表面活性剂对华根霉脂肪酶有机相酯合成活性的激活

利用表面活性剂结合无机盐与华根霉(Rhizopus chinensis)脂肪酶(RCL)共冻干,以提高RCL在有机相中的酯合成活性.结果表明,n-十二烷基-β-D-麦芽糖苷(DDM)可以显著激活RCL的酯合成活性.DDM激活效果的关键影响因素是DDM与RCL的摩尔比,而非DDM浓度;当二者摩尔比大于30时,RCL酯合成活性随着该比值的增大而提高.此外,添加适量无机盐可以进一步强化DDM对脂肪酶酯合成活性的激活效果.利用激活的RCL分别在有机溶剂和无溶剂体系中催化生物香料己酸乙酯和生物柴油油酸乙酯的合成,酯合成效率得到明显提高,表明该方法的有效性和适用性.     

华根霉菌丝体结合脂肪酶催化酯合成动力学拆分2-辛醇

研究了华根霉CCTCC M201021菌丝体结合脂肪酶(RCL)在非水相中催化酯合成动力学拆分外消旋2-辛醇的能力.发现RCL对该反应具有较好的光学选择性(E>100),辛酸和异辛烷分别是最佳的酰基供体和反应溶剂,体系水活度的减少对反应的光学选择性没有明显影响,但能显著提高反应初速度.在相同转化率下,通过添加3A分子筛降低体系水含量可使反应初始速度提高7.3倍.当底物浓度提高到0.230 mol/L,反应40 h时转化率达44.4%,产物酯的ee值为94.7%.与三种商品化脂肪酶进行了比较,发现在相同条件下RCL对2-辛醇的拆分不但具有较好的光学选择性(E=103.1),而且也表现出较高的反应初速度和转化率.     

华根霉全细胞脂肪酶催化合成生物柴油

比较了5种不同商品化脂肪酶和自制的华根霉CCTCCM201021全细胞脂肪酶(RCL)催化油脂合成生物柴油的转化效果,结果表明,RCL能有效应用于无溶剂体系催化合成生物柴油.在无溶剂体系中对该酶催化生物柴油的转酯化反应工艺进行优化,考察了甲醇用量、体系含水量、酶的添加量和反应温度对生物柴油收率的影响,使生物柴油最终收率大于86.0%.在有机溶剂体系中选择不同有机溶剂作为助溶剂进行转酯化反应,发现logP值在4.0～4.5的有机溶剂具有较好的转化效果.其中以正庚烷为助溶剂的转酯化反应具有最高的生物柴油收率86.7%.在无溶剂体系中RCL催化转化油酸和模拟高酸价油脂合成脂肪酸甲酯的研究表明,该酶具有很好的催化合成生物柴油的潜力.     

有机相中固定化脂肪酶催化合成植物甾醇酯

酶法合成植物甾醇酯具有反应条件温和、产物纯度和产量高等优点,但非水相酶催化的活性和稳定性普遍较低.本文以大孔树脂固定化脂肪酶为催化剂,并在催化过程中添加乳糖的类似物,构建了有机相高效合成植物甾醇酯的工艺过程.以酯化率为考察指标,对脂肪酶和反应溶剂进行筛选,对酯化条件进行优化,同时考察了糖的种类及添加量对酶催化性能的影响.结果表明,大孔树脂NKA吸附固定化的褶皱假丝酵母(Candida rugosa)脂肪酶(NKA-CRL)为最适宜的催化剂,以正己烷为反应介质,在酸醇摩尔比为2和添加酶蛋白质量7.5%的海藻糖的条件下,40°C反应10 h,酯化率达到96.6%.连续6次催化后,植物甾醇的酯化率仍维持在85.0%以上.     

脂肪酶催化酯化拆分与水解拆分2-甲基丁酸及其酯

2-甲基丁酸手件中心碳原子上的基团差异较小,是一种典型的酶法动力学拆分较为困难的风味化合物.本文分别在不同体系中比较了几种商品化脂肪酶和华根霉脂肪酶(RCL)催化酯化和水解反应对2-甲基丁酸及其酯的拆分,结果表明,RCL不仅在非水相中具有一定的选择性酯化能力,而且在水相中具有更强的选择性水解2-甲基丁酸乙酯的能力,在40℃下优先水解(S-型底物,反应10h后(R)-2-甲基丁酸乙酯的ee值为92.4%.进一步考察了温度对RCL催化酯化拆分与水解拆分的影响,结果表明,低温下反应的对映体选择性较高,在4℃下通过水解拆分获得的(R)-2-甲基丁酸乙酯的ee值町提高至95.0%.     

