纳豆激酶的研究与应用

纳豆激酶是一种枯草芽孢杆菌产生的丝氨酸蛋白酶 ,具有强烈的纤溶活性 ,有望开发成为新型口服溶栓药。综述了纳豆激酶的生化特性、生物学活性、结构与功能及开发应用前景等。     

一株高产纳豆激酶菌株的鉴定与分析

鉴定高产纳豆激酶菌株Td,分析纳豆激酶的分子特征。利用菌体形态、生理生化特征、以及分子生物学方法对菌株Td进行鉴定;并采用MALDI-TOF质谱测定与分析、SDS-PAGE和纤溶活性测定等方法检测纳豆激酶特性,利用PCR方法扩增纳豆激酶的基因全长。结合菌体形态、生理生化特征和16S r DNA、gyr A基因序列、DNA-DNA杂交率等实验结果,鉴定菌株Td为枯草芽孢杆菌枯草亚种(Bacillus subtilis subsp.subtilis);菌株Td发酵产生的纳豆激酶产量可达300 mg/L以上,占发酵液总蛋白的40%以上;纤溶活性达230 U/m L以上;氨基酸序列与subtilisin E的序列相似性最高;基因全长序列为1 143 bp。枯草芽孢杆菌枯草亚种Td是一株高产、高活性纳豆激酶的产生菌,具有优良的工业化开发价值。     

产纳豆激酶枯草芽孢杆菌种子液培养条件的优化

利用Plackett-Burman法(PB法),以紫外分光光度法作为测量方法对产纳豆激酶枯草芽孢杆菌种子液培养条件进行了优化。确定了产纳豆激酶枯草芽孢杆菌种子液的最佳氮源是大豆蛋白胨,优化出最佳种子液培养条件为牛肉膏0.5 g/100 mL、大豆蛋白胨1 g/100 mL、NaCl 0.75 g/100 mL、pH 7、培养温度37℃、培养时间8.5 h、接种量为3 mL/100 mL、装液量100 mL/250 mL。     

纳豆芽孢杆菌菌种纯化及不同氮源对其产纳豆激酶的影响

纳豆激酶(nattokinase, NK)是一种由纳豆芽孢杆菌发酵产生的丝氨酸蛋白酶,具有良好的纤溶活性。本研究从wako Nattokinase中分离纯化出高品质的纳豆芽孢杆菌,旨在探究最适宜该菌产纳豆激酶的发酵培养基氮源。研究人员选择了6种氮源对其进行发酵实验,通过连续测定发酵液的菌量、pH和纤溶活性以观察不同氮源对纳豆芽孢杆菌产纳豆激酶的影响。研究结果表明:最优氮源为乳清蛋白,在以此为氮源的培养基中发酵培养120 h后,纳豆激酶的纤溶活性高达1 757.79 U/mL。以乳清蛋白发酵培养基对纳豆芽孢杆菌进行发酵,不仅可以得到高活性的纳豆激酶,还可为纳豆激酶应用于食品、保健品领域提供思路。     

纳豆芽孢杆菌应用研究进展及开发前景

20世纪中期,有学者从日本的传统食物纳豆中成功分离出了纳豆芽孢杆菌,它是属于细菌科和芽孢杆菌属的范畴,对人体没有病原性危害,是一种安全菌株。作为一种众所周知的益生菌,纳豆芽孢杆菌已经证明有着多种益生功能。本综述介绍了纳豆芽孢杆菌在生物保鲜、畜禽生产及基因工程方面的应用,并对纳豆芽孢杆菌微生态制剂的开发前景作了展望。     

纳豆激酶基因工程研究进展

纳豆是日本的民间传统食品,1987年日本学者须见洋行从纳豆中分离提取和纯化出一种具有纤溶活性的蛋白激酶,称之为纳豆激酶(Nattokinase,NK)。它是由枯草芽孢杆菌(Bacillus subtilis)经发酵产生的一种丝氨酸蛋白酶,该酶能显著溶解体内外血栓,明显缩短优球蛋白的溶解时间,并能激活静脉内皮细胞产生纤维蛋白溶酶原激活剂(Sumi H,1987年)。     

纳豆激酶基因工程研究进展

纳豆激酶是由纳豆芽孢杆菌(Bacillussubtillisnatto)分泌的一种具有纤溶作用的碱性丝氨酸蛋白酶。对纳豆激酶基因的克隆与表达,基因与蛋白质结构以及基因工程纳豆激酶的特性和功能进行了综述。     

纳豆激酶的研究进展

纳豆激酶是一种由纳豆芽孢杆菌产生的具有强溶纤作用的碱性丝氨酸蛋白酶,具有安全性好、半衰期长、口服有效等优点.就纳豆激酶的结构、理化性质、功能、溶栓机制、酶活性测定、分离纯化以及纳豆激酶的应用现状及发展前景等方面进行了综述.     

