Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02

Two fibrinolytic enzymes (QK-1 and QK-2) purified from the supernatant of Bacillus subtilis QK02 culture broth had molecular masses of 42,000 Da and 28,000 Da, respectively. The first 20 amino acids of the N-terminal sequence are AQSVPYGISQ IKAPALHSQG. The deduced protein sequence and its restriction enzyme map of the enzyme QK-2 are different from those of other proteases. The enzyme QK-2 digested not only fibrin but also a subtilisin substrate, and PMSF inhibited its fibrinolytic and amidolytic activities completely; while QK-1 hydrolyzed fibrin and a plasmin substrate, and PMSF as well as aprotinin inhibited its fibrinolytic activity. These results indicated QK-1 was a plasmin-like serine protease and QK-2 a subtilisin family serine protease. Therefore, these enzymes were designated subtilisin QK. The sequence of a DNA fragment encoding subtilisin QK contained an open reading frame of 1149 base pairs encoding 106 amino acids for signal peptide and 257 amino acids for subtilisin QK, which is highly similar with that of a fibrinolytic enzyme, subtilisin NAT (identities 96.8%). Asp32, His64 and Ser221 in the amino acid sequence deduced from the QK gene are identical to the active site of nattokinase (NK) produced by B. subtilis natto.     

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.     

纳豆激酶基因在大肠杆菌中的表达及活性分析

目的:构建纳豆激酶基因的表达载体,鉴定其在大肠杆菌中的表达及表达产物的生物活性鉴定.方法:以纳豆芽胞杆菌基因组为模板,PCR技术克隆出纳豆激酶基因的成熟肽序列,分别克隆进具有信号肽的pMAL-p2x及无信号肽pMAL-c2x质粒中,经酶切和测序鉴定其正确性,分别将重组质粒转化至大肠杆菌中表达.结果:成功构建的两组重组质粒在IPTG诱导下,均能分别在37℃及16℃条件下表达出可溶性的融合蛋白,SDS-PAGE胶检测证实重组质粒在大肠杆菌中可表达出相对分子量约76kDa的纳豆激酶蛋白.纤维蛋白平板实验证明两种融合蛋白均有活性,且有信号肽的融合蛋白的酶活较无信号肽的融合蛋白高.结果:成功构建了两组重组纳豆激酶基因的表达质粒,且该两组重组基因在大肠杆菌中可溶性表达并具有生物活性,因此为下一步研究表达产物纳豆激酶的功能、应用和生产奠定了基础.     

纳豆激酶基因的克隆及其在毕赤酵母中的表达

纳豆激酶纳是从日本传统食品纳豆中发现的一类具有溶栓效果的蛋白酶,由于其具有安全,高效,作用时间长,易吸收,廉价等优点,现在正成为一个开发治疗血栓类疾病药物的研究热点。从本实验室保存的一株高溶栓的纳豆杆菌N07出发,提取总基因组DNA,利用PCR手段扩增获得了纳豆激酶长为825bp的成熟肽基因片段。构建重组表达质粒pPICZaA-NK,经EcoR I、Xba I双酶切、PCR、测序验证得出重组表达质粒上的外源基因即为825bp的目的片段;将重组质粒pPICZaA-NK用内切酶Sac I线性化后电击导入毕赤酵母X33,通过含Zeocin的YPDS平板筛选获得重组酵母。重组酵母在BMMY培养基中发酵培养,用1%甲醇诱导目的蛋白表达。用纤维蛋白平板法检测发现发酵上清具有纤溶活性,经硫酸铵盐析、透析、Sephadex-G50过柱等步骤分离得到纳豆激酶蛋白,进行SDS-PAGE鉴定表明,表达的纳豆激酶蛋白分子量为27KD。以尿激酶为标准,实验所得纳豆激酶发酵上清液溶栓活性约为195U/mL。成功的将纳豆激酶成熟肽基因在毕赤酵母X33中表达,为纳豆激酶基因工程进一步研究奠定基础。     

纳豆激酶的研究进展

纳豆激酶是一种由纳豆菌产生的具有强烈纤溶作用的丝氨酸蛋白酶,与传统的一些溶栓剂相比,其具有安全性好、成本低、口服有效等优点。就纳豆激酶的理化性质、制备过程、生物学功能、酶活测定方法及分子生物学研究等进行了综述。     

