定点突变提高豆豉纤溶酶的酶活力和底物特异性的研究

以pBE—DFE质粒为模板,采用反向长距离PCR技术对豆豉纤溶酶(Douchi Fibrinolytic Enzyme,DFE)基因进行定点突变,使位于酶的底物结合区第156位的谷氨酸和第166位的甘氨酸分别替换为丝氨酸和丙氨酸,使临近酶的底物结合区的第169位甘氨酸残基替换为丙氨酸,获得了三个突变体E156S,G166A和G169A.将含突变基因的表达载体转化枯草杆菌WB800,构建了豆豉纤溶酶基因突变体的表达菌株WB800(E156S);WB800(G166A)和WB800(G166A).将3种表达菌株进行液体发酵培养,结果发现发酵上清液中纤溶酶的纤溶活性为460,790和900U/mL,分别是未突变酶活性(660U/mL)的70%,115%和136%;3种突变酶对合成底物的H-D-Val-Leu-Lys-pNA的酰胺水解活性是未突变酶的17%,125%和121%.     

谷氨酸棒杆菌(Corynebacterium glutamicum)海藻糖合成酶的定点突变及其酶学性质研究

以已知晶体结构的Pseudomonas mesoacidophila MX-45菌株海藻酮糖合成酶（MutB）的晶体结构为模板,在SWISS-MODEL模建立谷氨酸棒杆菌（Corynebacterium glutamicum）海藻糖合成酶的立体结构,并对初始结构作能量优化,通过氨基酸序列比对,选择TreS-glu保守区内的氨基酸R245、D247、E289、F244和保守区外的氨基酸A288进行定点突变,并对突变酶F244C、F244L、F244W、F244Y、A288G、R245X、E289X、D247N、D247E进行纯化和酶学性质研究,比较突变子对酶活性和热稳定性的影响。结果表明,R245、E289突变为其它的19个氨基酸后酶活力全部丧失,D247E和D247N也丧失酶活,F244C、F244L、F244W、F244Y和A288G的比活力分别降低到TreS-glu的38%、24%、62%、64%和35%,A288突变成T288后没有酶活。与TreS-glu相比,F244C、F244W、A288G的Km值基本不变,F244L、F244Y对底物麦芽糖的亲和力降低,F244Y的最适反应温度和TreS-glu相同,均为27℃,而F244C、F244L、F244W和A288G的最适温度提高到32℃。与TreS-glu相比,突变酶的最适反应pH值均有所下降,其中F244C、F244Y和A288G的为7.5,比TreS-glu的8.0均下降了约0.5个单位,而F244L和F244W的为6.5,比TreS-glu的8.0均下降了近1.5个单位。与TreS-glu相比,突变酶的热稳定性均有不同程度提高,其中F244Y、F244W和A288G的Tm值比TreS-glu的提高约1℃,F244L提高约2℃,F244C提高了近4℃。     

环糊精水解酶cds1-3底物通道氨基酸的定点突变研究

本研究对具有大分子水解能力的环糊精水解酶cds1-3的蛋白结构进行分析,选取底物通道相关氨基酸进行定点突变。通过比较突变酶和野生酶的功能差异,定位决定cds1-3特殊功能的氨基酸。采用sybyl 1.2进行蛋白质底物结合分析,选取和多聚体形成、底物结合以及底物通道相关的氨基酸Glu66、Pro48、Phe289为突变位点,反向PCR构建pSE380/E66G、pSE380/P48H、pSE380/F289A表达质粒并进行表达,获得酶活突变体并与原始酶进行底物特异性比较分析。其结果显示,突变酶E66G降解大分子底物木薯淀粉和支链淀粉的相对酶活力分别提高26.96%和23.15%,而对小分子底物普鲁兰糖的水解能力下降13.14%。因此,cds1-3是一个能水解大分子底物的特殊环糊精水解酶,氨基酸Glu66是cds1-3水解大分子支链淀粉的关键氨基酸之一。     

