有机相中包衣脂肪酶催化酯化反应动力学

用合成的谷氨酸十二烷基酯核糖醇对来源于Candida rugosa的脂肪酶包衣后在有机溶剂中催化月桂酸和月桂醇的酯化反应。确定了最佳反应条件：制备包衣酶用缓冲液的最适PH为6．8，温度30℃，有机溶剂为异辛烷，酯化反应符合单底物醇抑制的Ping-Pang Bi-Bi机制。在最适反应条件下，月桂酸和月桂醇的米氏常数分别为29．1mmol.L^-1和71．4mmol.L^-1，月桂醇的抑制常数为11．3mmol.L^-1，最大反应速率为0．66mmol.min^-1.g^-1。动力学模型预测值与实验结果吻合良好。     

在有机溶剂中猪胰脂肪酶催化合成己酸乙酯的研究

研究了有机溶剂中猪胰脂肪酶催化正己酸和无水乙醇合成己酸乙酯的反应。以正己酸和无水乙醇在各种反应介质中进行酶催化反应,用气相色谱检测生成的己酸乙酯,考察反应介质、底物浓度、底物摩尔比、加酶量、反应时间对产率的影响。发现在高底物浓度和低底物浓度的条件下,有机溶剂介质对反应的影响是不同的。采用中等底物浓度,选择lgP值适中的溶剂为介质最合适。适当的反应条件为:以环己烷为反应介质,正己酸浓度0.6mol/L,n(正己酸)∶n(无水乙醇)=1∶1.25,加酶量15g/L,反应温度37℃,反应24h后,产率达到90.5%。该文的底物浓度较高,是文献[1,10,11,17]报道的1.5～3.0倍,获得了相近的产率。     

表面活性剂包衣酶用于催化有机溶剂中的反应

用表面活性剂直接对酶进行包衣后用于有机相中的酶催化反应是近 10年来发展起来的一种新方法 ,与通常有机相中的酶催化反应方法相比 ,表面活性剂包衣酶具有制备容易、催化活性高、有机溶剂选择范围广等显著优点。该文介绍了表面活性剂包衣酶的概念、制备方法、催化活性等 ,并探讨了今后的研究方向     

Rational Design of Lipase ROL to Increase Its Thermostability for Production of Structured Tags

1,3-regiospecific lipases are important enzymes that are heavily utilized in the food industries to produce structured triacylglycerols (TAGs). The Rhizopus oryzae lipase (ROL) has recently gained interest because this enzyme possesses high selectivity and catalytic efficiency. However, its low thermostability limits its use towards reactions that work at lower temperature. Most importantly, the enzyme cannot be used for the production of 1,3-dioleoyl-2-palmitoylglycerol (OPO) and 1,3-stearoyl-2-oleoyl-glycerol (SOS) due to the high melting points of the substrates used for the reaction. Despite various engineering efforts used to improve the thermostability of ROL, the enzyme is unable to function at temperatures above 60 °C. Here, we describe the rational design of ROL to identify variants that can retain their activity at temperatures higher than 60 °C. After two rounds of mutagenesis and screening, we were able to identify a mutant ROL_10x that can retain most of its activity at 70 °C. We further demonstrated that this mutant is useful for the synthesis of SOS while minimal product formation was observed with ROL_WT. Our engineered enzyme provides a promising solution for the industrial synthesis of structured lipids at high temperature.     

表面活性剂包衣脂肪酶在有机溶剂中催化酯合成

用合成的谷氨酸二烷基酯核糖醇对来源于Candidarugosa的脂肪酶进行了包衣 ,以月桂酸与月桂醇的酯化为模型反应 ,研究了各种操作条件对包衣酶活性的影响。结果表明 ,包衣酶制备过程中的缓冲溶液的最适 pH为 6.8,谷氨酸双十二烷基酯核糖醇的包衣效果最好 ,最适反应温度为 3 0℃ ,最佳溶剂为异辛烷。在 10h内底物转化率达 94%。     

Discovery of the Key Mutation Site Influencing the Thermostability of Thermomyces lanuginosus Lipase by Rosetta Design Programs

Lipases are remarkable biocatalysts and are broadly applied in many industry fields because of their versatile catalytic capabilities. Considering the harsh biotechnological treatment of industrial processes, the activities of lipase products are required to be maintained under extreme conditions. In our current study, Gibbs free energy calculations were performed to predict potent thermostable Thermomyces lanuginosus lipase (TLL) variants by Rosetta design programs. The calculating results suggest that engineering on R209 may greatly influence TLL thermostability. Accordingly, ten TLL mutants substituted R209 were generated and verified. We demonstrate that three out of ten mutants (R209H, R209M, and R209I) exhibit increased optimum reaction temperatures, melting temperatures, and thermal tolerances. Based on molecular dynamics simulation analysis, we show that the stable hydrogen bonding interaction between H198 and N247 stabilizes the local configuration of the 250-loop in the three R209 mutants, which may further contribute to higher rigidity and improved enzymatic thermostability. Our study provides novel insights into a single residue, R209, and the 250-loop, which were reported for the first time in modulating the thermostability of TLL. Additionally, the resultant R209 variants generated in this study might be promising candidates for future-industrial applications.     

