脂肪酶中,来自Rhizomucor miehei的固定化脂肪酶RM IM催化效果最好.以RM IM为催化剂,进一步考察了酶用量、底物摩尔比、水分添加量、反应温度、反应时间等因素对辛酸插入率的影响.结果表明:当大豆油500mg时,最佳的反应条件为:反应温度40℃,底物摩尔比6∶1(辛酸∶大豆油),固定化酶10%(底物重量百分比),水分添加量10%(酶的重量百分比),反应24h,辛酸的插入率为43%,质构脂质的脂肪酸分布最合理.     

水活度对全细胞脂肪酶GZUF36催化合成1,3-甘油二酯中的影响

1,3-甘油二酯是一种健康油脂,并且可用来合成药物的中间材料,但其在天然油脂中含量有限。所以本文用黑曲霉GZUF36全细胞脂肪酶法甘油解合成了1,3-甘油二酯,重点探讨了影响其合成的关键因素:水活度。控制水活度的方法包括用饱和盐溶液平衡酶粉、水合盐平衡反应体介质、水合盐控制水活度水合盐分别预平衡酶粉和反应介质。结果表明,水活度显著地影响1,3-甘油二酯的产量和脂肪酶对1,3-甘油二酯的选择性。过高或过低的水活度都不利于全细胞脂肪酶GZUF36催化甘油解反应合成1,3-甘油二酯,水活度过低会导致酶活力不高,水活度过高则易使催化反应向水解方向进行。当用aw=0.58的水合盐分别预平衡酶粉和反应介质时,1,3-甘油二酯的得率和脂肪酶对1.3-甘油二酯的选择性都为最优,分别是26.100%和83.056%。本文为进一步优化全细胞脂肪酶GZUF36催化的选择性合成1,3-甘油二酯中的反应体系奠定了基础。     

脂肪酶在手性药物制备中的应用

手性药物在新药中越来越受重视.生物催化具有反应条件温和、高度选择性、产物纯度高等优点.在有机相中,脂肪酶催化消旋体的拆分,为制备单异构体手性药物提供了重要的技术手段.现对利用脂肪酶的对映选择性、区域选择性技术制备手性醇、手性胺药物中间体的研究进展作一综述.     

生物催化法水解制备N-BOC-L-α-氨基丁酸

利用实验室筛选得到的高立体选择性脂肪酶产生菌株溜曲霉(A spergillus tamarii WYC3)不对称水解拆分N-BOC-DL-α-氨基丁酸甲酯得到N-BOC-L-α-氨基丁酸,对其反应条件进行研究,确定其最优反应条件为:0.1 mol/L pH 7.2的磷酸钾缓冲液,反应温度30℃,底物浓度52.24 mmol/L,0.1 g菌粉,V(底物)∶V(丙酮)=1∶8,200 r/min恒温水浴反应6h,可获得产物N-BOC-L-α-氨基丁酸,此时e.e.s值达到99.95％,对映体选择率E值为20.11,产率为36.21％.     

无溶剂体系合成1,3-甘油二酯用脂肪酶的筛选及其酯化性质

对不同来源的脂肪酶进行了系统的筛选,并根据位置专一性的差别将脂肪酶分为1,3 特异的、1,3 选择性和非选择性的3类.采用LipozymeRMIM在无溶剂系统中催化甘油、脂肪酸合成1,3 sn 甘油二酯.研究表明脂肪酸、底物醇种类、温度、水分活度对LipozymeRMIM的活力有明显的影响.     

脂肪酶催化玉米胚芽油与辛酸制备MLM型结构脂质

为研究无溶剂系统脂肪酶催化玉米胚芽油与辛酸酸解制备功能性脂质的可能性及反应条件,以玉米胚芽油为原料,比较6种脂肪酶(Lipozyme RM IM、Lipozyme TL IM、Novozym 435、Lipase AK、Lipase AY和Newlase F)对无溶剂体系中固定化脂肪酶催化玉米胚芽油与辛酸酸解制备MLM结构脂质的影响。研究结果表明:所选6种脂肪酶中,来自米黑根毛霉的固定化脂肪酶Lipozyme RM IM的催化效果最好。当玉米胚芽油与辛酸的比率为1∶4(物质的量比)、脂肪酶量为12%(底物质量分数)、反应时间16 h、反应温度50℃时,玉米胚芽油中辛酸插入率最高。在无溶剂系统,利用Lipozyme RM IM脂肪酶催化玉米胚芽油与辛酸进行酸解反应,可制备高品质的MLM型功能性脂质。     