基于枯草芽孢杆菌产物应用的基因组精简研究进展

枯草芽孢杆菌代谢产物众多,具有很强的应用开发前景,在工业上广泛应用。本文总结了聚谷氨酸、纳豆激酶和维生素K2三种热点代谢产物及其在医药、食品、农业上的应用。枯草芽孢杆菌是常用的基因工程菌,对其进行遗传改造的方法较多。本文综述了同源重组、位点特异性重组和CRISPR/Cas9三种基因组精简手段,总结了困扰枯草芽孢杆菌代谢产物开发应用与基因组精简的问题,推断未来发展的方向,期望推动枯草芽孢杆菌的开发与应用。     

植物内生枯草芽孢杆菌纳豆激酶同源基因的序列分析及其在大肠杆菌中的表达

利用基因组学和生物信息学的方法,从一种植物内生枯草芽孢杆菌菌株Bs0922的基因组DNA中克隆了纳豆激酶的同源基因NK-Bs0922,长度为1 149 bp。通过重组和转化在大肠杆菌BL21(DE3)中表达了一个大小为48.0 kD的重组蛋白。     

Changes in fecal microflora induced by intubation of mice with Bacillus subtilis (natto) spores are dependent upon dietary components.

We examined changes in mouse fecal microflora after various dietary components and Bacillus subtilis (natto) spores were delivered by intubation. The administration of intact spores of Bacillus subtilis (natto) did not affect fecal Enterobacteriaceae and Enterococcus spp. in all three diet groups; on the other hand, it did affect fecal Bacteroidaceae and Lactobacillus spp., depending upon the diets fed. The administration of autoclaved spores did not alter fecal microflora. In vitro cultures of Lactobacillus murinus obtained from mouse feces, together with Bacillus subtilis (natto) under aerobic conditions as a mixed culture, revealed that the growth of L. murinus was enhanced by the addition of intact spores of Bacillus subtilis (natto). This enhancement of growth was displayed only in media containing either sucrose, glucose, maltose, or fructose but not in media containing cornstarch, soluble starch, or microcrystalline cellulose. From these results it was evident that some metabolites of Bacillus subtilis (natto) produced during germination and (or) outgrowth of spores of this strain, requiring monosaccharides or oligosaccharides, participated in the enhancement of the growth of L. murinus.     

Histological alterations of intestinal villi in chickens fed dried Bacillus subtilis var. natto

Two experiments were conducted. In experiment 1, chickens were fed dried Bacillus subtilis var. natto for 3 or 28 days. Growth performance and internal organs were not different from controls, but feed efficiency tended to be improved in the 28-day feeding. In these birds, blood ammonia concentration was decreased (P<0.05). Blood glucose concentration, and amylase and lipase activity in the intestinal content were not significantly different among dietary groups. These results suggest that the B. subtilis natto depressed ammonia concentration. In experiment 2, chickens were fed dietary B. subtilis natto for 28 days. These birds had a tendency to display greater growth performance and intestinal histologies, such as villus height, cell area and cell mitosis, than the controls. Flat cell outline on the duodenal villus surface in controls developed large, protruded cell clusters and cell protuberances after feeding of dietary B. subtilis natto. These results indicate that intestinal function was activated by the depressed blood ammonia concentration in the body of the chicken. The present results may suggest that the B. subtilis natto has the potential to be a beneficial microorganism in chickens.     

