枯草杆菌蛋白酶E的蛋白质工程

用定点突变和随机突变的方法,对枯草杆菌碱性蛋白酶E基因进行改造。突变后的基因插入大肠杆菌-枯草杆菌穿梭质粒pBE-2中,在碱性和中性蛋白酶缺陷型的枯草杆菌DBl04中进行表达,得到突变种的碱性蛋白酶．它们的突变位点分别是(M222A)、(M222A、N118S)、(M222A、N118S、Q103R)、(M222A、N118S、Q103R、D60N)。各突变种酶的性质测定 结果表明．M222A突变使酶抗氧化,N118S突变使酶增加热稳定性,Q103R和D60N突变虽然能增加酶的比活,但使酶的热稳定性大大下降,尤其是D60N突变使酶变得极不稳定。野生型碱性蛋白酶与(M222A)突变种的等电点均为8．92．而M222A,N118S)。(M222A,N118S ,Ql03R)和(M222A,118S．Q103R,D60N)突变酶分别为8.88．9.10和9．17。用Nsuc-AAPF-pNA作为底物时酶反应景适pH值为7.5～9.5,而用酪蛋白底物时最适pH值为10～12。     

枯草杆菌蛋白酶E基因多位点定点突变库的构建及其筛选

利用化学合成的15个寡聚核苷酸片段作为诱变引物,同时对枯草杆菌蛋白酶E(Subtilisin E或Apr E)基因进行体外突变,获得了合全部突变位点各种随机组合突变的突变库,通过点杂交法和DNA序列分析肯定了该突变库的可靠性.从突变库中选择一单点突变(Met222Ala)和3点突变(Asn76Asp/Asn109Ser/Ile205Cys)的基因进行克隆、表达和产物的酶学性质研究,发现其抗氧化性和热稳定性分别比野生型的有显著提高,与文献报道的一致.表明了该突变库在枯草杆菌蛋白酶工程研究中的应用价值.     

甜菜夜蛾拓扑异构酶I氨基酸突变对其DNA解旋活性的影响

【目的】为了探究甜菜夜蛾 Spodoptera exigua 拓扑异构酶I（topoisomerase I, Top I）氨基酸突变对其DNA解旋活性的影响。【方法】通过克隆甜菜夜蛾 Top I 基因,构建原核表达载体,采用完全重叠PCR定点突变技术,向甜菜夜蛾Top I 的V420, L530, A653和S729（根据人Top I 氨基酸序列编号）4个位点引入突变,将改造成功的重组 Top I 基因转化至大肠杆菌BL21 (DE3)中,诱导重组蛋白表达、纯化,测定Top I突变对其解旋活性的影响。【结果】完全重叠PCR能实现甜菜夜蛾 Top I 定点突变。重组蛋白在体外得到稳定的表达,表达产物经SDS-PAGE电泳分析在96.0 kDa处出现特异性条带。通过对重组蛋白分离纯化并测定对质粒pBR322解旋酶活性,发现引入V420I, L530P和A653T突变后Top I的比活力显著降低,而引入S729T突变后比活力与野生型蛋白无显著差异。【结论】本研究证明在甜菜夜蛾Top I中引入V420I, L530P和A653T突变后,其对底物pBR322的解旋活性显著降低,为后期探索甜菜夜蛾Top I的定点突变与其对喜树碱及其衍生物敏感性的关系奠定了基础。     

枯草蛋白酶E的定点突变及其对酶性质的影响

用定点突变的方法研究S221C/P225A,N118S/S221C/P225A,D60N/S221C/P225A和Q103R/S221C/P225A突变对蛋白酶活性,酯酶活性与蛋白酶活性之比的影响。结果表明：S221C/P225A突变使蛋白酶活性比枯草蛋白酶E低73000多倍,酯酶活性与蛋白酶活性之比是Subtiligase的3倍;N118S/S221C/P225A突变使蛋白酶活性和酯酶活性分别比S221C/P225A突变下降3.6倍和15倍,酯酶与蛋白酶活性之比下降4倍,同时增加变体酶的热稳定性;D60N/N118S/S221C/P225A突变使蛋白酶活性比N118S/S221C/P225A突变体下降15倍,但对酯酶活性几乎没有影响,酯酶与蛋白酶活性之比增加14倍,分别是S221C/P225A突变体和Subtiligase的3.3倍和10.3倍;但是,Q103R/N118S/S221C/P225A突变使蛋白酶活性比N118S/S221C/P225A突变体增加5倍,酯酶活性下降55倍,酯酶与蛋白酶活性之比下降1000倍。     

