南极假丝酵母脂肪酶B的酿酒酵母表面展示及其催化己酸乙酯的合成

从南极假丝酵母(Candida antarctica)基因组克隆得到南极假丝酵母脂肪酶B(Candida antarctica Lipase B, CALB)全基因片段, 利用连接肽celA Linker将CALB与酿酒酵母细胞表面展示蛋白a-凝集素的C端连接融合, 构建表面展示载体pICAS-celAL-CALB, 转化酵母后获得重组酵母菌Saccharomyces cerevisiae pICAS-celAL-CALB。该重组酵母菌经葡萄糖诱导表达及分析, 表明CALB已在酿酒酵母细胞表面成功展示, 水解活力达26.26 u/(g·dry cell)。重组酵母菌经冻干能有效地实现在非水相中全细胞催化己酸和乙醇酯化合成己酸乙酯。反应物己酸与乙醇的摩尔比为1:1.25, 己酸乙酯的产率为98.0%, 具有较好的操作稳定性。     

展示南极假丝酵母脂肪酶B的毕赤酵母全细胞催化合成短链芳香酯

展示酶的酵母细胞作为全细胞催化剂,既具有固定化酶的优点,又有制备简单、成本较低的特点。本研究将细胞表面展示南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)的重组毕赤酵母用于非水相中催化合成短链芳香酯,通过滴定和气相色谱的方法测定底物酸的转化率,从底物的碳链长度、醇的结构、酵母冻干粉的添加量、底物浓度及底物的酸醇摩尔比等方面考察了展示CALB的毕赤酵母全细胞催化合成短链芳香酯的特性。研究结果表明:该全细胞催化剂可催化C10以下的酸和醇直接酯化合成多种短链芳香酯,酸的转化率达到90%以上;其中己酸和乙醇为酶的最适底物;酵母冻干粉的添加量20g/L(306.0U/g-drycell)、己酸浓度0.8mol/L、酸醇摩尔比1:1.1是合成己酸乙酯的最佳条件。在此条件下反应1.5h,己酸的转化率达到97.3%。在现有的关于脂肪酶非水相催化合成短链芳香酯的报道中,该全细胞催化剂显示出较好的底物耐受性以及较高的催化反应速率。因此,展示CALB的毕赤酵母全细胞催化剂在合成短链芳香酯方面具有较大的商业化应用潜能。     

酵母表面展示脂肪酶合成己二酸二异辛酯

展示酶的酵母细胞既具有固定化酶的优点,又有制备简单、成本较低的特点.采用表面展示南极假丝酵母脂肪酶B (Candida antarctica lipase B,CALB)的毕赤酵母细胞催化合成己二酸二异辛酯(Diisooctyl adipate,DIOA),对该反应体系进行优化,并实现了初步工艺放大制备.经条件优化后,在10mL反应体系中,DIOA的产率可达85.0％.该工艺放大到200mL反应体系时,DIOA产率可达97.8％.经减压蒸馏,DIOA纯度可达到98.2％.该酵母表面展示脂肪酶在合成绿色润滑油己二酸二异辛酯中具有良好应用前景.     

毕赤酵母表面展示南极假丝酵母脂肪酶B全细胞催化合成葡萄糖月桂酸酯

利用表面展示南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)的毕赤酵母细胞为全细胞催化剂,以葡萄糖为酰基受体,月桂酸为酰基供体,在非水相体系中催化合成糖酯。用硅胶柱层析对产物进行初提,再用制备液相色谱进一步分离纯化,并用高效液相色谱-质谱鉴定纯品性质。对该酶法合成糖脂反应体系进行了优化,其中考察了有机溶剂种类、复合溶剂体系中二甲基亚砜(DMSO)体积百分比、酶量、底物摩尔比、水活度和温度等几个影响酯化反应的因素。结果表明:在5mL反应体系中,以叔戊醇/二甲基亚砜(DMSO30%,V/V)为反应介质,添加初始水活度为0.11的全细胞催化剂0.5g,葡萄糖0.5mmol/L,月桂酸1.0mmol/L,60°C下反应72h后,葡萄糖月桂酸单酯的转化率达到48.7%。     

