一株纤溶酶高产菌株的发酵特性研究

筛选了一株产纤溶酶能力较强的芽孢细菌,本文对其液体发酵条件进行了优化,结果表明菌株产酶最佳条件为:可溶性淀粉4%,蛋白胨2%,柠檬酸铁铵0.1%,磷酸钙0.4%,接种量2%,pH 7.5,发酵时间96 h,装瓶量75/500(mL/mL),摇床温度37℃,转速150 rpm,在该条件下产酶酶活可达581.81 IU/mL。     

根霉纤溶酶发酵条件的优化

研究了不同培养基对中国根霉１２号（Ｒｈｉｚｏｐｕｓ ｃｈｉｎｅｓｉｓ１２）发酵生产纤溶酶的影响,发现Ｃ／Ｎ的降低有利于产酶,同时用正产实验给出了最佳培养条件为：以麸皮水＋胰蛋白胨＋豆渣为培养基,接种量２０％,转速１７０ｒ／ｍｉｎ;用响应面设计确定了最佳培养基为４．３６６６１％麸皮水＋０．９８９７５％胰蛋白胨＋４．８９８９５％豆渣;在最优条件下,纤溶酶的发酵效价可达３．０６Ｕ／ｍｌ。发现添加金属离子     

一株产纤溶酶菌株的分离鉴定及其纤溶组分分析

【目的】筛选性能良好的产纤溶酶菌株,对菌株进行多项分类鉴定,分析其纤溶酶系的组成特征及纤溶能力。【方法】通过酪蛋白培养基初筛,琼脂-纤维蛋白双层平板复筛,从海泥、土壤等环境中筛选纤维蛋白降解菌,以尿激酶为标准测定纤溶酶活性。通过形态学、生理生化特征研究,结合16S rDNA基因序列分析菌株种类及系统分类地位。通过SDS-PAGE和纤维蛋白酶谱法分析胞外纤溶酶系的组成特征。【结果】筛选到一株能降解纤维蛋白的细菌CNY16,鉴定其为沙福芽孢杆菌(Bacillus safensis)。该酶为胞外酶,SDS-PAGE和纤维蛋白酶谱结果表明该纤溶酶系有至少两种分子量大小不同的纤溶酶,分别约33 kD和23 kD。能有效溶解血块中纤维蛋白,并且对红细胞无降解作用。【结论】细菌CNY16是一株新的纤溶酶产生菌,纤溶酶活性及稳定性较好,具有潜在开发价值。为获取新型纤溶酶提供了一种新的菌源。     

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。     

海洋来源链霉菌MY0504产纤溶酶的发酵条件优化

【目的】以发酵液纤溶酶活力为指标,优化海洋来源的链霉菌菌株MY0504的发酵条件。【方法】在菌株生长曲线及单因素试验基础上,采用Plackett-Burman设计筛选影响纤溶酶活性的主要因素,进一步用最陡爬坡试验及Box-Behnken中心组合设计法优化发酵条件。【结果】纤溶酶活性最高的发酵条件为:葡萄糖21.68 g/L,酵母粉25.31 g/L,NaCl5.0 g/L,K_2HPO_4·3H_2O3.0 g/L,MgSO_4·7H_2O 0.5 g/L,FeSO_4·7H_2O 0.02 g/L,装液量50 mL(250 mL摇瓶),接种量10%(体积比),初始pH 7.5,温度24°C,转速200 r/min,培养时间4.5 d。发酵液纤溶酶活性可达2 190.6 U/mL。【结论】确定了MY0504菌株产纤溶酶的最优发酵条件,为该酶的进一步分离纯化及性质研究奠定基础。     

产纤溶酶枯草杆菌液体发酵工艺优化研究

采用单因子实验和正交实验对高产纤溶酶的枯草杆菌最佳发酵工艺进行了优化,结果表明,该菌株分泌的胞外酶具有较强的体外溶栓作用,产纤溶酶最佳的发酵条件为：3％可溶性淀粉,2％豆浆全汁(鲜豆),0．02％CaCl2,培养温度为37℃,初始pH8．3,装液量为250mL三角瓶50mL,发酵时间44h左右,Ca^2 、Mg^2 和Mn^2 对酶活力有促进作用,Cu^2 对酶活性有强烈抑制作用。     

少根根霉菌株发酵生产纤溶酶条件的研究

发酵条件优化可提高少根根霉菌株8B所产纤溶酶的活性。使用单因素试验和正交试验确定该茵发酵产酶的最佳条件。试验确定最优化发酵条件,培养基：麸皮水5g／L,尿素5g／L,胰蛋白胨0．5g／L,K2HPO4·3H2O 0．3g／L,MgSO4·7H2O 0.15g／L,pH5.5,摇床转速160r／min,30℃发酵56h。在此优化条件下培养,8B产纤溶酶活力达到345．41U／mL,是初始培养基发酵产酶活力的7．52倍。     