利用定点突变法研究精氨酸脱亚胺酶活性的影响机制

精氨酸脱亚胺酶(ADI)是一种针对精氨酸缺陷型癌症(如:肝癌、黑素瘤)的新药,目前处于临床三期试验。文中通过定点突变技术分析了精氨酸脱亚胺酶的特定氨基酸位点对酶活力的影响机制。针对已报道的关键氨基酸残基A128、H404、I410,采用QuikChange法进行定点突变,获得ADI突变株M1(A128T)、M2(H404R)、M3(I410L)和M4(A128T/H404R)。将突变株在大肠杆菌BL21(DE3)中进行重组表达,并对纯化获得的突变蛋白进行酶学性质研究。结果表明,突变位点A128T和H404R对ADI最适pH的提高,生理中性(pH 7.4)条件下的酶活力和稳定性的提高,以及Km值的降低均具有显著的作用。研究结果为阐明ADI的酶活力影响机制和蛋白质的理性改造提供了一定的依据。     

定点突变对酰胺水解酶DamH可溶性表达和酶活的影响

摘要:【目的】DamH是一种具有酯酶活性的酰胺水解酶,其非活性中心氨基酸残基的突变对重组酶可溶性表达和比酶活产生一定的影响。拟探索DamH的活性中心氨基酸残基构成,并对其非活性中心氨基酸残基突变对可溶性表达和比酶活的影响进行研究。【方法】通过重叠延伸的方法对DamH可能的活性中心氨基酸S149、E244和H274以及非活性中心氨基酸D165及N192进行定点突变,通过静息细胞测活验证了S149、E244和H274 在催化2-氯-N-(2’-甲基-6’-乙基苯基)乙酰胺(CMEPA)水解反应中的作用,通过Ni2+- NTA亲和层析对D165及N192突变子进行纯化,对突变株和野生型比酶活进行比较。【结果】研究表明S149A使DamH的CMEPA 水解酶活性下降为野生型的5%,E244A和H274A突变导致其失去活性;D165P和N192P突变影响到DamH的可溶性表达,表达量分别为野生型的28.2%和20.8%,突变子N192P、D165P比酶活分别为野生型比酶活的55.5%和49.7%。【结论】DamH催化酯类底物和芳基酰胺类底物可能共用同一活性中心S149、E244和H274,其两个α螺旋的转角处氨基酸侧链极性和刚性结构的改变对可溶性表达以及活性有很大的影响。     

K178M、S170F、G180V突变对木聚糖酶XynZF-2热稳定性的影响

利用生物信息学分析黑曲霉木聚糖酶Xyn ZF-2,选择α-螺旋178位点、170位点和180位点氨基酸进行定点突变(K178M,S170F,G180V)获得突变木聚糖酶基因xyn MFV,构建重组表达载体转化大肠杆菌E.coli BL21(DE3)诱导表达。酶学性质分析对比发现,突变酶最适温度(50℃)比原酶(40℃)提高了10℃;40℃条件下保温1 h突变酶Xyn MFV相对酶活力下降到热处理前的56.0%,原酶Xyn ZF-2下降到42.0%;45℃时突变酶Xyn MFV的半衰期t_(1/2)为21 min,与原酶Xyn ZF-2(t_(1/2)=7 min)相比较提高了14 min。结果表明,K178M、S170F、G180V突变木聚糖酶可以提高Xyn ZF-2最适温度和热稳定性。     