添加表面活性剂两步沉淀法制备甲醇催化剂

采用添加表面活性剂两步沉淀法制备了具有高表面铜相对浓度的超细甲醇合成催化剂。以组成为H2/CO/CO2/N2=66/27/3/4(体积比）的原料气对催化剂进行了活性评价。结果表明,该催化剂比传统并流沉淀法制备的铜基催化剂活性提高47.9%,比两步沉淀法和添加表面活性剂并流沉淀法制备的铜基催化剂活性分别提高9.3%和16.8%。利用SEM、XRD及XPS方法对催化剂的结构、形貌和表面金属组成进行了表征。     

界面活化的溶胶凝胶包埋Candida rugosa脂肪酶催化合成维生素E琥珀酸酯

采用界面活化的溶胶凝胶包埋Candida rugosa脂肪酶(CRL)催化合成了维生素E琥珀酸酯.考察了影响溶胶凝胶包埋固定化CRL的因素,获得的最佳固定化条件为:丙基三甲氧基硅烷/正硅酸四乙酯摩尔比为1/1,水与硅烷前体摩尔比为15,酶的添加量为0.5mg/ml,PEG400的添加量为12μl/ml溶胶. 溶胶凝胶包埋的CRL在50℃,18h后其活性仍然保持了70.58%,是游离酶的2.6倍,且稳定性得到了明显的改善.基于CRL的界面特性,采用五种表面活性剂对其进行界面活化.结果表明,采用橄榄油活化的溶胶凝胶包埋的CRL合成维生素E琥珀酸酯的酯化活力最高,相比原酶和未界面活化的溶胶凝胶包埋酶分别提高了6.7和1.43倍.     

脂肪酶催化缩聚法合成可完全降解的聚羟基丙酸酯(英文)

以3-羟基丙酸甲酯为聚合单体,建立了以固定化脂肪酶Novozym 435为催化剂的酶催化缩聚反应体系,合成可完全降解的高分子聚酯聚羟基丙酸酯,考察了反应条件和介质对反应性能的影响,结果表明,纯度大于95%的单体即可在温和条件下合成聚羟基羧酸酯;降低反应压力可有效提升产物产率和分子量.通过选择合适的有机溶剂介质和表面活性剂,可使产物分子量提升至13000(Mw)以上.脂肪酶催化剂重复利用能力优异,经6批次反应后,其相对活性保持在95%以上.     

来源于类芽孢杆菌属新型耐有机溶剂脂肪酶的克隆、表达与性质研究（英文）

脂肪酶(EC 3.1.1.3)全称为三酰基甘油水解酶,是一类能够将长链脂肪酸甘油酯水解成脂肪酸和二甘酯、单甘酯或甘油的酯键水解酶.它除了能够水解脂肪外,还具有催化酯化反应、酯交换反应、酸解反应、醇解反应以及氨解等反应的性质.在脂肪酶催化的反应中,通常用有机溶剂代替水.有机溶剂可以转移合成反应的平衡方向,通过溶剂工程修饰酶的选择性能够提高底物的溶解度、有机相产物的回收率、酶的热稳定性.但有机溶剂对酶活性和稳定性有不同程度的影响.因此,寻找在有机溶剂中表现出高活性和稳定性的脂肪酶是一个亟待解决的重要课题.由于微生物种类多、作用底物专一性强,且微生物来源的脂肪酶一般分泌到胞外,因此微生物脂肪酶是工业用脂肪酶的重要来源.目前,微生物脂肪酶的研究主要集中于根霉属(Rhizopus)、曲霉属(Aspergillus)、青霉属(Penicillium)、毛霉属(Mucor)、地霉属(Geotrichum)、假丝酵母属(Candida)、假单胞菌属(Pseudomonas)、伯克霍尔德菌属(Burkholderia)等具有工业应用价值的菌株.很少有类芽孢杆菌属所产脂肪酶进行相关酶学性质的研究.我们以Paenibacillus pasadenensis CS0611为出发菌株,在全基因序列草图中得到了一个新型脂肪酶基因lp2252.以Paenibacillus pasadenensis CS0611基因组为模板,设计特异性引物对目标序列进行扩增,并成功将其插入到表达载体p ET-28a中得到含有目的基因的重组质粒.在E.coli BL21(DE3)中,脂肪酶lp2252经0.1mmol/L的IPTG诱导后在20°C实现了高水平表达.重组脂肪酶的活性约为野生型的1631倍.用镍离子亲和层析柱快速、高效地纯化了两端带有组氨酸标签的重组脂肪酶,回收率为63.5%,纯化因子为10.78.纯化后的脂肪酶最适温度为50°C,在20-40°C范围内具有良好的稳定性.最适pH值为7,属于中性脂肪酶,同时在pH 3.0-8.0间具有较高稳定性.在金属离子如钙、镁离子和一些非离子表面活性剂的作用下,其活性有所提高.此外,纯化后的脂肪酶可被一系列水溶性有机溶剂激活,例如一些短链醇.而对某些水不溶性有机溶剂,其也具有高度的耐受性.综上所述,本文所涉新型脂肪酶在非水相催化领域具有广泛的应用和前景.     