新的纤溶酶产生菌培养条件及其酶性质研究

采用定向筛选方法,分离到一株能产生强烈分解纤维蛋白的枯草芽孢杆菌（Bacillus subtilis)EM29-5。对其生长,产酶条件,酶性质进行系统研究,结果表明：生长和产酶温度25－37℃,37℃时最好,生长最适pH6.4-7.0,产酶为pH7.0-8.0;12种碳源氮源均能利用,但对生长和产酶均有利的是大豆浸出淮EM29－5。纤溶酶为外分泌蛋白酶,发酵上清液经硫酸铵沉淀,透析脱盐,上离子交换柱和凝胶过滤柱后得纯酶。经SDS－PAGE电泳得单一条带,分子量为32500左右;等电聚焦电泳分析PI9．0左右;酶蛋白在pH5.0-10.0范围内稳定,作用最适pH9.0,为碱性蛋白酶;5mmPMSF能完全抑制酶活性,为氨酶蛋白酶;不能分解人工合成的氨基酸的酯,而能分解纤维蛋白（原）,特别对交联纤维蛋白具更高亲和性。     

华根霉产纤溶酶液体发酵条件的研究

研究了华根霉TCCC 41014产纤溶酶液态发酵的工艺条件,采用单因素实验对液态发酵培养基的碳源、氮源、初始pH值、温度和装液量进行了优化.结果表明,实验范围内华根霉TCCC41014液态发酵产纤溶酶的适宜培养基组成(g桙L):糊精80,蛋白胨60,豆粕60,初始pH为4.5.适宜培养条件:培养温度为29℃,装液量为5...     

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45℃,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-Succinvl-Ala-Ala-Pro-Phe-pNA,其米氏常数Km为O.23mmol/L,酶转换数Kcat为16.36 s-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4.70%;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.     

Enhanced production of fibrinolytic enzyme by a new Xanthomonas oryzae IND3 using low-cost culture medium by response surface methodology

Cardiovascular diseases (CVDs) cause high mortality throughout the world. Existing fibrinolytic agents are highly expensive and have many side effects. Microbial fibrinolytic enzymes are very much considered as novel therapeutic candidate for the treatment of CVDs. Reports on fibrinolytic enzyme from Xanthomonas sp. is lacking. This study reports fibrinolytic enzymes from Xanthomonas oryzae IND3 as it shows hyperactivity on fibrin-agarose plates. This organism utilized various agro-industrial wastes for enzymes production. Among all, cow dung enhanced more enzyme production, hence it was used as the low-cost substrate for statistical optimization of fibrinolytic protease in Solid state fermentation. Response surface methodology was employed to optimize the factors and enhanced yield by 4-fold. The interactions among the variables, viz, sucrose, yeast extract, and pH of the medium were investigated using Central Composite Design (CCD). The predicted fibrinolytic enzyme activity was 2340 U/g, and the observed fibrinolytic enzyme activity was 2294?±?12.8?U/g. The fibrinolytic enzyme degraded blood clot in vitro completely. This study is the first report on statistical optimization of fibrinolytic enzyme production in SSF from Xanthomonas sp. The crude extract has immense activity on proteinaceous wastes. The production of fibrinolytic protease using the low-cost substrate could reduce the production cost of enzyme.     

Optimization of conditions for acid protease partitioning and purification in aqueous two-phase systems using response surface methodology

Aspergillopepsin I, an acid protease, was purified using an aqueous two-phase system that comprised various combinations of polyethylene glycol (PEG), NaH₂PO₄ and NaCl. Partition of the enzyme depended upon the molecular mass of the PEG and the presence of NaCl. With PEG 1500, 4000 and 6000, the partition coefficients were increased by 1,500-, 1,800- and 560-fold compared to values without NaCl. The presence of NaCl (8.75%, w/w) increased purification by 3.8, 9.5 and 2.8 times into these respective PEGs. The optimal aqueous two-phase system for acid protease purification was developed using response surface methodology. This system contained 17.3% of PEG 4000 (w/w), 15% NaH₂PO₄ (w/w) and 8.75% NaCl (w/w) and provided the best partition coefficient (Ke > 1,100) and yield over 99% in the same phase. The optimal ATPS purification factor of acid protease was over 5.     

