根霉12#发酵产生纤溶酶的酶学性质

溶栓疗法是血栓性疾病安全有效的治疗手段 ,开发新型纤溶酶具有实际应用意义。分离自南方小酒药的根霉 12^#以豆粕和麸皮为原料可产生纤溶酶。已采用盐析 ,疏水层析、离子交换层析和凝胶层析方法对纤溶酶分离提纯。提纯的纤溶酶比活力 2143u/mg(尿激酶单位 ) ,有直接溶解血栓和激活纤溶酶原的双重溶栓作用 ,降解纤维蛋白α、β和γ肽链速度快 ;最适作用温度 4 5℃ ,适宜作用pH范围 6.8～8.8;等电聚焦方法测定该酶等电点 8.5± 0.1;只分解生色底物N-Succinyl-Ala-Ala-Pro-Phe-pNA ,其米氏常数K_m 为 0.23mmol/L ,酶转换数K_cat为 16.36s^-1;Molish实验和甲苯胺蓝实验均证明该酶为糖蛋白 ,地衣酚_硫酸法测得该酶含糖量 470% ;EDTA、PMSF、PCMB对该纤溶酶有抑制作用 ,说明活性中心含有巯基、金属和丝氨酸 ;N端12个氨基酸序列为NH2-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly,与其它生物来源的纤溶酶相比较没有同源性。根霉 12^#产生的纤溶酶为新型纤溶酶,有希望开发成溶栓药物。     

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.     

冬虫夏草固态培养菌丝中纤溶酶的纯化和酶学性质

【背景】血栓性疾病发病率逐年递增,发病人群呈现低龄化趋势,严重地影响着人们的身心健康。因此研发高效、安全、特异性强的溶栓药物具有重要的意义,是血栓性疾病预防与治疗研究领域的热点。【目的】对冬虫夏草菌丝固态培养过程中产生的纤溶酶进行分离纯化,并对纯化的纤溶酶进行酶学性质分析。【方法】通过硫酸铵盐析、阳离子交换色谱和Superdex 75凝胶过滤色谱分离纯化虫草纤溶酶。采用Bradford法测定样品中总蛋白质浓度,纤维蛋白平板法测定纤溶酶活性,Native-PAGE检测纯度,SDS-PAGE测定相对分子量。【结果】在固态培养中冬虫夏草菌丝可以产生至少两种纤溶酶,分别命名为OSP-1和OSP-2。纯化后OSP-1比活力达到4186.25U/mg,纯化倍数为41.69倍。OSP-1由两个亚基构成,相对分子量分别为27.60k D和23.83k D,是一种丝氨蛋白酶。酶学性质分析表明,该酶最适作用温度为40°C,最适pH为4.0。Cu2+可促进OSP-1酶活,而Zn2+会抑制酶活。除了具有较高的纤溶酶活性,OSP-1还可发挥激活纤维蛋白酶原的作用。在水解纤维蛋白原的过程中,该酶可依次降解γ、Aα、Bβ链。【结论】研究发现的OSP-1具有开发成新型溶栓药物的潜力。     

蛹虫草无性型深层培养液中纤溶酶的分离纯化和酶学性质研究

【目的】确立蛹拟青霉深层培养液中高纯度、高纤溶活性纤溶酶的分离纯化方法并测定其酶学性质。【方法】采用硫酸铵盐析、Sephadex G-25凝胶色谱、Phenyl-Sepharose HP疏水相互作用色谱、CM-Sepharose FF弱阳离子交换色谱和Superdex 75凝胶色谱对蛹拟青霉纤溶酶进行分离。用Lowry法测定蛋白质浓度,纤维蛋白平板法测定其纤溶活性,SDS-PAGE鉴定其纯度并确定其分子量,IEF法测定其等电点。【结果】研究发现,以蔗糖和豆饼为培养基主要基质时,蛹拟青霉深层培养可以产生至少两种纤溶酶。提纯后的纤溶酶Ⅱ比活力达到800.46 U/mg,总纯化倍数为30.07倍。纤溶酶Ⅱ的相对分子量和等电点分别为32 kD和9.3±0.2。纤溶酶Ⅱ是一种糖蛋白,总含糖量为0.98%(W/V)。该酶可以顺次降解人血纤维蛋白(原)的α、β和γ链。其最适作用pH及温度分别为7.4和41°C。Aprotinine与PMSF对该纤溶酶的活性完全抑制,推测此纤溶酶可能是一种丝氨酸蛋白酶。【结论】单一的高纤溶活性纤溶酶的获得和酶学性质的确定,为该酶开发成为新型溶栓药物提供了理论依据。     