用蛋白质工程的方法改良枯草杆菌蛋白酶E(Subtilisin E)的抗氧化性

以含有蛋白酶E基因(aprE)的单链M13mp18-aprE DNA为模板,合成的寡核苷酸5′-3′为诱变引物,用缺口双链法对aprE进行Met-222-Ala点突变。经菌落印迹杂交筛选,选出阳性噬斑。用SaⅡ酶解M13mp18-aprE得到aprE,将它和pPZW103重组,转化中性、碱性蛋白酶缺失宿主菌DB104。经含卡那霉素和脱脂奶粉板筛选和比较aprE限制性内切酶NcoⅠ和SacⅡ水解电泳图谱分析,完成构建一个分泌抗氧化的枯草杆菌蛋白酶E的工程菌PW8888。     

嗜热酯酶APE1547催化活性的定向进化研究

对来源于嗜热古菌Aeropyrum pernix的酯酶(APE1547)催化活性进行定向进化研究。利用APE1547特殊的稳定性,建立了准确的高通量高温酯酶筛选方法。对第一代随机突变库筛选获得了催化活性较野生型提高1.5倍的突变体M010,序列分析表明其氨基酸突变为R526S。从第二代突变库中筛选出的总活力提高5.8倍突变体M020,突变位点为R526S/E88G/A200T/I519L,其比活力与M010一致,但表达量比野生型提高约4倍。对M020酶学性质表征发现,其最适pH为8.5,比野生型向碱性偏移0.5;活性中心残基酸性基团的解离常数(pK1)由野生型的7.0提高至7.5。晶体结构分析表明,突变位点R526距离活性中心较近,将其突变为Ser降低了活性中心的极性,抑制了催化残基His的解离,使酸性基团的解离常数升高。     

集成干扰素突变体IIFN/165S的构建及其生物学评价

目的:通过定点突变,构建集成干扰素(IIFN/165S),以期获得高效的新型药物分子.方法:采用PCR体外定点突变技术,使集成干扰素IIFN基因的第165位密码子由CGT突变为AGT.扩增片段克隆入pET-23b表达载体,重组质粒转化大肠杆菌BL21(DE3).在LB培养基中培养,经IPTG诱导表达的IIFN/165S经包涵体变性、复性以及层析纯化后,经SDS-PAGE、Western blot和MALDI-TOF-MS分析,用WISH-VSV系统进行抗病毒活性测定同时应用流式细胞术检测细胞凋亡率.结果:IIFN/165S以包涵体形式表达.纯化后,IIFN/165S的纯度大于95％,分子量为18172,比活性(7.63±0.22)×108 IU/mg,诱导细胞凋亡率呈剂量依赖.结论:构建了IIFN/165S的表达载体,并成功地在大肠杆菌中表达,获得了高纯度高活性突变分子IIFN/165S.     

Nonfixed relationship of the Michaelis constant and maximum velocity with their corresponding rate constants

The Michaelis constant (K(m)) and V(mas) (E0k(cat)) values for two mutant sets of enzymes were studied from the viewpoint of their definition in a rapid equilibrium reaction model and in a steady state reaction model. The "AMP set enzyme" had a mutation at the AMP-binding site (Y95F, V67I, and V67I/L76V), and the "ATP set enzyme" had a mutation at a possible ATP-binding region (Y32F, Y34F, and Y32A/Y34A). Reaction rate constants obtained using steady state model analysis explained discrepancies found by the rapid equilibrium model analysis. (i) The unchanged number of bound AMPs for Y95F and the wild type despite the markedly increased K(m) values for AMP of the AMP set of enzymes was explained by alteration of the rate constants of the AMP step (k(+2), k(-2)) to retain the ratio k(+2)/k(-2). (ii) A 100 times weakened selectivity of ATP for Y34F in contrast to no marked changes in K(m) values for both ATP and AMP for the ATP set of enzymes was explained by the alteration of the rate constants of the ATP steps. A similar alteration of the K(m) and k(cat) values of these enzymes resulted from distinctive alterations of their rate constants. The pattern of alteration was highly suggestive. The most interesting finding was that the rate constants that decided the K(m) and k(cat) values were replaced by the mutation, and the simple relationships between K(m), k(cat), and the rate constants of K(m)1 = k(+1)/k(-1) and k(cat) = k(f) were not valid. The nature of the K(m) and k(cat) alterations was discussed.     

