可口革囊星虫体内纤溶酶的初步研究

目的从可口革囊星虫中寻找到纤溶酶,并对其进行初步研究。方法采用匀浆、抽提离心、Sephacryl S-300凝胶过滤等方法对可口革囊星虫纤溶酶初步分离,用纤维蛋白平板法和合成发色底物法检测其纤溶活性,并用该酶进行体外溶血凝块实验。结果可口革囊星虫内脏中存在纤溶酶。此酶既直接降解纤维蛋白又间接激活纤溶酶原,它对体外血凝块有明显溶解作用。结论可口革囊星虫内脏中存在着一种既具直接降解纤维蛋白作用又具激活纤溶酶原作用的纤溶酶。     

来自光裸方格星虫产纤溶酶蜡状芽孢杆菌的鉴定

对广西北部湾光裸方格星虫中产纤溶酶菌株进行筛选和鉴定,结合脱脂乳平板与纤维蛋白板筛选出产纤溶酶菌株,并对其粗酶液的血栓溶解机制进行了体外初步研究。结果显示,从光裸方格星虫获得5株产纤溶酶菌株,其中GXUSP-1菌株纤溶酶粗酶活力(相当尿激酶酶活力单位)为303.2 U/mL,经初步鉴定该菌为蜡状芽孢杆菌属,属于海洋共生菌,该菌株粗酶液能通过直接和间接两种方式溶解血栓,且体外溶解血栓作用显著。GXUSP-1具有良好的潜在工业应用价值。     

单环刺螠纤溶酶UFE-Ⅰ的性质和溶栓活性

单环刺螠纤溶酶UFE-Ⅰ的最适反应温度为45 ℃;最适反应pH为7.0;Mg2+、Mn2+和Fe2+是该纤溶酶的强激活剂;Fe3+、Cu2+、Ag+、Hg+和Pb2+对该纤溶酶具有一定的抑制作用;SBTI和PMSF,完全抑制UFE-Ⅰ,说明该酶为丝氨酸蛋白酶;糜蛋白酶抑制剂部分抑制UFE-Ⅰ,亮抑酶肽、抑蛋白酶肽、苯甲脒较弱的抑制UFE-Ⅰ.与蚓激酶类似,UFE-Ⅰ不仅具有直接的纤溶活力更具有纤溶酶原激活活力(84.0%),另外分别将蚓激酶原料与该酶加入到家兔血栓块中,37 ℃孵育3 h,结果表明该酶具有比蚓激酶更为强大的溶栓能力.     

一株产纤溶酶菌株的分离、鉴定及其酶活特征的初步研究

[目的]分离筛选并鉴定产纤溶酶的菌株.[方法]采用血粉培养基富集,琼脂糖-纤维蛋白平板筛选,从自然界中分离筛选出一株产纤溶活性物质的菌株.通过形态学特征、生理生化特征研究,并结合16S rRNA基因序列分析及分子系统发育树的构建结果,确定菌株的种类.[结果]从自然界分离筛到一株产纤溶酶的菌株EF608,经鉴定该菌株为粪肠球菌(Enterococcus faecalis). SDS-PAGE和纤维蛋白自显影表明该纤溶酶的分子量为37 kD,最适反应温度和pH分别为35℃和7.5,EDTA能完全抑制其纤溶活性,而PMSF对其活性无抑制作用.菌株EF608发酵液不仅可以直接水解纤维蛋白,而且具有体外溶栓的作用,对血红细胞没有溶解作用.[结论]筛选到一株具有纤溶活性的粪肠球菌——EF608,为获取新型纤溶酶提供了一种的新的菌源.     