全基因组重排育种技术提高产豆豉纤溶酶菌产酶量

枯草芽孢杆菌DC-12 是从豆豉里面筛选出来的,具有纤溶酶活性的菌株。本论文采用了全基因组重排技术提高DC－12的产酶量,首先通过对DC－12进行紫外诱变和亚硝酸诱变构建重组突变库,在研究其原生质体制备和再生的基础上,以其中4株诱变菌株作为直接亲本,采用电融合的方法进行两次多亲本的全基因组重排,结合双灭活的筛选方法,共筛选出2株酶活大大提高并能稳定遗传的菌株,使亲本菌株的酶活提高了4~5倍,最高达2710 IU/ml。     

Purification and biochemical characterization of a fibrinolytic enzyme from Bacillus subtilis BK-17

A fibrinolytic enzyme from Bacillus subtilis BK-17 has been purified to homogeneity by gel-filtration and ion-exchange chromatography. Compared to the crude enzyme extract, the specific activity of the enzyme increased 929-fold with a recovery of 29%. The subunit molecular mass of the purified enzyme was estimated to be 31 kDa by SDS–PAGE. The N-terminal amino acid sequence of the purified fibrinolytic enzyme was: A-Q-S-V-P-Y-G-V-S-Q-I-K-A-P-A-A-H-N. The sequence was highly homologous to the fibrinolytic enzymes nattokinase, subtilisin J and subtilisin E from Bacillus spp. However, there was a substitution of three amino acid residues in the N-terminal sequence. The amidolytic activity of the purified enzyme for several substrates was assessed. In comparison with nattokinase and CK (fibrinolytic enzyme from a Bacillus spp.), which showed strong fibrinolytic activity, the amidolytic activity of the enzyme for the synthetic substrate, kallikrein (H-D-Val-Leu-Arg-pNA, S-2266) increased 2.4- and 11.8-fold, respectively.     

纳豆激酶基因的克隆及其在枯草杆菌中的表达

为研究纳豆激酶（nattokinase,NK)的生物化学性质,以纳豆芽胞杆菌基因组DNA为模板,PCR扩增包含启动子及3‘端非翻译区的纳豆激酶全长基因序列。经鉴定后构建大肠杆菌－枯草杆菌穿梭表达质粒pBLNK,转化蛋白缺陷型枯草杆菌,筛选表达菌株。表达株培养15h后收集培养上清液,SDS－PAGE分析证实表达产物后,用超滤浓缩、分子筛、离子交换等步骤分离纯化重组NK,每升发酵液得纯度达95％的重组NK约100mg,比活性达12000u/mg。     

Secretory expression of a heterologous nattokinase in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Nattokinase has been reported as an oral health product for the prevention of atherosclerosis. We developed a novel strategy to express a nattokinase from Bacillus subtilis in a live delivery vehicle, Lactococcus lactis. Promoter P _nisZ and signal peptide SP_Usp were used for inducible and secretory expression of nattokinase in L. lactis. Western blotting analysis demonstrated that nattokinase was successfully expressed, and about 94% of the enzyme was secreted to the culture. The recombinant nattokinase showed potent fibrinolytic activity, equivalent to 41.7 urokinase units per milliliter culture. Expression and delivery of such a fibrinolytic enzyme in the food-grade vehicle L. lactis would facilitate the widespread application of nattokinase in the control and prevention of thrombosis diseases.     

Expression and purification of recombinant nattokinase in <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis> cells

A recombinant baculovirus, rv-egfp-NK, containing a reporter gene encoding the enhanced green fluorescent protein (EGFP), was used to express nattokinase (NK), a fibrinolytic enzyme, in Spodoptera frugiperda (SF-9) cells. The recombinant protein also included a histidine tag for purification using Ni(2+) resins. The recombinant NK, approximately 30 kDa, retained fibrinolytic activity (60 U/ml). The integration of the EGFP expression cassette in the Bac-to-Bac system is thus an effective method for the expression and purification of recombinant NK protein in Spodoptera frugiperda insect cells.     

纳豆激酶基因的克隆与表达

利用ＰＣＲ方法从分泌纳豆激酶的枯草杆菌基因组ＤＮＡ 中扩增得到了纳豆激酶基因,并测定其核苷酸序列．利用基因重组技术构建了纳豆激酶基因的表达载体,并在大肠杆菌中进行了表达．ＳＤＳ－聚丙烯酰胺凝胶电泳表明,表达蛋白占菌体蛋白的１５．２％ ,琼脂糖－纤维蛋白平板法测出表达产物具有溶解血栓活性．     

Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang.