Modifying the substrate specificity of staphylococcal lipases.

The lipase from Staphylococcus hyicus (SHL) displays a high phospholipase activity whereas the homologous S. aureus lipase (SAL) is not active or hardly active on phospholipid substrates. Previously, it has been shown that elements within the region comprising residues 254-358 are essential for the recognition of phospholipids by SHL. To specifically identify the important residues, nine small clusters of SHL were individually replaced by the corresponding SAL sequence within region 254-358. For cloning convenience, a synthetic gene fragment of SHL was assembled, thereby introducing restriction sites into the SHL gene and optimizing the codon usage. All nine chimeras were well-expressed as active enzymes. Eight chimeras showed lipase and phospholipase activities within a factor of 2 comparable to WT-SHL in standard activity assays. Exchange of the polar SHL region 293-300 by the more hydrophobic SAL region resulted in a 32-fold increased k(cat)/K(m) value for lipase activity and a concomitant 68-fold decrease in k(cat)/K(m) for phospholipase activity. Both changes are due to effects on catalytic turnover as well as on substrate affinity. Subsequently, six point mutants were generated; G293N, E295F, T297P, K298F, I299V, and L300I. Residue E295 appeared to play a minor role whereas K298 was the major determinant for phospholipase activity. The mutation K298F caused a 60-fold decrease in k(cat)/K(m) on the phospholipid substrate due to changes in both k(cat) and K(m). Substitution of F298 by a lysine in SAL resulted in a 4-fold increase in phospholipase activity. Two additional hydrophobic to polar substitutions further increased the phospholipase activity 23-fold compared to WT-SAL.     

Familial Mediterranean fever in Syrian patients: <Emphasis Type="Italic">MEFV</Emphasis> gene mutations and genotype–phenotype correlation

Familial Mediterranean fever is an autosomal recessive disorder characterized by recurrent attacks of abdominal pain, synovitis and pleuritis. MEFV gene mutations are responsible for the disease. The objective of this study was to identify the frequency and distribution of 12 MEFV mutations in 153 Syrian patients and perform a genotype–phenotype correlation in the patients’ cohort. Of the 153 unrelated patients investigated, 97 (63.4%) had at least one mutation. The most frequent mutation was M694V (36.5%), followed by V726A (15.2%), E148Q (14.5%), M680I (G/C) (13.2%), and M694I (10.2%) mutations. Rare mutations (R761H, A744S, M680I (G/A), K695R, P369S, F479L and I692del) were also detected in the patients. M694V was associated with the severe form of the disease. The identification of a significant number of FMF patients with no mutations or only one known mutation identified indicates the presence of new mutations in the MEFV gene which will be investigated in the future.     

Frequency of MEFV gene mutations in Hatay province,Mediterranean region of Turkey and report of a novel missense mutation (I247V)

In the present study, 1000 patients with clinical suspicion of FMF were retrospectively reviewed to determine the spectrum of MEFV gene mutations by using DNA sequence analysis between September, 2008 and April, 2012. Sixteen different mutations and 55 different genotypes were detected in 618 of 1000 patients. Among 16 different mutations, R202Q (21.35%) was the most frequently observed mutation; followed by E148Q (8.85%), M694V (7.95%), M680I (2.40%), V726A (1.85%), M694I (0.95%), A744S (0.80%), R761H (0.55%), P283L (0.35%), K695R (0.20%), E230K (0.15%), L110P (0.10%), I247V (0.05%), G196W (0.05%) and G304R (0.05%). In the present study, a novel missense mutation (I247V) and a silent variant (G150G) were identified in the MEFV gene. On the other hand, P238L, G632A and G304R mutations are the first cases reported from Turkey. Our results indicated that MEFV mutations are highly heterogeneous in our study population as in other regions of Turkey and mutation screening techniques such as PCR-RFLP, amplification refractory mutation system or reverse hybridization do not adequately detect uncommon or novel mutations. Therefore, it was proven that sequence analysis of the MEFV gene could be useful for detection of rare or unknown mutations.     