重组纤溶酶原激活剂及其突变体的表达研究

利用PCR技术 ,获得了人体组织型纤溶酶原激活剂tPAcDNA的缺失突变体———rPA ;在此基础上 ,运用定点突变技术 ,得到rPA的定点突变体———rPA(KHRR2 96～ 2 99AAAA) ,rPA(A473S)和二者的复合突变体rPA(KHRR2 96～2 99AAAA A473S) ;将rPA及其 3个突变体分别亚克隆至原核表达载体pET 2 8a( )中 ,获得表达载体pErA ,pErA(K) ,pErA(A)和pErA(KA) .酶切鉴定和序列分析结果均表明实现了实验设计的氨基酸突变 .表达载体转化大肠杆菌 ,经IPTG诱导、菌体裂解及SDS PAGE电泳分析发现 ,只缺失而无突变的pErA和突变的pErA(A)都未表达目的蛋白 ,突变体pErA(K)与pErA(KA)则获高水平表达 ;蛋白产量分别占菌体总蛋白的 35 .97%与 37.71% ,分子量均为 39.6kD .Westernblotting显示 ,表达产物与抗tPA抗体呈特异性阳性反应 .该产物经初步纯化后进行复性与活性 (mFAPA)测定 ,结果表明其复性产物具有明显的体外纤溶活性 .以上结果为rPA突变体的进一步纯化、体内活性研究以及规模化制备提供了基础     

白孢链霉菌(Streptomyces albosporeus)CT—86几丁质酶研究

应用磷酸膨胀几丁质双层平板,从土壤中篩选到48株分解几丁质的链霉菌。经紫外线诱变后,得到一株能强烈分解磷酸膨胀几丁质的突变株CT—86。在1～1.5％的磷酸膨胀几丁质、1％豆饼粉的营养盐培养基中,30℃培养8～10天,酶活达到0.4mg N—乙酰葡萄胺/h。在几丁质浓废为0.5～1％范围内,单位酶活随几丁质浓度的增加而提高。以葡萄糖和N—乙酰葡萄糖胺为碳源时,在上述培养条件下,无酶活捡出。磷酸膨胀几丁质为底物,酶作用的最适温度是48℃,最适pH为6.5;酶液在60℃保温2小时后,酶活力丧失70％左右。     

大肠杆菌holo-ACP突变体的表达纯化及相应acyl-ACP的性质初探

酰基-酰基载体蛋白（acyl-acyl carrier protein,acyl-ACP）作为酰基供体,在脂肪酸、聚酮等天然产物合成途径中具有重要作用.目前,常以酰基-辅酶A（acyl-CoA）来代替尚未商品化的acyl-ACP进行相关酶的体外活性研究.由于底物蛋白acyl-ACP的ACP部分与相关酶发生相互作用,影响酶的催化特性,这种替代无法真实认识相关酶的酰基转移特性.本文以大肠杆菌pET-28a（+）-ACP为模板,构建12株单点突变体,表达纯化出相应holo-ACP,并进一步合成C16:0-ACP和C18:1-ACP.高效液相色谱的结果表明,ACP单点突变会影响acyl-ACP整体的性质,其中T40残基的改变对acyl-ACP的疏水性、紫外吸收响应和稳定性均产生了显著影响.ACP突变体的性质表征为acyl-ACP与相关酶的互作机制研究奠定了基础.      

Burkholderic cepecia 1003产海因酶固定化条件

海因酶是利用海因酶法制备手性氨基酸的关键酶.采用Eupergit C、Eupergit C250L及其氨基化后的载体作为固定化介质,对酶液pH值、蛋白浓度、固定化时间以及EDC用量等参数对海因酶固定化过程的影响进行研究,并在实验范围内,得到了上述参数的最优值.研究表明,环氧基载体的最适固定化条件为：酶液pH值为7.0,酶液蛋白质量浓度为0.4mg/mL,固定化时间为20 h;氨基载体最适固定化条件为：酶液pH值为8.0,酶液蛋白质量浓度为0.4 mg/mL,固定化时间为10 h,EDC用量：Eupergit C（-NH2）为70μL,Eupergit C250L（-NH2）为120μL.其中以Eupergit C250L为固定化载体,海因酶的活性回收率可高达87%,且固定化酶的稳定性也较好,酶活半衰期长达2 500h左右,具有较好的稳定性.     

