P197E与ep8叠加突变对扩展青霉脂肪酶热稳定性的影响

为提高脂肪酶的热稳定性,作者利用重叠延伸PCR对扩展青霉脂肪酶(PEL)基因进行了体外定点突变,构建了P197E(即将第197位的脯氨酸突变为谷氨酸)与随机突变体ep8叠加突变的重组质粒pPIC3.5K-ep8-P197E。将该质粒电转化至毕赤酵母Pichiapastoris GS115中,进行异源表达。与野生型酶和单点突变酶PEL-ep8的酶学性质比较,结果表明:叠加突变体PEL-ep8-P197E在40°C温育处理30min后,残余酶活分别比野生型PEL和随机突变体PEL-ep8提高了42.13%和37.3%。叠加突变体PEL-ep8-P197E的Tm值为41.51°C,比野生型酶PEL提高了2.81°C,比随机突变体脂肪酶PEL-ep8提高了2.25°C。通过对脂肪酶PEL的叠加突变,提高了该酶的热稳定性,并为结构与功能的进一步研究提供了材料。     

K55R与ep8叠加突变对扩展青霉脂肪酶热稳定性的改善

利用重叠延伸PCR对扩展青霉脂肪酶(PEL)基因进行体外定点突变,构建了K55R与随机突变体ep8叠加突变的重组质粒pAO815-ep8-K55R。将该质粒电转化引入毕赤酵母(Pichia pastoris)GS115,进行异源表达。实验结果表明:该叠加突变体在毕赤酵母中获得了活性表达,得到表达产物脂肪酶PEL-ep8-K55R-GS。其表达量为508u/mL,分别约为野生型脂肪酶PEL-GS(627u/mL)的81%,随机突变脂肪酶PEL-ep8-GS(924u/mL)的55%;其比活力为2309.1u/mg,与随机突变脂肪酶PEL-ep8-GS和野生型脂肪酶PEL-GS的相仿。叠加突变脂肪酶PEL-ep8-K55R-GS的最适作用温度为37℃,与野生型脂肪酶PEL-GS和随机突变脂肪酶PEL-ep8-GS一致;其Tm值为41.0℃,比野生型脂肪酶PEL-GS提高了2.3℃,比随机突变脂肪酶PEL-ep8-GS提高了0.8℃。表明叠加突变脂肪酶PEL-ep8-K55R-GS的热稳定性有了进一步的提高。     

扩展青霉脂肪酶K56R叠加突变对热稳定性的影响

目的:扩展青霉脂肪酶随机突变体ep8是一株热稳定性比野生型有所提高的突变体.获得热稳定性提高的优良菌株.方法:在ep8的基础上利用重叠延伸PCR构建叠加突变重组质粒pPIC3.5K-ep8一K56R,将该质粒电转毕赤酵母(Pichia paaoris)GS115进行异源表达.结果:该叠加突变脂肪酶在毕赤酵母中获得了活性表达.15%SDS-PACE结果分析表明突变脂肪酶PEL-ep8-K56R-GS分子量与野生型PEL-GS一致,约为28kDa.叠加突变脂肪酶在37℃时酶活为852U/mL、野生型为760u/mL、随机突变体为824u/mL,叠加突变体酶活相比野生型提高了21.1%,相比随机突变体提高了3.4%.热稳定性分析数据表明叠加突变脂肪酶Tm值为40.1℃、野生型为38.7℃、随机突变体为39.9℃,Tm值相比野生型提高了1.4℃,相比随机突变体提高了0.2℃.     