纳豆芽胞杆菌16S rRNA基因的克隆及其系统进化分析

纳豆芽胞杆菌是从豆豉中分离出的一种具有益生功能的芽胞杆菌。该研究从纳豆芽胞杆菌提取基因组DNA,以芽胞杆菌16S rRNA基因的通用引物,用PCR方法成功扩增出纳豆芽胞杆菌的部分16S rRNA基因,所克隆序列长1 435 bp,G＋C含量为55%,该序列已被GeneBank收录,其编号为AY864812。BLAST分析结果显示,AY864812与GeneBank中收录的枯草芽胞杆菌16S rRNA基因同源性最高,其中与AY601722的同源性为100%.用Clustalx 1.8对相关序列进行系统进化分析,结果显示纳豆芽胞杆菌与枯草芽胞杆菌在进化关系上的地位最近,从分子水平上证实了纳豆芽胞杆菌是枯草杆菌的1个亚种。     

Divergent structure of the ComQXPA quorum-sensing components: molecular basis of strain-specific communication mechanism in Bacillus subtilis

In Bacillus subtilis, the ComQXPA quorum-sensing system controls cell density-dependent phenotypes such as the production of degradative enzymes and antibiotics and the development of genetic competence. Bacillus subtilis (natto) NAF12, a mutant defective in poly-gamma-glutamate (gamma-PGA) production, was derived from B. subtilis (natto) NAF4 by Tn917-LTV1 insertional mutagenesis. Determination of the mutant DNA sequences flanking the Tn917-LTV1 insert revealed that the insertion had inactivated comP in this mutant, indicating that gamma-PGA synthesis in B. subtilis (natto) is under the control of the ComP-ComA signal transduction system. A comparison of the amino acid sequences revealed striking variation in the primary structures of ComQ (44% identity), ComX (26%) and the sensor domain of ComP (36%) between B. subtilis (natto) NAF4 and B. subtilis 168. In contrast, the amino acid and nucleotide sequences of the kinase domains of ComP and of the ComA response regulator share 95% and 100% identity respectively. The comP genes of NAF4 and 168 restored the impaired competence of B. subtilis BD1658 (comP:cat) and gamma-PGA production of B. subtilis (natto) NAF12 (comP:Tn917-LTV1) to only 15% of the level achieved by the respective parent comP genes. However, when introduced together with the cognate comQ and comX genes, the comP genes restored the relevant defect of the heterologous comP mutants nearly to wild-type levels. Analogous to the comCDE system of Streptococcus strains and the agrBCDE system of Staphylococcus aureus, the concerted variation in the comQXP genes appears to establish specific intercellular communication between B. subtilis strains sharing the same pheromone system.     

Construction of a Bacillus subtilis (natto) with high productivity of vitamin K2 (menaquinone-7) by analog resistance

To invent a functional natto promoting bone formation, the construction of a strain with high productivity of vitamin K2 (menaquinone-7: MK-7), which is important in the carboxylation of a kind of bone protein participating in bone formation, osteocalcin, was investigated. To screen for a strain appropriate to making natto (a Japanese traditional fermented soybean food) with high productivity of MK-7, a combination of analog resistance to the compounds on the biosynthetic pathway of menaquinones with mutation was done. Consequently, strain OUV23481, with 2-fold higher productivity (1,719 microg/100 g natto) of MK-7 than that of a commercial strain, was constructed as a mutant with analog resistance to 1-hydroxy-2-naphthoic acid (HNA), p-fluoro-D,L-phenylalanine (pFP), m-fluoro-D,L-phenylalanine (mFP), and beta-2-thienylalanine (betaTA). This strain was classified as Bacillus subtilis (natto). The natto made using this strain was evaluated to have a good quality as natto in all the viewpoints of appearance, flavor, taste, texture, and stringiness.     

Functional myo-inositol catabolic genes of Bacillus subtilis Natto are involved in depletion of pinitol in Natto (fermented soybean)

Soybeans are rich in pinitol (PI; 3-O-methyl-D-chiro-inositol), which improves health by treating conditions associated with insulin resistance, such as diabetes mellitus and obesity. Natto is a food made from soybeans fermented by strains of Bacillus subtilis natto. In the chromosome of natto strain OK2, there is a putative promoter region almost identical to the iol promoter for myo-inositol (MI) catabolic genes of B. subtilis 168. In the presence of MI, the putative iol promoter functioned to induce inositol dehydrogenase, the enzyme for the first-step reaction in the MI catabolic pathway. PI also induced inositol dehydrogenase and the promoter was indispensable for the utilization of PI as well as MI, suggesting that PI might be an alternative carbon source metabolized in a way involving the MI catabolic genes. Natto fermentation studies have revealed that the parental natto strain consumed PI while a mutant defective in the iol promoter did not do so at all. These results suggest that inactivating the MI catabolic genes might prevent PI consumption, retaining it in natto for enrichment of possible health-promoting properties.     