基于脯氨酸理论提高枯草芽孢杆菌脂肪酶A的热稳定性

以枯草芽孢杆菌脂肪酶A(LipA)为研究对象,根据从RCSB数据库中获取的晶体结构,采用分子动力学模拟和分子生物学实验相结合的方法进行脂肪酶热稳定性位点突变的理性设计。首先,利用分子动力学模拟获得晶体结构中柔性较高的Loop区域;进而,结合"脯氨酸理论",将位于该区域附近的Gly残基突变为Pro,分析引入Pro突变对LipA热稳定性的影响,筛选得到Gly52和Gly158两个突变位点;最后,通过定点突变操作对突变株LipAG52P和LipAG158P进行热稳定性实验验证。结果显示,突变株LipAG52P、LipAG158P的比活力分别是野生型LipA的5.6倍和2.7倍,Tm值分别提高了15℃和7℃,催化效率分别提高了85%和22%。     

The Stabilizing Effects of Polyols and Sugars on Porcine Pancreatic Lipase

Porcine pancreatic lipase (PPL) has been used as a biocatalyst for many years and is one of the most widely used enzymes for biotechnological applications; however, it is a rather complex mixture with various active enzymes. The present study has been undertaken to determine the effects of polyols and sugars (cosolvents) on the thermal stability of PPL preparation. The thermal stability of PPL exposed to 60°C for 10 min was enhanced in the presence of cosolvents in terms of both residual specific activity and conformational stability. Thermal denaturation, changes in circular dichroism, fluorescence spectra, apparent kinetic parameters, activity, and preferential interaction parameter of PPL preparation are discussed in terms of contributions to the mechanism of thermal stability and the activity enhancement. Partial specific volume measurement of PPL in the presence of cosolvents is presented for the first time. The preferential interaction parameter (ξ ₃) was negative in all cosolvents used, and maximum hydration was observed in the presence of trehalose, where the preferential interaction parameter was −0.076 g/g. The observed increase in the thermal stability of PPL preparation in the presence of cosolvents is due to the preferential hydration of the enzyme.     

溶剂脱毒汽爆玉米秸秆对酶解以及发酵的影响

以脱毒对酶解及发酵的影响为研究对象,以酶解还原糖得率及发酵乙醇质量浓度为指标,采用溶剂萃取的方法对无催化汽爆玉米秸秆进行萃取脱毒。结果表明,酶解还原糖得率随着萃取剂及萃取方式介于34.85%和89.7%,酶解还原糖得率和发酵乙醇质量浓度与脱毒有机溶剂的沸点高度负相关,表明有机溶剂的残留是导致酶失活的主要原因。而对于所考察的溶剂,乙醇产率为0.47～0.49 g乙醇/g还原糖,表明有机溶剂残留对乙醇发酵并无显著影响。采用乙醚和丙酮的组合萃取,乙醇最高产率可以达到理论值的96.1%。     

Purification and Characterization of a Lipase with High Thermostability and Polar Organic Solvent‐Tolerance from Aspergillus niger AN0512

An extracellular lipase (EC 3.1.1.3, AN0512Lip) from Aspergillus niger AN0512 was purified and its characteristics were investigated. After the process of ammonium sulfate precipitation followed by ion‐exchange chromatography and gel filtration, the purified lipase was achieved with 203.6‐fold purification and 22.1 % recovery. AN0512Lip exhibited the highest activity at 50 °C and pH 5.0. It was thermostable and pH‐stable, as indicated by that more than 50 % activity retained at 60 °C for 20 h and more than 90 % activity retained at pH 3.0 for 20 h, respectively. AN0512Lip activity was stimulated by some divalent metal ions (especially Cu²⁺, Ca²⁺), while greatly suppressed by EDTA, indicating that AN0512Lip was a metal‐activated enzyme. Moreover, AN0512Lip exhibited high tolerance for various polar organic solvents with log P < 0.8, and the highest lipase activity (476 % of its original activity) was achieved after addition of 90 % (V/V) isopropanol to the reaction mixture. AN0512Lip also displayed 3‐regiospecificity and great affinity for the long‐chain fatty ester. The preliminary test showed that AN0512Lip was a candidate for enriching EPA and DHA in fish oil. All the unique properties, such as thermostability, Cu²⁺‐dependent, 3‐regiospecificity, and polar organic solvent‐tolerance, indicated that AN0512Lip could have potential applications in the food industry, even in organic synthesis and the pharmaceutical industry.     