Bacillus sp. strain CK 11-4, which produces a strongly fibrinolytic enzyme, was screened from Chungkook-Jang, a traditional Korean fermented-soybean sauce. The fibrinolytic enzyme (CK) was purified from supernatant of Bacillus sp. strain CK 11-4 culture broth and showed thermophilic, hydrophilic, and strong fibrinolytic activity. The optimum temperature and pH were 70 degrees C and 10.5, respectively, and the molecular weight was 28,200 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 14 amino acids of the N-terminal sequence of CK are Ala-Gin-Thr-Val-Pro-Tyr-Gly-Ile-Pro-Leu-Ile-Lys-Ala-Asp. This sequence is identical to that of subtilisin Carlsberg and different from that of nattokinase, but CK showed a level of fibrinolytic activity that was about eight times higher than that of subtilisin Carlsberg. The amidolytic activity of CK increased about twofold at the initial state of the reaction when CK enzyme was added to a mixture of plasminogen and substrate (H-D-Val-Leu-Lys-pNA). A similar result was also obtained from fibrin plate analysis.     

纳豆激酶液态发酵条件的研究

考察纳豆杆菌在7.5 L发酵罐中分批发酵产纳豆激酶的条件,纳豆激酶酶活采用四肽底物测定。结果表明:纳豆杆菌生长和产酶的适宜条件不一致。发酵过程中发酵罐搅拌转速控制为500 r/min不变,0～12 h时控制pH为8.0、温度为37℃;12～36 h调整pH为7.0、温度为30℃;16 h时补加外源C源,连续发酵36 h。该过程中发酵液中比酶活最高达到3 232 U/mL,与摇瓶发酵相比,比酶活提高了58%。     

Metabolic pathway analysis and kinetic studies for production of nattokinase in Bacillus subtilis

We have constructed a reaction network model of Bacillus subtilis. The model was analyzed using a pathway analysis tool called elementary mode analysis (EMA). The analysis tool was used to study the network capabilities and the possible effects of altered culturing conditions on the production of a fibrinolytic enzyme, nattokinase (NK) by B. subtilis. Based on all existing metabolic pathways, the maximum theoretical yield for NK synthesis in B. subtilis under different substrates and oxygen availability was predicted and the optimal culturing condition for NK production was identified. To confirm model predictions, experiments were conducted by testing these culture conditions for their influence on NK activity. The optimal culturing conditions were then applied to batch fermentation, resulting in high NK activity. The EMA approach was also applied for engineering B. subtilis metabolism towards the most efficient pathway for NK synthesis by identifying target genes for deletion and overexpression that enable the cell to produce NK at the maximum theoretical yield. The consistency between experiments and model predictions proves the feasibility of EMA being used to rationally design culture conditions and genetic manipulations for the efficient production of desired products.     

BSF1 fibrinolytic enzyme from a marine bacterium Bacillus subtilis A26: Purification,biochemical and molecular characterization

A novel fibrinolytic enzyme, subtilisin BSF1, from a newly isolated Bacillus subtilis A26 was purified, characterized and the gene was isolated and sequenced. The subtilisin BSF1 was purified to homogeneity by five-step procedure with a 4.97-fold increase in specific activity and 6.28% recovery. The molecular weight of the purified enzyme was estimated to be 28 kDa by SDS-PAGE and gel filtration. The purified enzyme exhibited high fibrinolytic activity on fibrin agar plates.Interestingly, the enzyme was highly active over a wide range of pH from 7.0 to 12.0, with an optimum at pH 9.0. The relative activities at pH 10.0 and 11.0 were 97.8% and 85.2% of that at pH 9.0. The optimum temperature for enzyme activity was 60 °C. The activity of subtilisin BSF1 was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The N-terminal amino acid sequence of the first 11 amino acids (aa) of the purified fibrinolytic enzyme was AQSVPYGISQI.The bsf1 gene encoding the subtilisin BSF1 was isolated and its DNA sequence was determined. The bsf1 gene consisted of 1146 bp encoding a pre-pro-protein of 381 amino acids organized into a signal peptide (29 aa), a pro-peptide (77 aa) and a mature domain (275 aa). The deduced amino acids sequence of the mature enzyme (BSF1) differs from those of nattokinase from B. subtilis natto and subtilisin DFE from Bacillus amyloliquefaciens DC-4 by 5 and 39 amino acids, respectively.     