DNA-methylases from different strains of Bacillus subtilis and Bacillus natto

DNA-methyltransferase activity has been detected in some of Bacillus subtilis and Bacillus natto strains. Two strains of Bacillus subtilis exhibited DNA-cytosine methyltransferase activity, and the strains of Bacillus natto exhibited DNA-adenine methyltransferase activity. A possible effect of DNA-methyltransferase specificity on transformation efficiency is discussed.     

Strain and plastic composite support (PCS) selection for vitamin K (Menaquinone-7) production in biofilm reactors

Menaquinone-7 (MK-7), a subtype of vitamin K, has received a significant attention due to its effect on improving bone and cardiovascular health. Current fermentation strategies, which involve static fermentation without aeration or agitation, are associated with low productivity and scale-up issues and hardly justify the commercial production needs of this vitamin. Previous studies indicate that static fermentation is associated with pellicle and biofilm formations, which are critical for MK-7 secretion while posing significant operational issues. Therefore, the present study is undertaken to evaluate the possibility of using a biofilm reactor as a new strategy for MK-7 fermentation. Bacillus species, namely, Bacillus subtilis natto, Bacillus licheniformis, and Bacillus amyloliquifaciens as well as plastic composite, supports (PCS) were investigated in terms of MK-7 production and biofilm formation. Results show the possibility of using a biofilm reactor for MK-7 biosynthesis. Bacillus subtilis natto and soybean flour yeast extract PCS in glucose medium were found as the most potent combination for production of MK-7 as high as 35.5 mg/L, which includes both intracellular and extracellular MK-7.     

Improved growth and viability of lactobacilli in the presence of Bacillus subtilis (natto), catalase, or subtilisin

In an effort to demonstrate the potential usefulness of Bacillus subtilis (natto) as a probiotic, we examined the effect of this organism on the growth of three strains of lactobacilli co-cultured aerobically in vitro. Addition of B. subtilis (natto) to the culture medium resulted in an increase in the number of viable cells of all lactobacilli tested. Since B. subtilis (natto) can produce catalase, which has been reported to exhibit a similar growth-promoting effect on lactobacilli, we also examined the effect of bovine catalase on the growth of Lactobacillus reuteri JCM 1112 and L. acidophilus JCM 1132. Both catalase and B. subtilis (natto) enhanced the growth of L. reuteri JCM 1112, whereas B. subtilis (natto) but not catalase enhanced the growth of L. acidophilus JCM 1132. In a medium containing 0.1 mM hydrogen peroxide, its toxic effect on L. reuteri JCM 1112 was abolished by catalase or B. subtilis (natto). In addition, a serine protease from B. licheniformis, subtilisin, improved the growth and viability of L. reuteri JCM 1112 and L. acidophilus JCM 1132 in the absence of hydrogen peroxide. These results indicate that B. subtilis (natto) enhances the growth and (or) viability of lactobacilli, possibly through production of catalase and subtilisin.     

Disruption of the cell wall lytic enzyme CwlO affects the amount and molecular size of poly-γ-glutamic acid produced by Bacillus subtilis (natto)

Poly-γ-glutamic acid (γPGA), a polymer of glutamic acid, is a component of the viscosity substance of natto, a traditional Japanese food made from soybeans fermented with Bacillus subtilis (natto). Here we investigate the effects of the cell wall lytic enzymes belonging to the D,L-endopeptidases (LytE, LytF, CwlO and CwlS) on γPGA production by B. subtilis (natto). γPGA levels in a cwlO disruptant were about twofold higher than that of the wild-type strain, whereas disruption of the lytE, lytF and cwlS genes had little effect on γPGA production. The molecular size of γPGA in the cwlO disruptant was larger than that of the wild-type strain. A complementary strain was constructed by insertion of the entire cwlO gene into the amyE locus of the CwlO mutant genome, and γPGA production was restored to wild-type levels in this complementary strain. These results indicated that the peptidoglycan degradation enzyme, CwlO, plays an important role in γPGA production and affects the molecular size of γPGA.