基于细胞性质分析不同有机溶剂对简单节杆菌C1,2脱氢反应的影响

通过对简单节杆菌细胞性质的研究,探讨了不同有机溶剂(乙醇、DMF、DMSO、甲醇)加入后醋酸可的松生物转化反应效率产生差异的原因。采用底物转化法检测生物转化率,采用平板活菌计数法计算细胞存活率,通过糖代谢活力保留值表征细胞的生理代谢活力,用原子力显微镜观察细胞超显微结构,以FDA法表征细胞膜通透性。在体积分数为4%的条件下,4种有机溶剂均能提高简单节杆菌生物转化醋酸可的松的初始转化速率和最终转化率,转化率从高到低依次为乙醇>DMSO>甲醇>DMF;4种溶剂对简单节杆菌存活率和糖代谢活力保留值的影响趋势基本一致,其中DMF最大,DMSO次之,乙醇和甲醇较小;4种溶剂中,甲醇对简单节杆菌细胞完整性的影响最大,乙醇次之,DMF和DMSO差别不明显;4种有机溶剂对简单节杆菌细胞膜通透性的影响大小依次为甲醇>乙醇>DMSO>DMF。上述结果表明,细胞生理代谢活力和细胞膜通透性的差异是造成转化率水平不同的重要原因。其中,细胞膜通透性的增强有利于底物分子更容易进入细胞与生物酶接触,进而提高转化率。     

几种有机溶剂对靶标病原菌生长影响的研究

采用离体和活体试验法测定了丙酮、乙醇、二甲基甲酰胺、二甲基亚砜等四种溶剂对小麦赤霉病菌、水稻纹枯病菌、油菜菌核病菌、黄瓜灰霉病菌、水稻稻瘟病菌、小麦白粉病菌生长的影响.结果表明,在离体测定中四种有机溶剂对靶标病原菌菌丝的生长都存在着不同程度的影响,以二甲基甲酰胺最为明显,抑制率达20%以上.在活体测定中,除了二甲基亚砜对小麦白粉病有一定影响外,四种有机溶剂对靶标病原菌无任何不良影响.     

苯基膦酸在有机溶剂中溶解度的测定及关联

采用本实验室自制的苯基膦酰二氯(DCPPO),在常温下缓慢水解后两次重结晶制得苯基膦酸(PPA),为白色结晶状粉末。通过酸度、质谱、核磁、熔点分析对产物进行了标定。采用滴定法测定了苯基膦酸在丙酮和苯中的溶解度,采用静态平衡法测定了在乙酸乙酯中的溶解度。并且对溶解度和温度进行了关联,回归了溶解度经验方程。     

Lipase purification by various techniques and its thermostabilization in presence of surface active additives

Crude porcine pancreatic lipase was purified by removing water-insoluble impurities (residual lipid material) associated with it by a conventional method, using hydrotropic additives and by liquid coacervate extraction. Maximum yield with good recovery of activity was obtained when hydrotropes were used to separate the associated lipids from lipase. The thermostability of the enzyme was also checked in the solutions of additives such as sodium butyl monoglycol sulfate (Na-BMGS), proline and Triton X-114. In Na-BMGS solutions above a concentration of 0·2 mol dm⁻³ the lipase activity decreased beyond 50°C whereas in 1 mol dm⁻³ proline solution it was retained even at 80°C, showing a good thermostabilizing effect. However, in the presence of Triton X-114 the enzyme was completely inactivated with increase in temperature. © 1998 SCI.     

表面活性剂包衣Candida rugosa脂肪酶在异辛烷中的稳定性

The stability of Candida rugosa lipase coated with glutamic acid didodecyl ester ribitol amide was investigated taking esterification of lauryl alcohol and lauric acid in isooctane as a model reaction.At 30℃,the half-life of the activity of the coated lipase was ca 10h,the enzyme activity became less changed after 12h and the residual activity was 39% of the initial value ,The coated lipase obeyed a first-order deactivation model with a deactivation energy of 29.9 J.mol^-1.     