腈水合酶在大肠杆菌中的表达纯化

微生物的腈水合酶是催化丙烯腈生成丙烯酰胺的重要生物催化剂,广泛应用于氨基酸、酰胺、羧酸及其衍生物的合成。通过PCR扩增得到诺卡氏菌的腈水合酶基因。实验先构建得到BL21-pGEX-4T-1/NHase重组菌,表达GST-NHase融合蛋白。由于GST的相对分子质量较大,阻碍了腈水合酶的β亚基与α亚基的正确结合,从而使腈水合酶表现不出酶活。于是又构建了BL21-pET-30c/NHase重组菌,IPTG诱导表达结果显示,腈水合酶α亚基基本没有得到表达,影响了腈水合酶的活性。经生物信息学分析发现,腈水合酶α亚基的起始密码子为gtg,可能不易被大肠杆菌的RNA聚合酶识别,于是通过点突变将gtg替换成atg,阳性重组子经IPTG诱导表达,结果显示腈水合酶β亚基和α亚基的表达量都有所提高。诱导表达的菌液经超声破碎,离心得到粗酶液,用EPI-30-ARG-IDA-Co2+亲和载体纯化带6×His tag的腈水合酶,气相色谱法检测腈水合酶的酶活达到200 U/mL。     

青霉素酰化酶研究——Ⅰ.抑制作用,纯化和性质

丙酮、乙醇和苯丙氨酸是大肠杆菌青霉素酰化酶的竞争性抑制剂,分别抑制模拟底物3-苯乙酰胺基6-硝基苯甲酸水解的活力。青霉素酰化酶粗酶液经硫酸铵盐析,pH5沉淀,SE-Sephadex C50层析,苯丙氨酸-Sepharose 4B疏水层析和DEAE-Sephadex A25层析可得纯酶,聚丙烯酰胺凝胶梯度电泳呈现一条带,比活为27.5u/mg蛋白。纯化总活力回收率为28.1%。青霉素酰化酶分子量约为90000,有两个亚基,分子量分别为70000和20000,其最适温度为40℃,最适pH为8.0,催化青霉素G水解的米氏常数为2.94mmol/L。     

分子动力学模拟探索乳克鲁维酵母来源的β-半乳糖苷酶热变性条件

为了研究乳克鲁维酵母中β-半乳糖苷酶可能的熔解温度，采用分子动力学模拟的方法，分别对4种不同温度条件下(35、50、65、80 ℃)的β-半乳糖苷酶进行了50 ns的计算模拟，分析了酶的构象变化以及酶活性中心的差异。研究在原子水平揭示了β-半乳糖苷酶的温度耐受等关键信息:35 ℃为最适酶活温度，该温度下的β-半乳糖苷酶的整体构象最稳定；该酶在50 ℃时的原子波动性显著增加，表明此温度可能趋近熔解温度临界值；蛋白在大于65 ℃条件下丧失柔性，说明蛋白已经变性；进一步的构象分析发现80 ℃高温会破坏β-D-半乳吡喃糖(GAL)结合位点微环境。     

Purification and cloning of amyloid precursor protein beta-secretase from human brain