理性设计提高酯合成催化反应脂肪酶的热稳定性

【背景】南极假丝酵母脂肪酶B (Candida antarctica lipase B,CALB)具有优异的酯合成活性,是在非水相催化中应用极为广泛的工业用酶。【目的】在保留CALB优秀催化性能的基础上,提高CALB的热稳定性。【方法】采用预测软件PoPMuSiC和FoldX计算CALB潜在热稳定性突变位点,并根据氨基酸残基的空间位置进一步筛选。利用重叠延伸PCR技术在基因calb中引入10个单点突变,于毕赤酵母GS115中表达。【结果】点突变A146G、A151P、L278M均能有效提高CALB的热稳定性。在单点突变的基础上,组合突变体A146G-L278M和A146G-L278M-A151P的热稳定性得到进一步提高。与野生型相比,突变体A146G-L278M和A146G-L278M-A151P的最适反应温度均提高了5°C,T_m值分别提高了3.3°C和4.2°C。此外,合成己酸乙酯的酶促反应动力学分析表明,相比于野生型,突变体A146G-L278M和A146G-L278M-A151P对己酸和乙醇均具有更高的亲和力,且对己酸的催化效率k_(catA)/K_(m A)是野生型的4.1倍。通过分子动力学模拟,从分子水平阐明了突变体A146G-L278M和A146G-L278M-A151P热稳定性提高的机制。【结论】本研究采用的理性设计策略对提高CALB的热稳定性是行之有效的,该策略可作为其他工业用酶提高热稳定性的参考。     

南极假丝酵母脂肪酶A的表面展示及酶学性质研究

采用a凝集素作为载体蛋白,首次将南极假丝酵母脂肪酶A展示在酿酒酵母细胞表面,通过MD平板筛选获得表面展示型的CALA酵母工程菌株。免疫荧光检测显示CALA被成功展示在酵母细胞壁表面,重组子经诱导后能在三丁酸甘油酯板上形成透明圈,说明展示的CALA具有活性。重组酵母在液体培养基培养72 h,活性达到最高,为80.4 U/g干细胞。酿酒酵母展示的CALA最适温度及pH值为70°C和pH 8.0。经50°C保温2 h,仍含有60%水解酶活力。展示的CALA在pH 7.0和pH 8.0溶液中比较稳定。经DMSO处理2 h,展示的CALA仍保持70%的活性。以上结果表明酵母展示的CALA可作为一种有潜质商业用途的全细胞催化剂。     

利用α凝集素在毕赤酵母表面展示脂肪酶

目的:将南极假丝酵母脂肪酶B(CALB)通过α凝集素3’末端功能区域展示在毕赤酵母表面。方法:采用PCR方法扩增得到CALB成熟肽编码基因,将其连接到α凝集素3’末端的上游再与穿梭载体pPIC9K连接,构建表面展示载体p KNS-CALB。检测其水解活力和相关酶学性质。结果:展示CALB的毕赤酵母在甲醇的诱导下,表现出水解活性,最高可达382 U/g干细胞。对展示CALB的酶学性质研究表明:其最适温度为45℃,最适pH为8.0,60℃水浴4 h后残留酶活力高于最大酶活力的50%,其水解对硝基苯酚丁酸酯的酶活力最高。结论:利用α凝集素成功将CALB展示于毕赤酵母表面,酶活力有较大提高。     

南极假丝酵母脂肪酶B催化3-(4-氟苯基)戊二酸酐不对称醇解

采用分子模拟技术,研究了南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)催化3-(4-氟苯基)戊二酸酐(3-FGA)不对称醇解的分子机制。首先借助力场修改的Autodock 4.2软件将过渡态底物与CALB进行对接,根据对接自由能差异解析了CALB催化3-FGA与不同醇反应的立体选择性差异,得到的S型底物结合能小于R型底物;其次,基于扭转角机制分析发现,S型底物扭转角小于R型底物,从分子水平上揭示了CALB对S型底物选择性优于R型底物的机制。     

毕赤酵母展示表达南极假丝酵母脂肪酶B

将南极假丝脂肪酶B(CALB)基因N端和C端,分别与酿酒酵母絮凝蛋白(Flo1p)絮凝结构域序列的N端(FS)和C端(FL)融合,构建成脂肪酶毕赤酵母表面展示载体KFS和KFL,并转化毕赤酵母GS115后获得重组子KFS-CALB和KFL-CALB。免疫荧光检测证实脂肪酶已展示于毕赤酵母细胞表面。甲醇诱导120 h后展示酶活性分别达到286 U/g干细胞和182 U/g干细胞。酶的热稳定性较游离酶有较大提高,50℃孵育4 h后KFS-CALB菌株的残留酶活力仍保持初始酶活力70%以上;KFL-CALB在50℃孵育2 h后的酶活力也达到初始酶活力50%,远远高于游离态的CALB,其在50℃孵育0.5 h后仅残留18%的初始酶活力。     