海南方格星虫纤溶酶分离纯化及部分基因克隆

采用硫酸铵盐析、DEAE-Sepharose FF离子交换、Sephadex G-50凝胶层析等方法从方格星虫(Sipunculus nudus)内脏组织中分离纯化出一种水解纤维蛋白的活性酶,命名为SNF1。经SDS-PAGE及native-PAGE分析其分子量为约24~26 k D的单链蛋白。MALDI-TOF MS分析分子质量为842.414 6的肽段与NCBI报道的方格星虫纤溶酶(gb|AEA06599.1|)肽段匹配。根据该纤溶酶cds基因序列设计引物,通过RT-PCR法扩增基因,并连接p MD19-T载体转化大肠杆菌E.coli DH5α感受态细胞。克隆转化子测序得到273 bp的纤溶酶基因序列,与该报道方格星虫纤溶酶基因序列相似度96%。本研究为此方格星虫纤溶酶c DNA全长序列的克隆及在原核或真核生物中表达的深入研究提供基础。     

光裸方格星虫纤溶酶SNFE体外溶栓作用及安全性初评

研究光裸方格星虫纤溶酶SNFE体外溶栓作用并初步评价药物的生物安全性。通过标准纤维蛋白平板和加热纤维蛋白平板实验、剩余可凝纤维蛋白原法、体外溶栓实验研究SNFE体外溶栓作用及作用方式;通过皮下出血实验及体外溶血实验初步评估SNFE的生物安全性。结果发现,SNFE在体外不仅具有激酶活性,且能直接溶解纤维蛋白原和纤维蛋白,对血凝块具有良好的溶栓作用,同时无出血活性及溶血作用,具有一定的开发价值。     

枯草芽孢杆菌BS-26菌株纤溶酶的性质分析及活性组分的分离纯化

[目的]溶栓疗法是血栓性疾病安全且有效的治疗手段,从微生物中寻找溶栓药物是一种理想有效的途径,枯草芽孢杆菌(Bacillus subtilis)BS-26菌株发酵液具有很强的体外纤溶活性,本文分析了发酵液中纤溶酶的性质并对活性组分进行了分离纯化.[方法]利用纤维蛋白平板法检测纤溶酶活性,利用硫酸铵分级盐析、DEAE-Sepharose Fast Flow阴离子交换层析和聚丙烯酰胺制备电泳等方法,进行分离纯化.[结果]此菌株产生的纤溶酶在50℃以下和pH5.0～11.0范围内具有较好的稳定性,最适作用温度为42℃;最适pH值为9.0;Mg2 、Ca2 对此酶有明显的激活作用,而Cu2 能完全抑制酶的活性;174.2μg/mL的苯甲基磺酰氟、1000μg/mL的鸡卵类粘蛋白和1000μg/mL大豆胰蛋白酶抑制剂能完全抑制酶活性,初步说明此酶属于丝氨酸蛋白酶类;体外溶纤作用表明,该酶溶解纤维蛋白的方式是直接溶解,而不是通过激活纤溶酶原.从该菌株的发酵液中获得了一种纤溶酶组分,比活力达8750 U/mg,回收率为3.2%,所获得样品纯度相对于发酵液提高了41倍,该酶在SDS-PAGE中是单肽链蛋白,分子量为32 kDa.[结论]获得了一种纤溶酶的单一组分,为纤溶酶发酵产品的大规模纯化及进一步研制和开发新的溶栓药物提供重要理论依据.     

重组枯草杆菌纤溶酶的酶学性质研究

对一种重组枯草杆菌纤溶酶 (rBSFE)的酶学性质进行了初步研究 ,结果表明 :酶作用最适温度为 35℃ ;最适反应pH为 8.0 ;酶活性能被PMSF抑制 ,是典型的丝氨酸蛋白酶。通过与尿激酶进行比较 ,发现该酶对纤维蛋白有直接降解作用 ,而对于纤维蛋白原的敏感性则低于尿激酶 ,提示该酶具有直接溶栓作用 ,又不致引起出血 ,是一种有潜力的新型溶栓剂。     