Lipase covalently attached to multiwalled carbon nanotubes as an efficient catalyst in organic solvent

Lipase was covalently attached to multiwalled carbon nanotubes (MWNTs). Structural changes of the lipase upon attachment onto MWNTs were analyzed through circular dichroism and FTIR spectroscopy. The conjugate was utilized for the resolution of a model compound (R,S)‐1‐phenyl ethanol, and the reaction medium was n‐heptane. The enzymatic resolutions were carried out at temperatures from 35 to 60°C. The results show that the lipase attached onto MWNTs has significantly affected the performance of the enzyme in terms of temperature dependence and resolution efficiency. The activity of MWNT–lipase was less temperature‐dependent compared with that of the native lipase. The resolution efficiency was much improved with MWNT–lipase. MWNT–lipase retained the selectivity of the native lipase for (R)‐1‐phenyl ethanol. The consecutive use of MWNT–lipase showed that MWNT–lipase had a good stability in the resolution of (R,S)‐1‐phenyl ethanol. © 2010 American Institute of Chemical Engineers AIChE J, 2010     

Enzymatic Synthesis of Glucose- and Xylose Laurate Esters Using Different Acyl Donors,Higher Substrate Concentrations,and Membrane Assisted Solvent Recovery

Glucose- and xylose laurate esters are enzymatically synthesized using equimolar substrate concentrations in 2-methyl-2-butanol, comparing free lauric acid with methyl- and vinyl-laurate as acyl donors. All reactions result in ≥70% acyl donor conversions after 72 h but the activated donors are also partially hydrolyzed to lauric acid, highlighting the difficulty in controlling water presence in this particular reaction system. The esterification of xylose generates a complex product profile, with several regioisomers of monoesters and diesters. The esterification of glucose is quite selective, forming mainly the 6-O monoester (≥96%) with a small presence of two diester isomers (4%). Increasing substrate concentration up to 800 millimoles kg⁻¹ results in lower conversion values (down to 58%) but shows that the reaction proceeds successfully even in the presence of high amounts of insoluble glucose. However, the reaction is less selective and the proportion of diester increases, becoming up to 46% (molar fraction) of the final product. Solvent recovery after esterification can be achieved by organic solvent nanofiltration through a polymeric membrane able to retain ≥80% of all reaction substrates and products. Practical Applications: The use of high substrate concentrations during the enzymatic synthesis of sugar ester biosurfactants leads to product titers that are more industrially appealing, without the need to find a solvent that can solubilize all initial substrate. The sustainability of the enzymatic conversion at mild temperatures can be enhanced by recycling of the reaction solvent through organic solvent nanofiltration, an energy efficient alternative to other traditional methods like distillation.     

Enhancing Thermostability and Organic Solvent Tolerance of ω‐Transaminase through Global Incorporation of Fluorotyrosine

Here, we have utilized the incorporation of non‐canonical amino acids as a tool kit to improve enzyme properties for organic synthesis applications. The global incorporation of 3‐fluorotyrosine (FY) into ω‐transaminase (ω‐TA) to give ω‐TA[FY] enhanced the thermostability and organic solvent tolerance without altering substrate specificity and enantioselectivity. Moreover, ω‐TA[FY] was able to completely convert 25 mM of acetophenone into (S)‐1‐phenylethylamine (ee>99%) in the presence of 20% DMSO (v/v) which is ∼2‐fold higher when compared to wild‐type ω‐TA.

     

Expression of an Organic Solvent Stable Lipase from Staphylococcus epidermidis AT2

An organic solvent tolerant lipase gene from Staphylococcus epidermidis AT2 was successfully cloned and expressed with pTrcHis2 in E. coli TOP10. Sequence analysis revealed an open reading frame (ORF) of 1,933 bp in length which coded for a polypeptide of 643 amino acid residues. The polypeptide comprised of a signal peptide (37 amino acids), pro-peptide and a mature protein of 390 amino acids. Expression of AT2 lipase resulted in an 18-fold increase in activity, upon the induction of 0.6 mM IPTG after a 10 h incubation period. Interestingly, this lipase was stable in various organic solvents (25% (v/v), mainly toluene, octanol, p-xylene and n-hexane). Literature shows that most of the organic solvent stable bacterial lipases were produced by Pseudomonas sp. and Bacillus sp., but very few from Staphylococcus sp. This lipase demonstrates great potential to be employed in various industrial applications.     

有机溶剂在药厂工作场所的使用限制

在医药工业中常使用大量的有机溶剂,生产工艺过程中有机溶剂属于化学性危险和有害因素。对照我国卫生标准工作场所有害因素职业接触限值及药典中残留溶剂规定,分析医药生产中使用有机溶剂可能存在的职业病危害因素。为了使发生危险的概率尽可能小,应该关注和探讨医药行业有机溶剂的职业危害。