MXR1基因对毕赤酵母工程茵代谢调控的影响

为了研究毕赤酵母中转录因子Mxrlp在毕赤酵母代谢调控中所起的作用,构建一株以南极假丝酵母脂肪酶B基因(CALB)作为报告基因,MXR1基因完全缺失的毕赤酵母基因工程菌株.将重组质粒pPIC9K-CALB转化毕赤酵母GS115,利用三丁酸甘油酯平板筛选得到分泌表达CALB的重组茵GS115/pPIC9K-CALB.通过重叠延伸PCR方法获得一段中间含有博来霉素抗性基因sh ble,两翼大约各有1 200 bp与毕赤酵母MXR1基因上下游同源的基因片段,将此片段用氯化锂法转化毕赤酵母细胞GS1 15/pPIC9 K-CALB后,利用博来霉素抗性及CALB酶活力丧失双重筛选的方法得到一株MXR1基因完全缺失的毕赤酵母基因工程菌株.该菌株在以甲醇为唯一碳源的培养基中不生长,在以乙醇、葡萄糖或者甘油为唯一碳源的培养基中生长缓慢.结果表明转录Mxrlp因子在毕赤酵母中的多条代谢途径中起着关键性的作用,主要涉及甲醇、乙醇、甘油和葡萄糖等代谢途径.     

Development of yeast cells displaying <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B and their application to ester synthesis reaction

We isolated the lipase B from Candida antarctica CBS 6678 (CALB CBS6678) and successfully constructed CALB-displaying yeast whole-cell biocatalysts using the Flo1p short (FS) anchor system. For the display of CALB on a yeast cell surface, the newly isolated CALB CBS6678 exhibited higher hydrolytic and ester synthesis activities than the well-known CALB, which is registered in GenBank (Z30645). A protease accessibility assay using papain as a protease showed that a large part of CALB, approximately 75%, was localized on an easily accessible part of the yeast cell surface. A comparison of the lipase hydrolytic activities of yeast whole cells displaying only mature CALB (CALB) and those displaying mature CALB with a Pro region (ProCALB) revealed that mature CALB is preferable for yeast cell surface display using the Flo1p anchor system. Lyophilized yeast whole cells displaying CALB were applied to an ester synthesis reaction at 60°C using adipic acid and n-butanol as substrates. The amount of dibutyl adipate (DBA) produced increased with the reaction time until 144 h. This indicated that CALB displayed on the yeast cell surface retained activity under the reaction conditions.     

Expression in Pichia pastoris of Candida antarctica lipase B and lipase B fused to a cellulose-binding domain

Candida antarctica lipase B (CALB) and C. antarctica lipase B fused to a cellulose-binding domain (CBD-CALB) were expressed functionally in the methylotrophic yeast Pichia pastoris. The cellulose-binding domain originates from cellulase A of the anaerobic rumen fungus Neocallimastix patriciarum. The genes were fused to the alpha-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. The recombinant proteins were secreted into the culture medium reaching levels of approximately 25 mg/L. The proteins were purified using hydrophobic interaction chromatography and gel filtration with an overall yield of 69%. Results from endoglycosidase H digestion of the proteins showed that CALB and CBD-CALB were N-glycosylated. The specific hydrolytic activities of recombinant CALB and CBD-CALB were identical to that reported for CALB isolated from its native source. The fusion of the CBD to the lipase resulted in a greatly enhanced binding toward cellulose for CBD-CALB compared with that for CALB.     