重组海蚯蚓纤溶酶的克隆、表达及活性鉴定北大核心CSCD

纤溶酶在溶栓治疗中起重要作用,能够溶解血凝块的主要成分纤维蛋白。采用RACE方法从海蚯蚓消化道组织中扩增出海蚯蚓纤溶酶编码序列,构建该基因原核表达载体,并进一步构建工程菌表达融合蛋白,经Ni2+树脂柱纯化后通过平板法检测该融合蛋白纤维蛋白酶原激活活性。结果获得海蚯蚓纤溶酶的c DNA序列和氨基酸序列,并成功构建重组表达载体p ET-21a-AFE,表达纯化出融合蛋白,该融合蛋白能够激活纤维蛋白酶原而溶解纤维蛋白。总之,本研究获得了海蚯蚓纤溶酶的c DNA序列和氨基酸序列,并初步证明其具有纤维蛋白酶原激活活性,为临床新型溶栓药物的开发提供实验基础。     

地鳖纤溶活性蛋白的纯化及性质研究

通过硫酸铵分段沉淀、DEAE-纤维素柱和SephadexG-75柱层析从雌地鳖(Eupolyphagesinensiswalker)体内分离纯化到一种相对分子质量约为41.3kD的纤溶活性蛋白,纤维蛋白平板测定表明,该蛋白具有纤溶作用,经SDS-PAGE电泳显示为一条带,含糖量为10.5%。其水解纤维蛋白的比活力为547.86u/mg。该成分受蛋白抑制剂和丝氨酸蛋白酶抑制剂PMSF的抑制,但EDTA对其影响不大,提示该成分属于丝氨酸蛋白酶类。该成分在40℃下基本稳定,最适温度40℃,最适pH为8.0,其激活纤维蛋白溶解酶(PLG)的机制与尿激酶(UK)有一定区别。推测其可能是一种新的地鳖纤溶酶组分。     

Relationship between enzyme heterozygosity and quaternary structure

The need for proteins to maintain particular quaternary structures constrains variability in amino acid sequence. Monomeric enzymes are then expected to be more variable than dimeric forms, which in turn are expected to be more variable than tetrameric forms. These predictions are confirmed by analysis of available data on enzyme variation. Theories relating enzyme heterozygosity to metabolic function are discussed in the light of these findings.Financial support for part of the work described in this article was derived from NERC Grant GR3/1558 to J. A. Beardmore.     

Detoxification,antioxidant, and digestive enzyme activities and gene expression analysis of Lymantria dispar larvae under carvacrol

Carvacrol is a terpene compound with various biological activities. However, few studies have specifically focused on its insecticidal activity and mechanism of carvacrol. The larvae of Lymantria dispar are seriously harmful herbivorous insect. This study measured the antifeedant, growth-inhibitory, and toxic effects of carvacrol on L. dispar larvae. To further clarify the insecticidal mechanism of carvacrol, the effects of carvacrol on detoxifying enzymes, antioxidative enzymes, digestive enzyme activities, and the mRNA expression of the above-mentioned enzyme genes were investigated. The results of the study showed that the median lethal concentration (LC₅₀) and the sublethal concentration (LC₂₀) of carvacrol were 1.120 mg/mL and 0.297 mg/mL, respectively, at 72 h. After LC₂₀ treatment of L. dispar larvae for 72 h, food intake and weight gain were significantly lower compared with the control. Enzyme activity assays showed that carvacrol significantly inhibited the activities of carboxylesterase (CarE), glutathione S-transferase (GST), and acetylcholinesterase (AchE), and the inhibition rate of AchE activity was highest (66.51%). Carvacrol also activated the activities of superoxide dismutase (SOD) and catalase (CAT), while it inhibited the activities of lipase (LIP) and amylase (AMS), and first inhibited and then activated protease. In addition, qRT-PCR tests showed that carvacrol affected the mRNA expression levels of CarE, GST, AchE, SOD, CAT, LIP, AMS, and protease. This study helps to clarify the insecticidal mechanism of carvacrol on L. dispar larvae.     