豆豉纤溶酶的研究现状

豆豉纤溶酶是从中国传统风味食品豆豉中分离得到的一种由杆菌产生的丝氨酸蛋白酶,具有极强的纤溶活性.综述了豆豉纤溶酶的产生菌种、发酵工艺、酶学性质、分离纯化、作用机理以及分子生物学特性等研究现状.豆豉纤溶酶在溶栓的过程中有着溶栓能力强,无毒副作用,原材料丰富,在体内半衰期长等优点,因此该酶有着广阔的应用发展前景.     

蚯蚓纤溶酶分子生物学进展

蚯蚓纤溶酶是近年发现的一种新型的溶解血栓物质,属丝氨酸蛋白酶,不同种属的蚯蚓中均可分离到,具纤溶活性和溶栓活性。有较好的热稳定性,多为单体酶,多数兼有纤溶活性和纤溶酶原激活活性。不同种属的蚯蚓分离的纤溶酶性质上有一定差别。已获得多种纤溶酶的N端序列及部分核酸序列,相互之间及与某些蛋白酶之间有一定的同源性。纤溶酶通过降解目的蛋白的特定位点而起作用 。     

蚯蚓纤溶酶分子生物学进展

蚯蚓纤溶酶是近年发现的一种新型的溶解血栓物质,属丝氨酸蛋白酶,不同种属的蚯蚓中均可分离到,具纤溶活性和溶栓活性。有较好的热稳定性,多为单体酶,多数兼有纤溶活性和纤溶酶原激活活性。不同种属的蚯蚓分离的纤溶酶性质上有一定差别。已获得多种纤溶酶的N端序列及部分核酸序列,相互之间及与某些蛋白酶之间有一定的同源性。纤溶酶通过降解目的蛋白的特定位点而起作用。     

食品纤溶酶研究概况

溶栓疗法是血栓性疾病安全有效的治疗手段,开发安全高效、廉价的新型纤溶酶对于预防与治疗血栓性疾病具有重要意义。近年来,在许多亚洲传统发酵食品中如日本纳豆、韩国大豆酱、中国豆豉、发酵虾酱等中均发现有丰富的纤溶酶资源。本文重点介绍传统发酵食品中纤溶酶的研究概况及其开发前景。     

豆豉纤溶酶--一种潜在的新型溶栓药物

豆豉纤溶酶是一种潜在的新型溶栓药物。该文介绍豆豉纤溶酶产生菌的筛选、诱变;纤溶酶的分离纯化、酶学性质及其分子生物学的研究等：并简述了纤溶酶活性的测定方法;提出了豆豉纤溶酶的研究方向及前景。     

根霉液态发酵产生纤溶酶的培养条件优化

目的:目前临床使用的溶栓药物疗效肯定,但还存在许多缺陷,而且价格昂贵,因此研制新型溶栓药物的需求迫切。方法:研究了根霉Rhizopus chinensisYY-15液体摇瓶发酵产生纤溶酶的工艺条件。采用单因素试验对液体发酵培养基的碳源、氮源、碳氮比、初始pH进行了优化;采用正交试验对发酵时间、接种量进行了研究。结果:实验范围内菌株液体发酵产纤溶酶的适宜培养基组成为:麸皮水浓度3%(w/v),豆粕浓度5%(w/v),初始培养基pH5.0。适宜培养条件为接种量6%,培养时间72h。优化条件下的摇瓶液体发酵纤溶酶产量平均达98.31 U/ml。     

Purification and characterization of a novel fibrinolytic enzyme from Rhizopus chinensis 12