K55R与ep8叠加突变对扩展青霉脂肪酶热稳定性的改善

利用重叠延伸PCR对扩展青霉脂肪酶（PEL）基因进行体外定点突变,构建了K55R与随机突变体ep8叠加突变的重组质粒pAO815-ep8-K55R。将该质粒电转化引入毕赤酵母（Pichia pastoris）GS115,进行异源表达。实验结果表明：该叠加突变体在毕赤酵母中获得了活性表达,得到表达产物脂肪酶PEL-ep8-K55R-GS。其表达量为508u/mL,分别约为野生型脂肪酶PEL-GS（627u/mL）的81％,随机突变脂肪酶PEL-ep8-GS（924u/mL）的55%;其比活力为2309.1u/mg,与随机突变脂肪酶PEL-ep8-GS和野生型脂肪酶PEL-GS的相仿。叠加突变脂肪酶PEL-ep8-K55R-GS的最适作用温度为37℃,与野生型脂肪酶PEL-GS和随机突变脂肪酶PEL-ep8-GS一致;其T_m值为41.0℃,比野生型脂肪酶PEL-GS提高了2.3℃,比随机突变脂肪酶PEL-ep8-GS提高了0.8℃。表明叠加突变脂肪酶PEL-ep8-K55R-GS的热稳定性有了进一步的提高。     

扩展青霉脂肪酶ep8与R182K叠加突变体的构建

利用重叠延伸PCR法对扩展青霉碱性脂肪酶(PEL)基因进行体外定点突变,并将含突变基因的重组质粒pAO815-ep8-R182K在毕赤酵母(Pichiapastoris)GS115中进行表达。叠加突变体PEL-ep8-R182K表达产物与野生型PEL、PEL-ep8比较实验表明:叠加突变体表达蛋白PEL-ep8-R182K最适反应温度与野生型PEL、PEL-ep8一致,均为40℃;热稳定性与野生型相似,比PEL-ep8降低2.25℃。但是,在比活上,PEL-ep8-R182K与PEL-ep8、野生型PEL相比,其比酶活分别提高了14.03%和3.86%。     

基于易错PCR定向进化扩展青霉 Penicillium expansum FS1884碱性脂肪酶

为改善扩展青霉FS1884碱性脂肪酶的活性及酶学性质,利用连续两轮易错PCR对扩展青霉FS1884脂肪酶基因PEL进行随机突变,在大肠杆菌JM109中构建突变文库。含突变脂肪酶基因的重组质粒电击转化巴斯德毕赤酵母GS115,经过YPOM板初筛和橄榄油检验板复筛,获得一株酶活性提高的脂肪酶突变体:PEL-ep25-GS。与野生型脂肪酶PEL-GS相比,在最适温度40℃、pH9.4时突变体的酶活力是野生型酶的1.3倍。测序结果表明:该突变体第253位氨基酸发生了突变,由赖氨酸变成蛋氨酸。     

基于多重计算设计策略提高枯草芽孢杆菌脂肪酶的热稳定性

提高酶的热稳定性是生物催化领域的热点和难点,计算机辅助的理性设计相比于传统的定向进化更加高效,在酶工程领域中的应用越来越广泛和深入。文中以枯草芽孢杆菌脂肪酶A为模式蛋白,首先,利用Rosetta-VIP计算设计对酶的结构空腔进行分析,选择了16个有利于结构空腔填充(ΔΔE0)的单点突变,并以突变位点的溶剂可及表面积和进化保守性为二次筛选依据,测定了其热稳定性与酶活性。有6个单点突变体(F17A、V74I、L114P、I135V、M137A、I157L)的热稳定性得到了提高,其中Tm值最大提高3.18℃。结果表明,单点突变体满足ΔΔE越低、蛋白溶剂可及表面积减少且符合序列保守性,则得到保留原有酶活力的正向突变的可能性越大。此外,将热稳定性提高的6个单点突变进行迭代组合突变,两点组合突变体的Tm最大提高4.04℃,三点组合突变体的Tm最大提高5.13℃,四点组合突变体的Tm提高了7.30℃,六点组合突变体的Tm提高了7.43℃。因此,基于酶的分子结构的空腔分析、溶剂可及表面积及氨基酸序列保守性计算的多重虚拟筛选方法,可有效提高酶的热稳定性。     