离子液体中糖酯类化合物的酶法合成

糖酯作为一种非离子型表面活性剂, 由于特殊的两亲结构, 已广泛应用于医药、食品及化妆品工业. 除作为表面活性剂外, 一些糖酯及其衍生物还具有抗菌及抗肿瘤等生物活性. 酶法合成糖酯反应条件温和, 选择性高. 有机介质中酶促糖酯合成的困难在于糖类化合物的低溶解度. 离子液体作为新型绿色反应介质, 用于糖酯类化合物的酶法合成具有许多优点. 综述了近年来该领域的研究进展.     

有机相中脂肪酶催化糖酯合成的研究

研究了有机相中脂肪酶催化以单糖和乙酸乙烯酯或乙酸酐为底物的糖脂合成反应。建立了定性、定量检测糖酯的方法，考察了７种脂肪酶催化糖酯合成的活力，发现来自假单孢菌属的ＰＳＬ１的活力最高。研究了ＰＳＬ１对不同单糖底物的选择性，发现对甘露醇的选择性最好，转化率可达９５％。研究了反应体系中的含水量对糖酯合成的影响，探讨了酶浓度和温度对反应的影响。     

纳米-微米复合孔泡沫陶瓷固定化脂肪酶

考察了泡沫陶瓷的孔径分布和表面性质对脂肪酶固定化的影响.研究表明,泡沫陶瓷的纳米孔孔径分布非常适合脂肪酶的固定化,对固定化酶的催化效率有决定性的影响.经1h的定化,泡沫陶瓷固定化酶的活性达商业化硅藻土固定化酶的1.33倍,体积活力为其2.63倍,蛋白载量为45.36mg/g陶瓷,比活为1215.39U/g,活力回收为41.2%.泡沫陶瓷固定化脂肪酶在有机相乙酸乙酯合成中表现优良,连续使用5次,每次反应3h,乙酸转化率均在93%左右.     

十六烷基三甲基溴化铵/山梨醇酐硬脂酸酯微乳液凝胶固定化脂肪酶的催化活性及立体选择性

制备了能在水溶液中长时间稳定存在的十六烷基三甲基溴化铵/山梨醇酐硬脂酸酯（CTAB/Span-60）微乳液凝胶（MBG）,确定了Span-60在乳化剂EM（正丁醇与Span-60的混合物）中的质量分数范围;分别以正己酸与正己醇的酯化反应、α-单硬脂酸甘油酯的水解反应、消旋布洛芬与正辛醇的不对称酯化反应为指示反应,研究了CTAB/Span-60 MBG固定化脂肪酶的催化活性及立体选择性。 结果表明,Span-60在EM中的质量分数小于57%时可形成机械强度较好的CTAB/Span-60 MBG;其固定化脂肪酶在有机溶剂中的酯化活性随EM中Span-60含量的增加先是逐渐增大,35%时最大,后又逐渐小幅度降低,在所考察的Span-60含量范围内均比在CTAB MBG中高;在水溶液中固定化脂肪酶能顺利催化α-单硬脂酸甘油酯的水解反应,24 h后反应转化率不再随反应时间的延长而增加,其水解活性在重复使用9次后仅降低13.68%,表明CTAB/Span-60 MBG固定化脂肪酶能够顺利进行分离并重复使用;此体系的脂肪酶也选择性地催化生成S-构型布洛芬辛酯,产物对映体过量值（eee）随反应的进行缓缓下降,但降幅不大,即其立体选择性要比在CTAB MBG中高。 因此,CTAB/Span-60 MBG作为脂肪酶固定化载体既可用于有机溶剂中又可用于水溶液中的生物合成与生物转化反应,扩大了微乳液凝胶固定化脂肪酶的应用范围。     

Immobilization and Properties of Lipase from Candida rugosa on Electrospun Nanofibrous Membranes with Biomimetic Phospholipid Moities