新型固定化Aspergillus oryzae脂肪酶催化合成1,3-二油酸-2-棕榈酸甘油三酯

旨在评价自制固定化sn-1,3专一性脂肪酶Aspergillus oryzae L03(AOL)替代商业固定化脂肪酶酸解法合成1,3-二油酸-2-棕榈酸甘油三酯(1,3-dioleoyl-2-palmitoylglycerol,OPO)的可行性。以棕榈硬脂和油酸为原料,在正己烷体系中,得到AOL最适反应参数为:反应温度55℃,反应时间1 h,棕榈硬脂和油酸的底物摩尔比为1∶8,正己烷与底物混合物质量比1∶6,AOL水分含量3.5%(w/w)。该条件下,OPO含量44.9%,三棕榈酸甘油酯(PPP)含量为3.23%,sn-2位棕榈酸占所有棕榈酸比例为68.86%,符合国家标准。同时,AOL和LipozymeRM IM、LipozymeTL IM两种商品酶对比,AOL的催化速率最快,1 h达到最大OPO生产峰值。经过8次循环使用,AOL具有良好的操作稳定性。研究表明,自制的新型廉价固定化酶AOL具有良好的应用前景。     

无溶剂体系酶促酸解合成富含1，3-二硬脂酸-2-油酸甘油三酯的工艺研究

优化了无溶剂体系中固定化1,3-特异性脂肪酶催化高油酸葵花籽油与硬脂酸合成富含1,3-二硬脂酸-2-油酸甘油三酯(SOS)的合成工艺。以SOS体系含量为考察指标,考察了酶的种类、底物摩尔比、反应温度、反应时间的影响。确定无溶剂体系下SOS合成的最佳工艺条件为:特异性脂肪酶为NS40086,酶添加量12%(以底物总质量计),底物(高油酸葵花籽油与硬脂酸)摩尔比1∶12,反应时间4 h,反应温度70℃。在最佳工艺条件下反应产物中SOS含量为66.1%,SSO含量为7.1%,SOO含量为13.5%。     

Enzymatic synthesis of structured lipids using a novel cold-active lipase from Pichia lynferdii NRRL Y-7723

Structured lipids (SL) were synthesized by the acidolysis of borage oil with caprylic acid using lipases. Six commercial lipases from different sources and a novel lipase from Pichia lynferdii NRRL Y-7723 were screened for their acidolysis activities and Lipozyme RM IM and NRRL Y-7723 lipase were selected to synthesize symmetrical SL since recently NRRL Y-7723 lipase was identified as a novel cold-active lipase. Both lipases showed 1,3-regiospecifity toward the glycerol backbone of borage oil. The effects of enzyme loading and temperature on caprylic acid incorporation into the borage oil were investigated. For Lipozyme RM IM and NRRL Y-7723 lipase, the incorporation of caprylic acid increased as enzyme loading increased up to 4% of total weight of the substrate, but significant increases were not observed when enzyme loading was further increased. The activity of NRRL Y-7723 lipase was higher than that of Lipozyme RM IM in the temperature range between 10 and 20 °C.     

Transesterification of conjugated linoleic acid and tricaprylin by lipases in organic solvents

Lipase-catalyzed transesterification of tricaprylin with conjugated linoleic acid (CLA) ethyl ester was performed to produce triacylglycerols containing conjugated linoleic acid. Six commercially available lipases, and seven solvents were screened for their ability to incorporate CLA into tricaprylin. The transesterification reaction was performed by incubating a 1:2 mole ratio of tricaprylin and CLA ethyl ester in 3 ml solvents at 55°C, and the products were analyzed by gas chromatography. Three lipases, Novozym 435, Lipozym IM and lipase PS-C were chosen to allow a comparison of transesterification activity in our model reaction. The highest CLA incorporation with Novozym 435 and Lipozym IM was achieved in hexane while isooctane produced the highest CLA incorporation with lipase PS-C. Lipase PS-C gave higher CLA incorporation into tricaprylin when fatty acid was used as the acyl donor than other lipases did. Lipozym IM and lipase PS-C had not restrict specificity to sn-1, 3 positions, even though they had high specificity at sn-1, 3 positions. Novozym 435 among lipases tested was the most effective on the incorporation of CLA into tricaprylin.     