基于结构基础的嗜热细菌贝斯其热解纤维素菌木聚糖酶CbXyn10C的热稳定性分子改良

【背景】作为降解木聚糖的核心酶种,木聚糖酶可以有效促进木质纤维素的消化水解,在动物养殖领域应用广泛。来源于嗜热细菌贝斯其热解纤维素菌(Caldicellulosiruptorbescii)的GH10家族木聚糖酶Cb Xyn10C最适温度为85℃,在80℃条件下具有良好的热稳定性,具有饲料工业应用潜力。【目的】为满足饲料制粒尤其是水产饲料加工过程的工艺要求,进一步提高木聚糖酶Cb Xyn10C的热稳定性并阐明其耐热机理。【方法】以Cb Xyn10C晶体结构为基础,采用刚性氨基酸引入、疏水作用网络重排2种策略对其热稳定性进行理性设计,获得在100℃条件下比活提高的单点突变体后,通过有益突变位点叠加策略进一步提升酶的热稳定性,最后采用分子动力学模拟技术分析其热稳定性提高的分子机制。【结果】共获得了4个稳定性提高的单点突变体A45P、T69P、F309V和A325P,其中突变体A45P效果最优。随着在A45P基础上另外3个突变位点的叠加,酶的热稳定性在不损失酶活的前提下得到了逐步提升。获得的四点突变体A45P/F309V/A325P/T69P的耐热性最好,其最适反应温度和熔解温度Tm值较野生型...     

D92P点突变对扩展青霉碱性脂肪酶最适作用温度的改善

利用重叠延伸PCR法对扩展青霉碱性脂肪酶(PEL)基因进行体外定点突变,并构建了含突变基因的重组质粒pPIC3．5K—lip-D92P。将该质粒在毕赤酵母GS115菌株中表达。与野生型表达产物PEL-GS相比较,突变体表达产物PELD92P—GS最适作用温度为45℃,比野生型提高了5℃;其热稳定性与野生型相当;突变体在40℃下的表达量为109U／mL,约为野生型的29％。结果分析表明,Pro替代Asp^92后,可能是由于Pro一级结构的特点,使酶结构更加稳定,在高温下更适于与底物结合。     

E83V对扩展青霉脂肪酶最适作用温度的影响*

利用重叠延伸PCR法对扩展青霉碱性脂肪酶(PEL)基因作了体外定点突变,获得了最适作用温度有所提高的突变体。含突变基因的重组质粒pPIC3.5K-lip-E83V在Pichiapastoris GS115中表达。对突变体表达产物PEL-E83V-GS与野生型表达产物PELGS作了比较:前者最适作用温度为45℃,比野生型提高了5℃;其热稳定性基本不变;突变体在37℃下的表达量为188U/mL,约为野生型的80%。最适作用温度的提高可能是由于83位亲水性的Glu用疏水性的Val取代,增加了脂肪酶     

Improvement of the optimum temperature of lipase activity for Rhizopus niveus by random mutagenesis and its structural interpretation

Random mutagenesis was used to improve the optimum temperature for Rhizopus niveus lipase (RNL) activity. The lipase gene was mutated using the error-prone PCR technique. One desirable mutant was isolated, and three amino acids were substituted in this mutant (P18H, A36T and E218V). The wild-type and this randomly mutated lipase were both purified and characterized. The specific activity of the mutant lipase was 80% that of the wild-type. The optimum temperature of the mutant lipase was higher by 15 degrees C than that of the wild-type. To confirm which substitution contributed to enhancing the optimum temperature for enzymic activity, two chimeric lipases from the wild-type and randomly mutated gene were constructed: chimeric lipase 1 (CL-1; P18H and A36T) and chimeric lipase 2 (CL-2; E218V). Each of the chimeric enzymes was purified, and the optimum temperature for lipase activity was measured. CL-1 had a similar optimum temperature to that of the wild-type, and CL-2 had a higher temperature like the randomly mutated lipase. The mutational effect is interpreted in terms of a three-dimensional structure for the wild-type lipase.     