Inducible catalase in Pseudomonas fluorescens

The catalase activity of a non-proliferating suspension of Pseudomonas fluorescens doubled after six hours incubation in a 50 mM phosphate buffer medium (pH 7.3). The same effect was observed in a peptone medium. The increased activity was due to induced enzyme synthesis, and not to activation of preexisting catalase. Induced catalase was separated by electrophoresis from deuterium labelled constitutive catalase. The enzyme was also induced under anaerobic conditions in phosphate buffer or in culture when nitrate was supplied as an electron acceptor. Induction was considerably increased by the addition of various nucleotides and amino acids to the incubation medium.     

Biosynthesis of the C-terminal amide in peptide hormones

Recent developments in the study of peptide amidation are reviewed. The main areas covered are assay procedures, purification of amidating enzymes, co-fact0rs and regulation; mechanism and specificity of the amidating reaction, and multiple forms of the amidating enzyme and glycosylation. Discussion is presented on aspects that are poorly understood and new areas open to investigation are indicated.     

Partial purification and kinetic properties of three different d-glucosamine 6-P:N-acetyltransferase forms from human placenta

Three distinct forms of -glucosamine 6-P (Gm 6-P):N-acetyltransferases (EC 2.3.1.4) were partially purified from human placental homogenates by carboxy methyl-Sephadex chromatography. Purification of forms I and II were 13.5-fold, while that of form III was 114-fold. All three forms had a pH optimum value of 9.7 in glycine–NaOH buffer. Enzymes II and III had a K_m value for Gm 6-P of 3.0 mM, which was less than half of that observed for form I (7.1 mM). The corresponding K_m values for acetyl CoA were 0.157 (form I), 0.187 (form II) and 0.280 mM (form III), respectively. Activities of all three forms were inhibited at high concentrations of either substrate. These enzymes were inhibited from 82 to 92% by 2.5 mM p-chloromercuribenzoate. The inhibition was largely reversible by inclusion of 2.5 mM dithiothreitol in the incubation mixtures. There was no requirement for divalent cations, as demonstrated by lack of inhibition of enzyme activity by ethylene diamine tetraacetate. The results are discussed in terms of differences among the enzyme properties of human placental, rodent and porcine liver forms.     

Metal-Induced reversible structural interconversion of human mitochondrial NAD(P)+-dependent malic enzyme

Human mitochondrial NAD(P)+-dependent malic enzyme was strongly inhibited by Lu3+. The X-ray crystal structures indicated a structural change between the metal-free and Lu3+-containing enzymes (Yang Z, Batra R, Floyd DL, Hung HC, Chang GG, Tong L. Biochem Biophys Res Commun 2000;274:440-444). We characterized the reversible slow-binding mechanism and the structural interconversion between Mn2+- and Lu3+-containing human mitochondrial malic enzymes. When Lu3+ was added, the activity of the human enzyme showed a downward curve over time, similar to that of the pigeon enzyme. The rate of the transformation (k(obs)) from the initial rate to the steady-state rate increased hyperbolically with the concentration of Lu3+, suggesting the involvement of an isomerization step. Lu3+ had a much higher affinity for the isomerized form (K*(i,Lu (app)) = 4.8 microM) than that of the native form (K(i,Lu (app)) = 148 microM). When an excess of Mn2+ was added to the Lu3+-inhibited enzyme, assays of the kinetic activity showed an upward trend, indicating reactivation. This result also indicated that the reactivation was a slow process. Fluorescence quenching experiments confirmed that the Lu3+-induced isomerization was completely reversible. The dynamic quenching constants for the metal-free, Mn2+-containing, and Lu3+-containing enzyme were 3.08, 3.07, and 3.8 M(-1), respectively. When the Lu3+-containing enzyme was treated with excess Mn2+, the dynamic quenching constant returned to the original value (3.09 M(-1)). These results indicated that binding of Mn2+ did not induce any conformational change in the enzyme. The open form transformed to the closed form only after substrate binding. Lu3+, on the other hand, transformed the open form into a catalytically inactive form. Excess Mn2+ could replace Lu3+ in the metal binding site and convert the inactive form back into the open form. This reversible process was slow in both directions because of the same but opposite structural change involved.     