CLEAs of Candida antarctica lipase B (CALB) with a bovine serum albumin (BSA) cofeeder core: Study of their catalytic activity

Highly active CALB cross-linked enzyme aggregates (CLEAs) were synthesized using a layered methodology based on the synthesis of a cross-linked protein cofeeder core over which an external layer of lipase was later cross-linked. The layered CALB CLEAs were characterized in terms of their catalytic activity in three different test reactions: esterification of oleic acid and ethanol in absence of solvents, esterification of oleic acid and heptanol in organic medium, and hydrolysis of triolein in emulsioned medium. The impact of the cross-linker/protein mass ratio on CLEAs activity, and its evolution with storage time were evaluated in the solventless synthesis of ethyloleate. The amount of cross-linker used showed to be a key parameter for the evolution of the catalytic activity of CLEAs during storage. Under the best conditions found, hyperactivated CALB CLEAs with up to 188% of recovered activity in ethyl oleate synthesis were obtained. In terms of hydrolytic activity mature layered CALB CLEAs showed a retained activity of 68%. The assay of dried mature layered CALB CLEAs in heptyl oleate synthesis showed catalytic activities much higher than the one exhibited by free CALB, reaching 1 h-fatty acid conversions of 14% and 2%, respectively. The high catalytic activity shown by layered CALB CLEAs, suggests that they are an interesting alternative specially for the catalysis of fatty acid esterifications in both organic and solventless medium.     

Evaluation of immobilized lipases on poly-hydroxybutyrate beads to catalyze biodiesel synthesis

Five microbial lipase preparations from several sources were immobilized by hydrophobic adsorption on small or large poly-hydroxybutyrate (PHB) beads and the effect of the support particle size on the biocatalyst activity was assessed in the hydrolysis of olive oil, esterification of butyric acid with butanol and transesterification of babassu oil (Orbignya sp.) with ethanol. The catalytic activity of the immobilized lipases in both olive oil hydrolysis and biodiesel synthesis was influenced by the particle size of PHB and lipase source. In the esterification reaction such influence was not observed. Geobacillus thermocatenulatus lipase (BTL2) was considered to be inadequate to catalyze biodiesel synthesis, but displayed high esterification activity. Butyl butyrate synthesis catalyzed by BTL2 immobilized on small PHB beads gave the highest yield (≈90 mmol L(-1)). In biodiesel synthesis, the catalytic activity of the immobilized lipases was significantly increased in comparison to the free lipases. Full conversion of babassu oil into ethyl esters was achieved at 72 h in the presence of Pseudozyma antarctica type B (CALB), Thermomyces lanuginosus lipase (Lipex(?) 100 L) immobilized on either small or large PHB beads and Pseudomonas fluorescens (PFL) immobilized on large PHB beads. The latter preparation presented the highest productivity (40.9 mg of ethyl esters mg(-1) immobilized protein h(-1)).     

Expression in Pichia pastoris of Candida antarctica Lipase B and Lipase B Fused to a Cellulose-Binding Domain

Candida antarctica lipase B (CALB) and C. antarctica lipase B fused to a cellulose-binding domain (CBD-CALB) were expressed functionally in the methylotrophic yeast Pichia pastoris. The cellulose-binding domain originates from cellulase A of the anaerobic rumen fungus Neocallimastix patriciarum. The genes were fused to the α-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. The recombinant proteins were secreted into the culture medium reaching levels of approximately 25 mg/L. The proteins were purified using hydrophobic interaction chromatography and gel filtration with an overall yield of 69%. Results from endoglycosidase H digestion of the proteins showed that CALB and CBD-CALB were N-glycosylated. The specific hydrolytic activities of recombinant CALB and CBD-CALB were identical to that reported for CALB isolated from its native source. The fusion of the CBD to the lipase resulted in a greatly enhanced binding toward cellulose for CBD-CALB compared with that for CALB.     

Combination of site-directed mutagenesis and yeast surface display enhances <Emphasis Type="Italic">Rhizomucor miehei</Emphasis> lipase esterification activity in organic solvent

To increase the activity of Rhizomucor miehei lipase (RML) in organic solvent, multiple sequence alignments and rational site-directed mutagenesis were used to create RML variants. The obtained proteins were surface-displayed on Pichia pastoris by fusion to Flo1p as an anchor protein. The synthetic activity of four variants showed from 1.1- to 5-fold the activity of native lipase in an esterification reaction in heptane with alcohol and caproic acid as substrates. The increase in esterification activity may be attributed to the four mutations changing the flexibility of RML or facilitating the reaction. In conclusion, this method demonstrated that multiple sequence alignments and rational site-directed mutagenesis combined with yeast display technology is a faster and more effective means of obtaining high-efficiency esterification lipase variants compared with previous similar methods.     