Engineering Streptomyces clavuligerus deacetoxycephalosporin C synthase for optimal ring expansion activity toward penicillin G

The deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was engineered with the aim of enhancing the conversion of penicillin G into phenylacetyl-7-aminodeacetoxycephalosporanic acid, a precursor of 7-aminodeacetoxycephalosporanic acid, for industrial application. A single round of random mutagenesis followed by the screening of 5,500 clones identified three mutants, G79E, V275I, and C281Y, that showed a two- to sixfold increase in the k(cat)/K(m) ratio compared to the wild-type enzyme. Site-directed mutagenesis to modify residues surrounding the substrate resulted in three mutants, N304K, I305L, and I305M, with 6- to 14-fold-increased k(cat)/K(m) values. When mutants containing all possible combinations of these six sites were generated to optimize the ring expansion activity for penicillin G, the double mutant, YS67 (V275I, I305M), showed a significant 32-fold increase in the k(cat)/K(m) ratio and a 5-fold increase in relative activity for penicillin G, while the triple mutant, YS81 (V275I, C281Y, I305M), showed an even greater 13-fold increase in relative activity toward penicillin G. Our results demonstrate that this is a robust approach to the modification of DAOCS for an optimized DAOCS-penicillin G reaction.     

Key NAD+-binding residues in human 15-hydroxyprostaglandin dehydrogenase

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase/reductase (SDR) family, catalyzes the first step in the catabolic pathways of prostaglandins and lipoxins, and is believed to be the key enzyme responsible for the biological inactivation of these biologically potent eicosanoids. The enzyme utilizes NAD(+) specifically as a coenzyme. Potential amino acid residues involved in binding NAD(+) and facilitating enzyme catalysis have been partially identified. In this report, we propose that three more residues in 15-PGDH, Ile-17, Asn-91, and Val-186, are also involved in the interaction with NAD(+). Site-directed mutagenesis was used to examine their roles in binding NAD(+). Several mutants (I17A, I17V, I17L, I17E, I17K, N91A, N91D, N91K, V186A, V186I, V186D, and V186K) were prepared, expressed as glutathione S-transferase (GST) fusion enzymes in Escherichia coli, and purified by GSH-agarose affinity chromatography. Mutants I17E, I17K, N91L, N91K, and V186D were found to be inactive. Mutants N91A, N91D, V186A, and V186K exhibited comparable activities to the wild type enzyme. However, mutants I17A, I17V, I17L, and V186I had higher activity than the wild type. Especially, the activities of I17L and V186I were increased nearly 4- and 5-fold, respectively. The k(cat)/K(m) ratios of all active mutants for PGE(2) were similar to that of the wild type enzyme. However, the k(cat)/K(m) ratios of mutants I17A and N91A for NAD(+) were decreased 5- and 10-fold, respectively, whereas the k(cat)/K(m) ratios of mutants I17V, N91D, V186I, and V186K for NAD(+) were comparable to that of the wild type enzyme. The k(cat)/K(m) ratios of mutants I17L and V186A for NAD(+) were increased over nearly 2-fold. These results suggest that Ile-17, Asn-91, and Val-186 are involved in the interaction with NAD(+) and contribute to the full catalytic activity of 15-PGDH.     

Thermal stable and oxidation-resistant variant of subtilisin E

A remarkable thermal stable and oxidation-resistant mutant was obtained using the random mutagenesis PCR technique on the mutant M222A gene of subtilisin E. Sequencing analysis revealed an A was replaced by G at nucleotide 671 of the subtilisin E gene, converting the asparagine codon (AAT) to serine codon (AGT) at position 118. The half-life of M222A/N118S enzyme activity, when heated at 65 degrees C, was approximately 80 min while the half-life of M222A and wild-type subtilisin E were 13 min and 15 min, respectively. This suggested the stability of the M222A/N118S mutant was five times greater than that of the wild-type enzyme. The mutant was also as oxidation resistant as the mutant M222A of subtilisin E. These results indicated the M222A/N118S mutant is both an oxidation-resistant and a heat-stable variant of subtilisin E.