Significantly enhanced stability of glucose dehydrogenase by directed evolution

An NaCl-independent stability-enhanced mutant of glucose dehydrogenase (GlcDH) was obtained by using in vitro directed evolution. The family shuffling method was applied for in vitro directed evolution to construct a mutant library of GlcDH genes. Three GlcDH-coding genes from Bacillus licheniformis IFO 12200, Bacillus megaterium IFO 15308 and Bacillus subtilis IFO 13719 were each cloned by direct PCR amplification into the p Trc99A expression vector and expressed in the host, Escherichia coli. In addition to these three GlcDH genes, a gene encoding a previously obtained GlcDH mutant, F20 (Q252L), derived from B. megaterium IWG3, was also subjected to directed evolution by the family shuffling method. A highly thermostable mutant, GlcDH DN-46, was isolated in the presence or absence of NaCl after the second round of family shuffling and filter-based screening of the mutant libraries. This mutant had only one novel additional amino acid residue exchange (E170K) compared to F20, even though DN-46 was obtained by family shuffling of four different GlcDH genes. The effect of temperature and pH on the stability of the GlcDH mutants F20 and DN46 was investigated with purified enzymes in the presence or absence of NaCl. In the absence of NaCl, F20 showed very poor thermostability (half-life =1.3 min at 66 degrees C), while the half-life of isolated mutant DN-46 was 540 min at 66 degrees C, i.e., 415-fold more thermostable than mutant F20. The activity of the wild-type and F20 enzymes dropped critically when the pH value was changed to the alkaline range in the absence of NaCl, but no such decrease was apparent with the DN-46 enzyme in the absence of NaCl.     

Highly potent fibrinolytic serine protease from Streptomyces

We introduce a highly potent fibrinolytic serine protease from Streptomyces omiyaensis (SOT), which belongs to the trypsin family. The fibrinolytic activity of SOT was examined using in vitro assays and was compared with those of known fibrinolytic enzymes such as plasmin, tissue-type plasminogen activator (t-PA), urokinase, and nattokinase. Compared to other enzymes, SOT showed remarkably higher hydrolytic activity toward mimic peptides of fibrin and plasminogen. The fibrinolytic activity of SOT is about 18-fold higher than that of plasmin, and is comparable to that of t-PA by fibrin plate assays. Furthermore, SOT had some plasminogen activator-like activity. Results show that SOT and nattokinase have very different fibrinolytic and fibrinogenolytic modes, engendering significant synergetic effects of SOT and nattokinase on fibrinolysis. These results suggest that SOT presents important possibilities for application in the therapy of thrombosis.     

Rapid Hydrolysis Pathway for a Tertiary Alcohol Ester through Intramolecular Transesterification in Rats

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for nattokinase, a subtilisin-like fibrinolytic enzyme in the fermented soybean food, natto. The assay method was developed using a combination of a murine anti-nattokinase monoclonal antibody coated on the microtiter well as a catching antibody and rabbit anti-nattokinase polyclonal antibody as a peroxidase-conjugated detecting antibody. The assay sensitivity was 0.1 ng/ml and cross-reactivity with subtilisin BPN′ and subtilisin Carlsberg was 0.0002% and 0.00002%, respectively. The ELISA value reflected the fibrinolytic activity of nattokinase. The correlation coefficient between nattokinase measured by a fibrinolytic assay and by the ELISA in 14 commercially available natto extracts was 0. 967, suggesting that nattokinase is the only fibrinolytic enzyme in natto.     

纳豆激酶的分离纯化研究

从实验室保存的一株高产纳豆激酶菌株出发,进行发酵产酶,确立具有纤溶活力的纳豆激酶的分离纯化工艺,主要通过硫酸铵分级盐析法和Phenyl Sepharose疏水柱层析进行分离纯化,用纤维蛋白平板法测定酶活力,用SDS-PAGE电泳验证为电泳纯,分子量为28kD。其有望开发为新型的口服溶栓药物。     

枯草杆菌DC-2纳豆激酶基因的克隆及其融合蛋白在E.coli中的表达

纳豆激酶是一种纤维蛋白溶解酶 ,有望开发成为新型的溶栓药物 .从中国豆豉中分离的具有较强纤溶活性的枯草杆菌DC 2中提取总DNA ,根据纳豆激酶 (NK)基因序列设计引物 ,用PCR法扩增NK基因 .序列分析表明 ,NK基因成熟肽编码区含有 82 5bp ,编码 2 75个氨基酸残基 ,与文献报道的序列分别有 93 4 %和 94 5 %同源性 .将NK基因插入载体pGEX 4T1构建表达质粒pGEX NK ,转化大肠杆菌JM10 9后 ,经 1mmol LIPTG诱导 4h ,发现大量NK融合蛋白表达 ,并形成包涵体 .SDS PAGE分析表明 ,NK融合蛋白作为包涵体的分子量为 5 3kD .凝胶自动扫描结果显示 ,NK融合蛋白约占菌体可溶性蛋白的 2 6 % .