Kinetic Properties and Enantioselectivity of The Lipases Produced by Four Aspergillus Species

Four high lipase-producing Aspergillus species, selected in our laboratory, were compared in terms of their stability and reactivity for enantioselective esterification between (R, S)-2-octanol with octanoic acid in n-hexane. We determined the pH and temperature reactions dependences of lipases activities, and we found that these enzymes exhibited various pH sensitivities. The optimum pH observed for Aspergillus terreus lipase was 5.5, for A. niger and A. oryzae lipases in the range of 6.0 to 6.5 and pH 7.0 for A. flavus lipase. Good stability was observed at pH ranging from 5.0 to 8.5 after 24 hours at 40° C, and the optimum activity was observed at 35-40° C for all lipases tested. The lipases from A. terreus and A. niger were highly thermostable, retaining 60% and 50% activity at 60° C after 1 hour, respectively. The lipases from A. niger and A. terreus lipases provided the best results in terms of enantioselectivity (E) in the esterification of (R, S)-2-octanol with octanoic acid in n-hexane (E = 4.9 and E = 4.5, respectively). These properties make these lipases good candidates for biocatalysis in organic media.     

Effect of Treatment with Compressed Propane on Lipases Hydrolytic Activity

The objective of this work was to assess the influence of compressed propane treatment on the hydrolytic activity of three lipases (Amano PS, Amano AY30, and a non-commercial lipase from Yarrowia lipolytica) free, resuspended, and in immobilized form. To evaluate the effect of process variables on the lipase activity, a semi-factorial experimental design with two levels and four variables was employed for free and immobilized lipases and a full 2² experimental design was carried out for lipases in solution. The residual activity was defined as the ratio of lipase activity before and after treatment with pressurized propane. For free and immobilized lipases, an enhancement in residual lipase activity in most of the experimental conditions investigated was observed. In the case of resuspended lipases, it is shown that enzyme kinetics is sensitive to treatment with compressed propane resulting in remarkable gains and losses of enzyme activity. In a general way, the results showed that the enzyme activity changes significantly depending on the enzyme, the presentation form, and the experimental conditions investigated, allowing the selection of operational conditions in terms of temperature, pressure, exposure time, and depressurization rate for advantageous application of these biocatalysts in hydrolysis reactions.     

Kinetic Properties and Enantioselectivity of The Lipases Produced by Four Aspergillus Species

Four high lipase-producing Aspergillus species, selected in our laboratory, were compared in terms of their stability and reactivity for enantioselective esterification between (R, S)-2-octanol with octanoic acid in n-hexane. We determined the pH and temperature reactions dependences of lipases activities, and we found that these enzymes exhibited various pH sensitivities. The optimum pH observed for Aspergillus terreus lipase was 5.5, for A. niger and A. oryzae lipases in the range of 6.0 to 6.5 and pH 7.0 for A. flavus lipase. Good stability was observed at pH ranging from 5.0 to 8.5 after 24 hours at 40° C, and the optimum activity was observed at 35–40° C for all lipases tested. The lipases from A. terreus and A. niger were highly thermostable, retaining 60% and 50% activity at 60° C after 1 hour, respectively. The lipases from A. niger and A. terreus lipases provided the best results in terms of enantioselectivity (E) in the esterification of (R, S)-2-octanol with octanoic acid in n-hexane (E = 4.9 and E = 4.5, respectively). These properties make these lipases good candidates for biocatalysis in organic media.     

金华火腿加工过程中内源脂肪酶活力变化特点研究

以浙江兰溪两头乌杂交猪后腿为原料,按传统工艺加工金华火腿。分别在原料、腌制结束、晒腿结束、成熟中期、成熟结束、后熟-1和后熟-2七个工艺点随机取5条腿的股二头肌为样品,分别测定中性脂肪酶、酸性脂肪酶的活力变化,以及测定pH5.8时脂肪酶综合活力的变化;同时,利用响应曲面法研究温度、盐含量、硝酸盐含量、亚硝酸盐含量对脂肪酶活力的影响,并利用所得回归方程预测金华火腿加工过程中脂肪酶的实际表现活力。结果表明,加工过程中,脂肪酶活力持续降低,在后熟-1工艺点,酸性脂肪酶和中性脂肪酶活力残留分别是8.16%和3.0%,至后熟-2已测不到酶活;温度、盐含量及其交互项是脂肪酶活力的显著影响因子(P<0.05),硝酸盐和亚硝酸盐含量对脂肪酶活力影响不显著(P>0.05)。加工过程中,预测的脂肪酶实际表现活力在成熟中期工艺点之前低于酸性脂肪酶及中性脂肪酶的潜在活力,之后则高于潜在活力,其变化规律与酸性和中性脂肪酶完全不同。     

Lipase-catalysed production of triacylglycerols enriched in pinolenic acid at the sn-2 position from pine nut oil