A novel fibrinolytic enzyme from Rhizopus chinensis 12 was purified through ammonium sulfate precipitation, hydrophobic interaction, ionic exchange, and gel filtration chromatography. The purification protocol resulted in a 893-fold purification of the enzyme, with a final yield of 42.6%. The apparent molecular weight of the enzyme was 18.0 kDa, determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and 16.6 kDa by gel filtration chromatography, which revealed a monomeric form of the enzyme. The isoelectric point of the enzyme estimated by isoelectric focusing electrophoresis was 8.5±0.1. The enzyme hydrolyzed fibrin. It cleaved the , , and chains of fibrinogen simultaneously, and it also hydrolyzed casein and N-succinyl-Ala-Ala-Pro-Phe-pNA. The enzyme had an optimal temperature of 45°C, and an optimal pH of 10.5. EDTA, PCMB, and PMSF inhibited the activity of the enzyme, and SBTI, Lys, TPCK, and Aprotinine had no obvious inhibition, which suggested that the activity center of the enzyme had hydrosulfuryl and metal. The first 12 amino acids of the N-terminal sequence of the enzyme were S-V-S-E-I-Q-L-M-H-N-L-G and had no homology with that of other fibrinolytic enzyme from other microbes.     

Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi,a traditional Chinese soybean food

Bacillus amyloliquefaciens DC-4, which produces a strongly fibrinolytic enzyme, was isolated from douchi, a traditional Chinese soybean-fermented food. A fibrinolytic enzyme (subtilisin DFE) was purified from the supernatant of B. amyloliquefaciens DC-4 culture broth and displayed thermophilic, hydrophilic and strong fibrinolytic activity. Subtilisin DFE was demonstrated to be homogeneous by SDS-PAGE and isoelectric focusing electrophoresis, and has molecular mass of 28000 Da and a pI of 8.0. The optimal reaction pH value and temperature were 9.0 and 48 degrees C, respectively. Subtilisin DFE not only hydrolyzed fibrin but also several synthetic substrates, particularly Suc-Ala-Ala-Pro-Phe-pNA, and phenylmethylsulfony fluoride can completely inhibit its fibrinolytic activity. These results indicated that subtilisin DFE is a subtilisin-family serine protease, similar to nattokinase from Bacillus natto. The first 24 amino acid residues of the N-terminal sequence of subtilisin DFE were AQSVPYGVSQIKAPALHSQGFTGS, which is identical to that of subtilisin K-54, and different from that of NK and CK. Results from subtilisin DFE gene sequence analysis showed that subtilisin DFE is a novel fibrinolytic enzyme.     

Purification and characterization of a novel fibrinolytic protease from <Emphasis Type="Italic">Fusarium</Emphasis> sp. CPCC 480097

A novel fibrinolytic enzyme from Fusarium sp. CPCC 480097, named Fu-P, was purified to electrophoretic homogeneity using ammonium sulfate precipitation and ion exchange and gel filtration chromatography. Fu-P, a single protein had a molecular weight of 28 kDa, which was determined by SDS-PAGE and gel filtration chromatography. The isoelectric point of Fu-P determined by isoelectric focusing electrophoresis (IEF) was 8.1, and the optimum temperature and pH value were 45°C and 8.5, respectively. Fu-P cleaved the α-chain of fibrin (ogen) with high efficiency, and the β-chain and γ-γ (γ-)-chain with lower efficiency. Fu-P activity was inhibited by EDTA and PMSF, and the enzyme exhibited a high specificity for the chymotrypsin substrate S-2586. Fu-P was therefore identified as a chymotrypsin-like serine metalloprotease. The first 15 amino acids of the N-terminal sequence of Fu-P were Q-A-S–S-G-T-P-A-T-I-R-V-L-V–V and showed no homology with that of other known fibrinolytic enzymes. This protease may have potential applications in thrombolytic therapy and in thrombosis prevention.     

Process optimization for production and purification of novel fibrinolytic enzyme from Stenotrophomonas sp. KG-16-3

Intravascular thrombosis is a major cardiovascular complication responsible for high mortality worldwide. Existing thrombolytic agents are expensive and have various side effects. As a consequence, researchers continue to search for better thrombolytic agents. Fibrinolytic proteases especially those of microbial origin are considered as potential therapeutic candidates for thrombosis. The current study reports fibrinolytic protease from a bacterial isolate Stenotrophomonas sp. KG-16-3, as it exhibits high fibrinolytic activity on fibrin agarose plate. Studies on fibrinolytic protease from Stenotrophomonas sp. are lacking. So, a detailed study was conducted for the production and purification of fibrinolytic protease. Optimizing process parameters using the Design of Experiments method enhanced the yield by 1.5-fold. The fibrinolytic enzyme was purified by ammonium sulfate precipitation, ion-exchange and gel-filtration chromatography resulting in 7.1-fold purification and 16.7% yield with specific activity of 383.8?U/mg. The purified enzyme exhibited higher fibrinolytic activity than plasmin and had a molecular weight of 39?kDa. Optimal activity of the enzyme was observed at 50?°C and pH 10. The enzyme exhibited stability up to 60?°C, over pH 7–10 and in the presence of different metal ions and solvents. The activity of the enzyme was significantly reduced in the presence of phenylmethyl sulfonyl fluoride, iodoacetic acid and 1,10-phenanthroline, suggesting that the enzyme belonged to the serine–cysteine metalloprotease category. The present study is the first ever report on the Design of Experiments based optimization of fermentation conditions for the production of fibrinolytic protease from Stenotrophomonas sp.     