高产洛伐他汀棒曲霉菌株的筛选、鉴定及发酵条件优化

从不同生境(食品、土壤、空气、有机质等)收集到的自然发酵样品中分离得到150株曲霉属菌株.用高效液相色谱(HPLC)法检测发酵液中洛伐他汀(Lovastatin)含量,筛选获得1株稳定高产Lovastatin的曲霉菌株(编号:Ac-32).根据菌落形态特征并结合18 S rDNA测序,鉴定其为棒曲霉(Aspergillus clavatus).通过摇瓶发酵单因素实验优化了碳氮源种类、碳氮源含量、碳氮比(C/N)、发酵温度、初始pH、转速、种龄和接种量,确定了棒曲霉菌株Ac-32摇瓶发酵产Lovastatin的适宜条件为:乳糖为碳源、蛋白胨为氮源、碳源含量为100 g/L、氮源含量为12 g/L、碳氮比(C/N)为15:1.8、温度28℃、转速180 r/min、初始pH 5.2、种龄4 d、接种量6%.采用Minitab 17软件的P-B实验设计法,筛选对Lovastatin产量有显著影响的因素为:温度、pH、碳源含量和氮源含量.根据P-B实验结果,运用响应面法分析,确定棒曲霉菌株Ac-32产Lovastatin的最优条件为:碳源含量100 g/L,氮源含量11.8 g/L,温度28℃,pH 5.2.在此条件下,Lovastatin最高产量为236.221μg/mL.     

Optimization of production conditions for mushroom polysaccharides with high yield and antitumor activity

Single factor experiment and response surface methodology were conducted to optimize the production conditions for polysaccharide with higher antitumor activity from mushroom fermentation broth. Based on the results of single factor experiments, a central composite design was applied to the following optimization and second-order polynomial regression models were established. As the yield and antitumor activity of the polysaccharides were considered with equal weight, the optimal conditions were that ethanol concentration and pH of the broth was adjusted to 85.00% and 7.70 respectively, then precipitated at 12 °C for 1 h and dried the precipitates at 40 °C. The verification experiments completed under the optimal conditions gave the polysaccharides yield of 9.843 ± 0.217 g/l, and the cancer cell growth inhibitory rate of 83.69 ± 0.167% by the polysaccharides, which were in close agreement with values predicted by the models (RSD < 5%).     

Crystal structure of earthworm fibrinolytic enzyme component a: revealing the structural determinants of its dual fibrinolytic activity

Earthworm fibrinolytic enzyme component A (EFEa) from Eisenia fetida is a strong fibrinolytic enzyme that not only directly degrades fibrin, but also activates plasminogen. Proteolytic assays further revealed that it cleaved behind various P1 residue types. The crystal structure of EFEa was determined using the MIR method and refined to 2.3A resolution. The enzyme, showing the overall polypeptide fold of chymotrypsin-like serine proteases, possesses essential S1 specificity determinants characteristic of elastase. However, the beta strand at the west rim of the S1 specificity pocket is significantly elongated by a unique four-residue insertion (Ser-Ser-Gly-Leu) after Val217, which not only provides additional substrate hydrogen binding sites for distal P residues, but also causes extension of the S1 pocket at the south rim. The S2 subsite of the enzyme was partially occluded by the bulky side-chain of residue Tyr99. Structure-based inhibitor modeling demonstrated that EFEa's S1 specificity pocket was preferable for elastase-specific small hydrophobic P1 residues, while its accommodation of long and/or bulky P1 residues was also feasible if enhanced binding of the substrate and induced fit of the S1 pocket were achieved. EFEa is thereby endowed with relatively broad substrate specificity, including the dual fibrinolysis. The presence of Tyr99 at the S2 subsite indicates a preference for P2-Gly, while an induced fit of Tyr99 was also suggested for accommodation of bigger P2 residues. This structure is the first reported for an earthworm fibrinolytic enzyme component and serine protease originating from annelid worms.     

枯草芽孢杆菌B47菌株高产抗菌物质的培养基及发酵条件优化

枯草芽孢杆菌Bacillus subtilis B47菌株为番茄内生细菌, 也是玉米小斑病拮抗菌, 能产生对玉米小斑病菌有强烈抑制作用的抗菌物质。以B47菌株发酵液的无菌滤液对玉米小斑病菌的抗菌活性为检测指标, 测定B47菌株产抗菌物质培养所需的最佳碳、氮源和无机盐, 并通过正交试验法对该菌株产抗菌物质的培养基配方和摇瓶发酵条件进行优化。研究结果表明, B47菌株产抗菌物质最佳碳、氮源和无机盐分别为蔗糖、酵母浸膏和MgSO4·7H2O, 最优培养基是YSB (Yeast extract-sucrose-beef extract)培养基, 其配方为: 蔗糖2%, 酵母浸膏2%, 牛肉浸膏1.5%, MgSO4?7H2O 0.06%, FeSO4·7H2O 0.000 9%, 最优发酵条件组合为: 30 °C, pH 7.0, 170 r/min摇床培养6 d, 接种量为1%, 装液量为40 mL/200 mL。