BACKGROUND: The purpose of this study was to produce triacylglycerols (TAGs) enriched in pinolenic acid (PLA) at the sn‐2 position using the principle of acyl migration, from the pine nut oil containing PLA esterified exclusively at the sn‐3 position. RESULTS: Two types of lipase‐catalysed reactions, i.e. redistribution and reesterification of fatty acids, were successively performed using seven commercially available lipases as biocatalysts. Of the lipases tested, Novozym 435 and Lipozyme TL IM were effective biocatalysts for positioning PLA at the sn‐2 location. These biocatalysts were selected for further evaluation of the effects of reaction parameters, such as temperature and water content on the migration of PLA residues to the sn‐2 position and TAG content. For both lipases, a significant decrease in TAG content was observed after the lipase‐catalysed redistribution of fatty acids for both lipases. The reduced TAG content could be enhanced up to approx. 92%, through lipase‐catalysed re‐esterification of the hydrolysed fatty acids under vacuum. CONCLUSION: TAG enriched in PLA at the sn‐2 position was synthesised from pine nut oil via lipase‐catalysed redistribution and re‐esterification of fatty acid residues using Lipozyme TL IM and Novozym 435 as biocatalysts. Copyright © 2011 Society of Chemical Industry     

Chemoenzymatic synthesis of structured triacylglycerols with conjugated linoleic acids (CLA) in central position

A chemoenzymatic process for the production of structured triacylglycerols (TAG) containing CLA at sn2 position and lauric acid at external ones is proposed. First, castor bean oil was chemically dehydrated and isomerised to obtain a new modified oil with very high proportion of CLA (>95%). Then, this new oil was used for enzymatic transesterification allowing the grafting of lauric acid at external positions of the TAG backbone by using 1,3 regioselective enzymes. Among these, Aspergillus niger lipase was not satisfactory giving very low lauroyl incorporation (<5%) On the contrary, lipases from Thermomyces lanuginosa (Lipozyme TL IM) and from Carica papaya latex allowed good reaction yields. The effect of the type of acyl donor was studied. With alkyl esters T. lanuginosa lipase provided a final incorporation of 58.9% after 72 h corresponding to 88.4% transesterification yield. Concerning C. papaya lipase, incorporation of lauroyl residues was lower than Lipozyme TL IM. This lipase exhibited higher performance with lauric acid accounting for 44.7% lauroyl incorporation at the end of reaction for a 67.1% transesterification yield. The effect of the substrates mole ratio was also evaluated. It was observed that a 1:3 TAG/acyl donor mole ratio was the most efficient for both lipases. Finally, fatty acids regiodistribution of the newly formed structured TAG was determined. With Lipozyme TL IM, the proportion of lauric acid incorporated at the sn2 position did not exceed 5.4% after 72 h while with C. papaya lipase a more pronounced incorporation of lauroyl residues at the central position (8.8%) was observed.     

酶法制备葛根素的丙酯衍生物

本文研究了不同种类的水解酶催化非水相葛根素丙酰化反应的催化效率,并研究以Novozym 435脂肪酶催化葛根素丙酰化反应为模型,几个关键反应因素对该模型的影响规律。研究发现固定化脂肪酶Novozym 435、Lipozyme IMTL、Lipozyme IMRM均能高效催化葛根素丙酰化反应,且转化率高达98%以上,但另外几种游离脂肪酶催化葛根素丙酰化反应活性及效率就特别低。相同条件下,猪胰脂肪酶催化葛根素丙酰化反应的转化率为67%左右;胰脂肪酶和CRL脂肪酶表现很低的催化活性,肽酶和蛋白酶没有表现催化活性。以四氢呋喃为反应溶剂,2 mg/m L Novozym 435脂肪酶,底物:酰基供体之比为1:30,水分含量为0的反应条件下,反应6 h,底物转化率达到99.5%。反应产物经分离纯化后进行了结构鉴定,高效液相色谱、质谱、傅里叶红外光谱结果表明,非水相酶催化葛根素酰化反应主要生成单酯,所得产物酯为葛根素丙单酯,区域选择性达98%。     

Production of highly enantioselective (−)-menthyl butyrate using Candida rugosa lipase immobilized on epoxy-activated supports

Optically active (−)-menthyl butyrate was synthesized by enantioselective esterification of racemic (±)-menthol and butyric anhydride using lipase from Candida rugosa immobilized onto epoxy-activated supports of Eupergit C and Eupergit C 250 L through physical adsorption method. The effects of various temperature, storage condition, stability in organic solvent and lipase recyclability were investigated for their influence on the enzymatic enantioselective formation of (−)-menthyl butyrate. The immobilized lipases retained high catalytic activity of up to 31% yield and 100% enantiomeric excess of the desired product, and showed better stability compared to the native lipase. They also exhibited about 50% retained activity even after incubation at higher temperatures, storage at room temperature and after long incubation in hexane. Immobilized lipases also showed considerably efficient reusability.