Proteolytic processing of the amyloid precursor protein (APP) generates amyloid beta (Abeta) peptide, which is thought to be causal for the pathology and subsequent cognitive decline in Alzheimer's disease. Cleavage by beta-secretase at the amino terminus of the Abeta peptide sequence, between residues 671 and 672 of APP, leads to the generation and extracellular release of beta-cleaved soluble APP, and a corresponding cell-associated carboxy-terminal fragment. Cleavage of the C-terminal fragment by gamma-secretase(s) leads to the formation of Abeta. The pathogenic mutation K670M671-->N670L671 at the beta-secretase cleavage site in APP, which was discovered in a Swedish family with familial Alzheimer's disease, leads to increased beta-secretase cleavage of the mutant substrate. Here we describe a membrane-bound enzyme activity that cleaves full-length APP at the beta-secretase cleavage site, and find it to be the predominant beta-cleavage activity in human brain. We have purified this enzyme activity to homogeneity from human brain using a new substrate analogue inhibitor of the enzyme activity, and show that the purified enzyme has all the properties predicted for beta-secretase. Cloning and expression of the enzyme reveals that human brain beta-secretase is a new membrane-bound aspartic proteinase.     

Structural basis for recognition and repair of the endogenous mutagen 8-oxoguanine in DNA

Spontaneous oxidation of guanine residues in DNA generates 8-oxoguanine (oxoG). By mispairing with adenine during replication, oxoG gives rise to a G x C --> T x A transversion, a frequent somatic mutation in human cancers. The dedicated repair pathway for oxoG centres on 8-oxoguanine DNA glycosylase (hOGG1), an enzyme that recognizes oxoG x C base pairs, catalysing expulsion of the oxoG and cleavage of the DNA backbone. Here we report the X-ray structure of the catalytic core of hOGG1 bound to oxoG x C-containing DNA at 2.1 A resolution. The structure reveals the mechanistic basis for the recognition and catalytic excision of DNA damage by hOGG1 and by other members of the enzyme superfamily to which it belongs. The structure also provides a rationale for the biochemical effects of inactivating mutations and polymorphisms in hOGG1. One known mutation, R154H, converts hOGG1 to a promutator by relaxing the specificity of the enzyme for the base opposite oxoG.     

Detection of single base substitutions in total genomic DNA

Certain single base substitutions causing genetic diseases or resulting in polymorphisms linked to mutant alleles, alter a restriction enzyme cleavage site and can therefore be detected in total genomic DNA using DNA blots. Many base substitutions do not lead to an altered restriction site, but these can be detected using synthetic oligonucleotides as hybridization probes if the DNA sequence surrounding the base substitution is known. In the case of beta-thalassaemia, where 22 different single base mutations have been identified, a large number of probes would be required for diagnosis. An approach which was used to detect mutations in viral DNA involves the S1 nuclease treatment of heteroduplexes formed between wild-type and mutant DNA. Although certain single base mismatches are cleaved by S1 nuclease (ref. 11 and T. Shenk, personal communication), many other mismatches examined by this procedure are not cleaved (B. Seed, personal communication; R.M.M., unpublished data). Heteroduplexes between mutant and wild-type subgenomic fragments of double-stranded reovirus RNA migrate slower than the corresponding homoduplexes in polyacrylamide gels containing 7 M urea, but it is not known whether this method is applicable to DNA heteroduplexes containing single base mismatches. Here we describe a procedure that involves the electrophoretic separation of DNA heteroduplexes in a well-characterized gel system. We show that four different human beta-thalassaemia alleles with known single base mutations can be detected with as little as 5 micrograms of total genomic DNA. The method should be useful in the localization and diagnosis of mutations associated with genetic diseases.     

Structure of the C2 domain of human factor VIII at 1.5 A resolution

Human factor VIII is a plasma glycoprotein that has a critical role in blood coagulation. Factor VIII circulates as a complex with von Willebrand factor. After cleavage by thrombin, factor VIIIa associates with factor IXa at the surface of activated platelets or endothelial cells. This complex activates factor X (refs 6, 7), which in turn converts prothrombin to thrombin in the presence of factor Va (refs 8, 9). The carboxyl-terminal C2 domain of factor VIII contains sites that are essential for its binding to von Willebrand factor and to negatively charged phospholipid surfaces. Here we report the structure of human factor VIII C2 domain at 1.5 A resolution. The structure reveals a beta-sandwich core, from which two beta-turns and a loop display a group of solvent-exposed hydrophobic residues. Behind the hydrophobic surface lies a ring of positively charged residues. This motif suggests a mechanism for membrane binding involving both hydrophobic and electrostatic interactions. The structure explains, in part, mutations in the C2 region of factor VIII that lead to bleeding disorders in haemophilia A.     