Degradation of triglycerides by a pseudomonad isolated from milk: the roles of lipase and esterase studied using recombinant strains over-producing, or specifically deficient in these enzymes

The roles of lipase and esterase in causing hydrolytic spoilage of milk by a highly lipolytic psychrotrophic strain of Pseudomonas fluorescens , LS107d2, has been studied. Strains of LS107d2 have been constructed that over-produce, or are specifically deficient in, a lipase (encoded by lip A ) and an esterase (encoded by est A ). Southern blot analysis reveals that LS107d2 contains only one esterase and one lipase (encoded by est A and lip A ) and this was confirmed by the phenotypes of mutants on triolein and tributyrin-containing agar. Analysis of broth cultures showed that the lipase is secreted into the culture medium; in contrast, the esterase is not secreted. Free fatty acid (FFA) levels in whole milk cultures of wild-type, over-producing and the mutant strains of LS107d2 have been examined. From these studies it is concluded that esterase is not involved in the accumulation of FFA by hydrolysing short chain fatty acid esters; that the highly lipolytic phenotype of LS107d2 is due solely to a single secreted lipase; and that the main FFA accumulated in milk cultures of LS107d2 are C4, C16, C18 and C18: 1. Evidence is also presented demonstrating that FFA degradation, as well as production, determines the level of FFA in milk contaminated with lipolytic organisms.     

Biochemical characterization of recombinant acetyl xylan esterase from Aspergillus awamori expressed in Pichia pastoris: mutational analysis of catalytic residues

We engineered an acetyl xylan esterase (AwaxeA) gene from Aspergillus awamori into a heterologous expression system in Pichia pastoris. Purified recombinant AwAXEA (rAwAXEA) displayed the greatest hydrolytic activity toward alpha-naphthylacetate (C2), lower activity toward alpha-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. Putative catalytic residues, Ser(119), Ser(146), Asp(168) and Asp(202), were substituted for alanine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the four mutant enzymes were examined. The S119A and D202A mutant enzymes were catalytically inactive, whereas S146A and D168A mutants displayed significant hydrolytic activity. These observations indicate that Ser(119) and Asp(202) are important for catalysis. The S146A mutant enzyme showed lower specific activity toward the C2 substrate and higher thermal stability than wild-type enzyme. The lower activity of S146A was due to a combination of increased K(m) and decreased k(cat). The catalytic efficiency of S146A was 41% lower than that of wild-type enzyme. The synthesis of ethyl acetate was >10-fold than that of ethyl n-hexanoate synthesis for the wild-type, S146A and D168A mutant enzymes. However, the D202A showed greater synthetic activity of ethyl n-hexanoate as compared with the wild-type and other mutants.     

Altering the stability of the Cdc8 overlap region modulates the ability of this tropomyosin to bind co-operatively to actin and regulate myosin

Tm (tropomyosin) is an evolutionarily conserved α-helical coiled-coil protein, dimers of which form end-to-end polymers capable of associating with and stabilizing actin filaments, and regulating myosin function. The fission yeast Schizosaccharomyces pombe possesses a single essential Tm, Cdc8, which can be acetylated on its N-terminal methionine residue to increase its affinity for actin and enhance its ability to regulate myosin function. We have designed and generated a number of novel Cdc8 mutant proteins with N-terminal substitutions to explore how stability of the Cdc8 overlap region affects the regulatory function of this Tm. By correlating the stability of each protein, its propensity to form stable polymers, its ability to associate with actin and to regulate myosin, we have shown that the stability of the N-terminal of the Cdc8 α-helix is crucial for Tm function. In addition we have identified a novel Cdc8 mutant with increased N-terminal stability, dimers of which are capable of forming Tm polymers significantly longer than the wild-type protein. This protein had a reduced affinity for actin with respect to wild-type, and was unable to regulate actomyosin interactions. The results of the present paper are consistent with acetylation providing a mechanism for modulating the formation and stability of Cdc8 polymers within the fission yeast cell. The data also provide evidence for a mechanism in which Tm dimers form end-to-end polymers on the actin filament, consistent with a co-operative model for Tm binding to actin.     