Display of Candida antarctica lipase B on Pichia pastoris and its application to flavor ester synthesis

Two alternative cell-surface display systems were developed in Pichia pastoris using the α-agglutinin and Flo1p (FS) anchor systems, respectively. Both the anchor cell wall proteins were obtained originally from Saccharomyces cerevisiae. Candida antarctica lipase B (CALB) was displayed functionally on the cell surface of P. pastoris using the anchor proteins α-agglutinin and FS. The activity of CALB displayed on P. pastoris was tenfold higher than that of S. cerevisiae. The hydrolytic and synthetic activities of CALB fused with α-agglutinin and FS anchored on P. pastoris were investigated. The hydrolytic activities of both lipases displayed on yeast cells surface were more than 200 U/g dry cell after 120 h of culture (200 and 270 U/g dry cell, respectively). However, the synthetic activity of CALB fused with α-agglutinin on P. pastoris was threefold higher than that of the FS fusion protein when applied to the synthesis of ethyl caproate. Similarly, the CALB displayed on P. pastoris using α-agglutinin had a higher catalytic efficiency with respect to the synthesis of other short-chain flavor esters than that displayed using the FS anchor. Interestingly, for some short-chain esters, the synthetic activity of displaying CALB fused with α-agglutinin on P. pastoris was even higher than that of the commercial CALB Novozyme 435.     

Hydrolysis and Esterification with Lipase from Candida Cylindracea. Influence of the Reaction Conditions and Acid Moiety on the Enantiomeric Excess

The enantiomeric ratio for hydrolysis and synthesis of 1-phenyl ethanol esters of straight chain aliphatic carboxylic acids catalyzed by Candida cylindracea lipase was determined. A distinct maximum in enantiomeric ratio was observed for valeric and caproic acid in the hydrolytic direction. No significant maximum could be determined in the esterification reaction. Even though the enzyme provided larger enantiomeric ratios in the synthetic direction the enantiomeric excess of the alcohol was not higher. The enantiomeric excess was depressed by racemization reactions in the esterification as the reaction approached thermodynamic equilibrium at an insufficient conversion. While choosing the optimal chain length of the acyl donor is important in hydrolytic reactions it seems to be of greater value to raise the equilibrium conversion in the esterification reactions.     

Synthesis of phosphatidylcholine with defined fatty acid in the sn-1 position by lipase-catalyzed esterification and transesterification reaction

The incorporation of caproic acid in the sn-1 position of phosphatidylcholine (PC) catalyzed by lipase from Rhizopus oryzae was investigated in a water activity-controlled organic medium. The reaction was carried out either as esterification or transesterification. A comparison between these two reaction modes was made with regard to product yield, product purity, reaction time, and byproduct formation as a consequence of acyl migration. The yield in the esterification and transesterification reaction was the same under identical conditions. The highest yield (78%) was obtained at a water activity (a(w)) of 0.11 and a caproic acid concentration of 0.8 M. The reaction time was shorter in the esterification reaction than in the transesterification reaction. The difference in reaction time was especially pronounced at low water activities and high fatty acid concentrations. The loss in yield due to acyl migration and consequent enzymatic side reactions was around 16% under a wide range of conditions. The incorporation of a fatty acid in the sn-1 position of PC proved to be thermodynamically much more favorable than the incorporation of a fatty acid in the sn-2 position.     

Synthetic resin-bound truncated Candida antarctica lipase B for production of fatty acid alkyl esters by transesterification of corn and soybean oils with ethanol or butanol

A gene encoding a synthetic truncated Candida antarctica lipase B (CALB) was generated via automated PCR and expressed in Saccharomyces cerevisiae. Western blot analysis detected five truncated CALB variants, suggesting multiple translation starts from the six in-frame ATG codons. The longest open reading frame, which corresponds to amino acids 35-317 of the mature lipase, appeared to be expressed in the greatest amount. The truncated CALB was immobilized on Sepabeads? EC-EP resin and used to produce ethyl and butyl esters from crude corn oil and refined soybean oil. The yield of ethyl esters was 4-fold greater from corn oil than from soybean oil and was 36% and 50% higher, respectively, when compared to a commercially available lipase resin (Novozym 435) using the same substrates. A 5:1 (v/v) ratio of ethanol to corn oil produced 3.7-fold and 8.4-fold greater yields than ratios of 15:1 and 30:1, respectively. With corn oil, butyl ester production was 56% higher than ethyl ester production. Addition of an ionic catalytic resin step prior to the CALB resin increased yields of ethyl esters from corn oil by 53% compared to CALB resin followed by ionic resin. The results suggest resin-bound truncated CALB has potential application in biodiesel production using biocatalysts.