A fibrinolytic enzyme from the medicinal mushroom Cordyceps militaris

In this study we purified a fibrinolytic enzyme from Cordyceps militaris using a combination of ion-exchange chromatography on a DEAE Sephadex A-50 column, gel filtration chromatography on a Sephadex G-75 column, and FPLC on a HiLoad 16/60 Superdex 75 column. This purification protocol resulted in a 191.8-fold purification of the enzyme and a final yield of 12.9 %. The molecular mass of the purified enzyme was estimated to be 52 kDa by SDS-PAGE, fibrin-zymography, and gel filtration chromatography. The first 19 amino acid residues of the N-terminal sequence were ALTTQSNV THGLATISLRQ, which is similar to the subtilisin-like serine protease PR1J from Metarhizium anisopliae var. anisopliase. This enzyme is a neutral protease with an optimal reaction pH and temperature of 7.4 and 37 degrees , respectively. Results for the fibrinolysis pattern showed that the enzyme rapidly hydrolyzed the fibrin alpha-chain followed by the gamma-gamma chains. It also hydrolyzed the beta-chain, but more slowly. The Aalpha, Bbeta, and gamma chains of fibrinogen were also cleaved very rapidly. We found that enzyme activity was inhibited by Cu2+ and Co2+, but enhanced by the additions of Ca2+ and Mg2+ ions. Furthermore, fibrinolytic enzyme activity was potently inhibited by PMSF and APMSF. This enzyme exhibited a high specificity for the chymotrypsin substrate S-2586 indicating it 's a chymotrypsin-like serine protease. The data we present suggest that the fibrinolytic enzyme derived from the edible and medicinal mushroom Cordyceps militaris has fibrin binding activity, which allows for the local activation of the fibrin degradation pathway.     

Enzymatic properties and identification of a fibrinolytic serine protease purified from Bacillus subtilis DC33

A novel fibrinolytic enzyme subtilisin FS33 was purified from Bacillus subtilis DC33, isolated from a traditional flavour-rich food in China. The purified subtilisin FS33 was a single chain protein with a molecular mass of 30 kDa measured by SDS-PAGE. After activated SDS-PAGE, the enzyme band exhibited strong fibrinolytic activity on the fibrin plate. Subtilisin FS33 was temperature-stable below 60°C over the pH range 5–12, with a maximum activity at pH 8.0, but the activity completely disappeared after 10 min above 65°C. The NH₂-terminal amino acid sequence of the enzyme was different from that of other known fibrinolytic enzymes, such as NK, CK, SMCE, KA38, subtilisin E, subtilisin DFE and Katsuwokinase. The amidolytic activities of subtilisin FS33 were inhibited completely by phenylmethanesulfonyl fluoride (PMSF) and soybean trypsin inhibitor (SBTI). EDTA did not affect the enzyme activity, and none of the ions tested activated the activity. Therefore, the enzyme was thought to be a subtilisin-like serine protease. The enzyme degraded the Bβ-chains of fibrin(ogen) very rapidly and then degraded the Aα-chain and at least five fragments from fibrin(ogen) were obtained after hydrolysis. Subtilisin FS33 was also able to cleave blood clots in the absence of endogenous fibrinolytic factors.     