诱变选育脂肪酶高产菌株及其脂肪酶固定化

以紫外和微波复合诱变选育脂肪酶产生菌 Rhizopus sp. RXF12,获得高产突变株RZ13,其脂肪酶摇瓶发酵单位是出发株的2.62倍。菌株经多次传代,遗传性状稳定。对RZ13菌株的发酵条件进行了正交优化,在25 ℃、pH 8.0的条件下,接入5 %(v/v)的RZ13菌株单孢子悬液 (107个/ml) 振荡培养84 h,达到RZ13菌株最佳产酶状态,脂肪酶活可达95.08 U/ml。考察了脂肪酶性质,在低于40 ℃,pH 7.0~9.0范围内脂肪酶活稳定。经载体筛选及固定化过程优化,选用镁铝水滑石25℃吸附4 h,对RZ13脂肪酶进行了固定化。结果表明,固定化酶的最适作用温度为35~55℃,pH为7.5~9.0,较游离酶的均有较大扩展。     

Improvement of the Optimum Temperature of Penicillium expansum Lipase by Site-Directed Mutagenesis

In order to improve the optimum temperature of lipases,the Penicillum expansum lipase (PEL) gene was mutated by site-directed mutagenesis using overlap extension PCR technique.The recombinant plasmid containing mutant E83 V pPIC3.5K-lip-E83V was expressed in Pichia pastoris GS 115.Comparison experiments of the mutant PEL-E83 V-GS and the wild-type PEL-GS showed that the optimum temperature (45℃) of the mutant was 5℃ higher than that of the wild type.The thermostability of the mutant was similar to that of the wild type.The enzymatic activity of the mutant was 188 U/ml at 37℃,which was 80% that of the wild type in the same conditions.Hydrophobic interaction may be enhanced in the surface region by the hydrophilic amino acid Glu substituted with the hydrophobic amino acid Val,and may be responsible for the improvement of the optimum temperature.     

Probing the structural basis for the difference in thermostability displayed by family 10 xylanases

Thermostability is an important property of industrially significant hydrolytic enzymes: understanding the structural basis for this attribute will underpin the future biotechnological exploitation of these biocatalysts. The Cellvibrio family 10 (GH10) xylanases display considerable sequence identity but exhibit significant differences in thermostability; thus, these enzymes represent excellent models to examine the structural basis for the variation in stability displayed by these glycoside hydrolases. Here, we have subjected the intracellular Cellvibrio mixtus xylanase CmXyn10B to forced protein evolution. Error-prone PCR and selection identified a double mutant, A334V/G348D, which confers an increase in thermostability. The mutant has a Tm 8 degrees C higher than the wild-type enzyme and, at 55 degrees C, the first-order rate constant for thermal inactivation of A334V/G348D is 4.1 x 10(-4) min(-1), compared to a value of 1.6 x 10(-1) min(-1) for the wild-type enzyme. The introduction of the N to C-terminal disulphide bridge into A334V/G348D, which increases the thermostability of wild-type CmXyn10B, conferred a further approximately 2 degrees C increase in the Tm of the double mutant. The crystal structure of A334V/G348D showed that the introduction of Val334 fills a cavity within the hydrophobic core of the xylanase, increasing the number of van der Waals interactions with the surrounding aromatic residues, while O(delta1) of Asp348 makes an additional hydrogen bond with the amide of Gly344 and O(delta2) interacts with the arabinofuranose side-chain of the xylose moiety at the -2 subsite. To investigate the importance of xylan decorations in productive substrate binding, the activity of wild-type CmXyn10B, the mutant A334V/G348D, and several other GH10 xylanases against xylotriose and xylotriose containing an arabinofuranose side-chain (AX3) was assessed. The enzymes were more active against AX3 than xylotriose, providing evidence that the arabinose side-chain makes a generic contribution to substrate recognition by GH10 xylanases.