两种贵金属萃取剂萃取性能的研究

以N ,N二苄基甘氨酸和N ,N二苯基甘氨酸为固定相 ,以不同浓度的盐酸和高氯酸为展开剂 ,研究了它们对 8种贵金属的萃取行为 .实验表明 :N ,N二苯基甘氨酸的萃取能力较N ,N二苄基甘氨酸大 ;两种萃取剂在盐酸体系中的萃取能力比在高氯酸体系中大 .     

一种利用甘氨酸氧化酶生产乙醛酸的方法

为实现生物法工业化生产乙醛酸,采用基因工程技术对甘氨酸氧化酶（ThiO）进行表达优化,使用SDS-PAGE电泳手段验证ThiO的可溶性表达,通过全细胞转化法检测重组菌体生产乙醛酸的能力,并优化了反应条件,在放大体系中考察其应用性。结果表明：构建的菌株Bacillus subtilis 168（pP43NMK-ThiO）...     

阿维拉霉素高产菌株的推理选育

为获得阿维拉霉素高产菌株,在研究菌株Streptomyces viridocbromogenes96(SV-96)葡萄糖及氨盐代谢的基础上,采用“NTG随机诱变 氨基乙酸和2-脱氧-D葡萄糖联合抗性”的试验方案进行阿维拉霉素高产菌株的推理选育,获得了氨基乙酸和2-脱氧-D-葡萄糖联合抗性高产突变株SV-GT-596.该菌株遗传性能良好,阿维拉霉素产量高达56.4mg/L,较出发菌株提高了162.3%.实验表明,氨基乙酸和2-脱氧-D-葡萄糖抗性推理选育可克服随机筛选的盲目性,提高筛选工作效率.     

Structure of a mutant of tyrosyl-tRNA synthetase with enhanced catalytic properties

One surprising outcome of applying the techniques of protein engineering to the enzyme tyrosyl-transfer RNA synthetase was that the enzyme's activity could actually be increased by a specific sequence change. In particular, altering residue threonine 51 to a proline (mutant TP51) increased the enzyme's affinity for tyrosyl adenylate complexes. The non-additive effect of combining the TP51 mutation with a second sequence alteration (histidine 48 to a glycine) suggested that the effect of the TP51 change might be mediated by a structural change involving the peptide backbone. To address the question of the mechanism by which the TP51 change increases the activity of tyrosyl-tRNA synthetase we have determined the structure of the mutant enzyme. We find the change has a purely local effect on the structure of the enzyme, and conclude that the increased activity of the TP51 mutant probably results from the replacement of the polar threonine residue by a non-polar group: in the wild-type enzyme substrate binding is disfavoured by the displacement of solvent from the vicinity of threonine 51. This unfavourable effect is absent in the TP51 mutant.     

分光光度法测定草甘膦生产废水中草甘膦和甘氨酸的含量

在甘氨酸存在下用紫外分光光度计法测定草甘膦含量,所测废水体系中草甘膦质量分数为1.10%,该法RSD为0.836%,线性相关系数为0.999,表观摩尔吸光系数为6.53×103L/（mol.cm）.用可见光分光光度计法测定甘氨酸含量,考察了组分、缓冲液用量、显色剂配比、甘氨酸浓度等因素对分析的影响,测得废水中甘氨酸质量分数为1.58%,标准偏差为2.13%,线性相关系数为0.998,表观摩尔吸光系数为1.32×103L/（mol.cm）,分析实验值与实际值相吻合.