Purification and characterization of fibrinolytic metalloprotease from Perenniporia fraxinea mycelia

In this study we purified and characterized a fibrinolytic protease from the mycelia of Perenniporia fraxinea. The apparent molecular mass of the purified enzyme was estimated to be 42 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), fibrin zymography and size exclusion using fast protein liquid chromatography (FPLC). The first 20 amino acid residues of the N-terminal sequence were ASYRVLPITKELLPPEFFVA, which shows a high degree of similarity with a fungalysin metallopeptidase from Coprinopsis cinerea. The optimal reaction pH value and temperature were pH 6.0 and 35–40 °C, respectively. Results for the fibrinolysis pattern showed that the protease rapidly hydrolyzed the fibrin α-chain followed by the β-chain. The γ–γ chains were also hydrolyzed, but more slowly. The purified protease effectively hydrolyzed fibrinogen, preferentially digesting the Aα-chains of fibrinogen, followed by Bβ- and γ-chains. We found that protease activity was inhibited by Cu²⁺, Fe³⁺, and Zn²⁺, but enhanced by the additions of Mn²⁺, Mg²⁺ and Ca²⁺ metal ions. Furthermore, the protease activity was inhibited by EDTA, and it was found to exhibit a higher specificity for the chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The mycelia of P. fraxinea may thus represent a source of new therapeutic agents to treat thrombosis.     

Purification and characterization of a fibrinolytic enzyme from Streptomyces sp. XZNUM 00004

A fibrinolytic enzyme (SFE1) from Streptomyces sp. XZNUM 00004 was purified to electrophoretic homogeneity with the methods including ammonium sulfate precipitation, polyacrylamide gel, DEAE-Sepharose Fast Flow anion exchange and gel-filtration chromatography. The molecular weight of SFE1 was estimated to be 20 kDa by SDS-PAGE, fibrin zymography, and gel filtration chromatography. The isoelectric point was 4.9. K (m) and V (max) values were 0.96 mg/ml and 181.8 unit/ml, respectively. It was very stable at pH 5.0-8.0 and below 65 °C. The optimum pH for enzyme activity was 7.8. The optimum temperature was 35 °C. The fibrinolytic activity of SFE1 was enhanced by Na(+), K(+), Mn(2+), Mg(2+), Zn(2+) and Co(2+). Conversely, Cu(2+) showed strong inhibition. Furthermore, the fibrinolytic activity was strongly inhibited by PMSF, and partly inhibited by EDTA and EGTA. SFE1 rapidly hydrolyzed the Aα-chain of fibrinogen, followed by the Bβ-chain and finally the γ-chain. The first 15 amino acids of the N-terminal sequence were APITLSQGHVDVVDI. Additionally, SFE1 directly digested fibrin and not by plasminogen activators in vitro. SFE1 can be further developed as a potential candidate for thrombolytic therapy.     

Screening and characterization of a novel fibrinolytic metalloprotease from a metagenomic library

A metagenomic library was constructed using total genomic DNA extracted from the mud in the west coast of Korea and was used together with a fosmid vector, pCC1FOS in order to uncover novel gene sources. One clone from approximately 30,000 recombinant Escherichia coli clones was identified that showed proteolytic activity. The gene for the proteolytic enzyme was subcloned into pUC19 and sequenced, and a database search for homologies revealed it to be a zinc-dependent metalloprotease. The cloned gene included the intact coding gene for a novel metalloproteinase and its own promoter. It comprised an open reading frame of 1,080 base pairs, which encodes a protein of 39,490 Da consisting of 359 amino acid residues. A His-Glu-X-X-His sequence, which is a conserved sequence in the active site of zinc-dependent metalloproteases, was found in the deduced amino acid sequence of the gene, suggesting that the enzyme is a zinc-dependent metalloprotease. The purified enzyme showed optimal activity at 50°C for 1 h and pH 7.0. The enzyme activity was inhibited by metal-chelating reagents, such as EDTA, EGTA and 1,10-phenanthroline. The enzyme hydrolyzed azocasein as well as fibrin. Thus, the enzyme could be useful as a therapeutic agent to treat thrombosis. The sequence reported in this paper has been deposited in the GenBank database (Accession number: EF100137).     

Purification and characterization of rhizopuspepsin isozymes from a liquid culture of Rhizopus chinensis

1. Rhizopuspepsin has been purified from liquid cultures of Rhizopus chinensis. 2. Purification by ammonium sulfate precipitation, affinity chromatography on pepstatin Sepharose and low/high resolution isoelectric focusing produced five isoelectric forms. 3. The two major isozymes pI 5.1 and 5.8 did not differ significantly in amino acid composition, molecular weight and enzyme activity. 4. Three minor isozymes were partially purified as pI 7.35, 7.41 and 7.9.