Reported here is a protocol to fabricate a biocatalyst with high enzyme loading and activity retention, from the conjugation of electrospun nanofibrous membrane having biomimetic phospholipid moiety and lipase. To improve the catalytic efficiency and activity of the immobilized enzyme, poly（acrylonitrile-co-2-methacryloyloxyethyl phosphorylcholine）s（PANCMPCs） were, respectively, electrospun into nanofibrous membranes with a mean diameter of 90 nm, as a support for enzyme immobilization. Lipase from Candida rugosa was immobilized on these nanofibrous membranes by adsorption. Properties of immobilized lipase on PANCMPC nanofibrous membranes were compared with those of the lipase immobilized on the polyacrylonitrile（PAN） nanofibrous and sheet membranes, respectively. Effective enzyme loading on the nanofibrous membranes was achieved up to 22.0 mg/g, which was over 10 times that on the sheet membrane. The activity retention of immobilized lipase increased from 56.4% to 76.8% with an increase in phospholipid moiety from 0 to 9.6%（molar fraction） in the nanofibrous membrane. Kinetic parameter Km was also determined for free and immobilized lipase. The Km value of the immobilized lipase on the nanofibrous membrane was obviously lower than that on the sheet membrane. The optimum pH was 7.7 for free lipase, but shifted to 8.3-8.5 for immobilized lipases. The optimum temperature was determined to be 35 ℃ for the free enzyme, but 42-44℃ for the immobilized ones, respectively. In addition, the thermal stability, reusability, and storage stability of the immobilized lipase were obviously improved compared to the free one.     

Lipase-catalyzed optical resolution of racemic naproxen in biphasic enzyme membrane reactors

A lipase-immobilized membrane reactor was applied for the optical resolution of racemic naproxen, and the effect of various operation conditions on reaction rate and enantio-selectivity was examined. The membrane reactor consisted of an organic phase dissolving naproxen ester, a lipase-immobilized polyamide membrane, and an aqueous phase to recover the reaction products. The lipase immobilized in the membrane reactor showed a stable activity for more than 200 h of continuous operation, while the native lipase in emulsioned stirred tank reactor quickly lost its activity showing an half-life time of about 2 h. When crude lipase was used, the biphasic enzyme membrane reactor gave smaller enantio-selectivity compared to the native lipase in the emulsioned tank reactor, whilst the use of pure lipase gave similar results to the native lipase. This paper discusses that other hydrolases present in the crude enzyme powder caused the decrease of enantio-selectivity. Enantio-selectivity depended on the substrate concentration, amount of enzyme loaded in the membrane and immobilization site. In fact, these parameters affect the organic/aqueous interface that plays an important role in the enhancement of enantio-selectivity.     

有机相中α-氰基-3-苯氧基苄醇乙酯的酶促醇解反应

研究了有机相中脂肪酶催化α-氰基-3-苯氧基苄醇乙酯的醇解化反应。制备α -氰基-3-苯氧基苄醇。考察了酶、溶剂、醇、醇用量、溶剂水含量以及底物浓度等 因素对反应的影响，结果表明Novozym435脂肪酶催化活性最高，经实验确定的最佳 条件为：脱水甲苯为溶剂，正辛醇为酰基受体，正辛醇、酯的摩尔比为1.5:1，酶 量为8 mg/mL时的最佳底物浓度为108.13 mmol/L，在上述条件下反应30 h酯的转化 率 > 96%。     

Carbon-carbon bonds by hydrolytic enzymes

Enzymes are efficient catalysts in synthetic chemistry, and their catalytic activity with unnatural substrates in organic reaction media is an area attracting much attention. Protein engineering has opened the possibility to change the reaction specificity of enzymes and allow for new reactions to take place in their active sites. We have used this strategy on the well-studied active-site scaffold offered by the serine hydrolase Candida antarctica lipase B (CALB, EC 3.1.1.3) to achieve catalytic activity for aldol reactions. The catalytic reaction was studied in detail by means of quantum chemical calculations in model systems. The predictions from the quantum chemical calculations were then challenged by experiments. Consequently, Ser105 in CALB was targeted by site-directed mutagenesis to create enzyme variants lacking the nucleophilic feature of the active site. The experiments clearly showed an increased reaction rate when the aldol reaction was catalyzed by the mutant enzymes as compared to the wild-type lipase. We expect that the new catalytic activity, harbored in the stable protein scaffold of the lipase, will allow aldol additions of substrates, which cannot be reached by traditional aldolases.     

Immobilized Lipase from Candida sp. 99–125 on Hydrophobic Silicate: Characterization and Applications

Lipase Candida sp. 99–125 has been proved to be quite effective in catalyzing organic synthesis reactions and is much cheaper than commercial lipases. Mesoporous silicates are attractive materials for the immobilization of enzymes due to their unique structures. The present research designed a hydrophobic silicate with uniform pore size suitable for the comfort of lipase Candida sp. 99–125 for improving its activity and stability. The resulting immobilized lipase (LP@PMO) by adsorption was employed to catalyze hydrolysis, esterification, and transesterification reactions, and the performances were compared with the lipase immobilized on hydrophilic silicate (LP@PMS) and native lipase. The LP@PMO showed as high activity as that of native lipase in hydrolysis and much increased catalytic activity and reusability in the reactions for biodiesel production. Besides, LP@PMO also possessed better organic stability. Such results demonstrate that immobilization of lipase onto hydrophobic supports is a promising strategy to fabricate highly active and stable biocatalysts for applications.