Engineered and Native Coenzyme B12-dependent Isovaleryl-CoA/Pivalyl-CoA Mutase

Adenosylcobalamin-dependent isomerases catalyze carbon skeleton rearrangements using radical chemistry. We have recently demonstrated that an isobutyryl-CoA mutase variant, IcmF, a member of this enzyme family that catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA also catalyzes the interconversion between isovaleryl-CoA and pivalyl-CoA, albeit with low efficiency and high susceptibility to inactivation. Given the biotechnological potential of the isovaleryl-CoA/pivalyl-CoA mutase (PCM) reaction, we initially attempted to engineer IcmF to be a more proficient PCM by targeting two active site residues predicted based on sequence alignments and crystal structures, to be key to substrate selectivity. Of the eight mutants tested, the F598A mutation was the most robust, resulting in an ∼17-fold increase in the catalytic efficiency of the PCM activity and a concomitant ∼240-fold decrease in the isobutyryl-CoA mutase activity compared with wild-type IcmF. Hence, mutation of a single residue in IcmF tuned substrate specificity yielding an ∼4000-fold increase in the specificity for an unnatural substrate. However, the F598A mutant was even more susceptible to inactivation than wild-type IcmF. To circumvent this limitation, we used bioinformatics analysis to identify an authentic PCM in genomic databases. Cloning and expression of the putative AdoCbl-dependent PCM with an α₂β₂ heterotetrameric organization similar to that of isobutyryl-CoA mutase and a recently characterized archaeal methylmalonyl-CoA mutase, allowed demonstration of its robust PCM activity. To simplify kinetic analysis and handling, a variant PCM-F was generated in which the αβ subunits were fused into a single polypeptide via a short 11-amino acid linker. The fusion protein, PCM-F, retained high PCM activity and like PCM, was resistant to inactivation. Neither PCM nor PCM-F displayed detectable isobutyryl-CoA mutase activity, demonstrating that PCM represents a novel 5′-deoxyadenosylcobalamin-dependent acyl-CoA mutase. The newly discovered PCM and the derivative PCM-F, have potential applications in bioremediation of pivalic acid found in sludge, in stereospecific synthesis of C5 carboxylic acids and alcohols, and in the production of potential commodity and specialty chemicals.     

酶制剂研制的国内外进展和发展策略

简述了酶工程的研究范围,国际上酶制剂工业发展的研究,特点。概述了近年来我国酶制剂工业发展中存在的问题,包括产品结构,技术含量,研发能力。提出我国酶制剂工业今后发展的一些建议：充分发挥资源优势,加强现代生物技术的应用,调整酶制剂产品的结构。     

Comparative studies of suidatrestin, a specific inhibitor of trehalases

Suidatrestin, isolated from a Streptomyces strain, was characterized as a new trehalase inhibitor. Its inhibitory potential was 7 to 50-fold higher than that of validamycin when tested against insect, fungal and mammalian trehalases. The kinetic properties of suidatrestin were studied in vitro with trehalases from flight muscle mitochondria of the fly, Protophormia terraenovae, from larval midgut of the moth, Spodoptera littoralis, and from porcine kidney, as well as with maltase from yeast. Suidatrestin was inactive on maltase but inhibited all trehalases with IC₅₀ values of 0.08–0.1 μM; K_i values ranged from 0.02 to 0.05 μM. The very low K_i/K_m ratios (3.9×10⁻⁶–4.9×10⁻⁶) indicated excellent in vitro inhibitory action of suidatrestin. When injected into larvae of S. littoralis, suidatrestin required high and repetitive doses which lead to reversible inhibition of larval growth only. Consecutive omission of the inhibitor even stimulated weight increase above that of controls. Significant mortality was achieved at a rather high dose only. Injection of a growth-inhibiting dose of suidatrestin did not change hemolymph osmolality as a measure of sugar concentration. The discrepancy between in vitro and in vivo potency of suidatrestin may be understood once its chemical structure is fully known.