S-腺苷甲硫氨酸合成酶对埃博霉素生物合成的影响

【目的】研究S-腺苷甲硫氨酸合成酶(SAMs)对埃博霉素生物合成的影响。【方法】通过向发酵培养基中添加抑制剂和促进剂,比较分析纤维堆囊菌中SAMs的活性变化以及埃博霉素的产量变化。【结果】在埃博霉素的合成期,SAMs的活性较高。加入抑制剂吲哚乙酸(IAA)之后,SAMs的活性和埃博霉素的产量都不同程度的降低,而加入促进剂对甲苯磺酸钠(p-TSA-Na)之后,SAMs的活性和埃博霉素的产量在不同程度上都有提高。在纤维堆囊菌的次级代谢中,SAMs活性与埃博霉素的生物合成量呈正相关。【结论】S-腺苷甲硫氨酸合成酶在纤维堆囊菌的埃博霉素生物合成过程中发挥了重要的作用。     

响应面法优化纤维堆囊菌SoF5-76产埃博霉素B发酵培养基

以纤维堆囊菌SOF5-76为试验菌株,用响应面分析法对其产埃博霉素B的培养基进行优化,以提高埃博霉素B的产量。在单因素试验的基础上,利用Plackett-Burman筛选出对埃博霉素B产量有显著影响的3个因素为马铃薯淀粉、脱脂奶粉和无水氯化钙,在此基础上通过最陡爬坡试验逼近最佳响应面区域;再运用Box-Behnken试验设计和响应面分析法进行回归分析,确定重要因素的最优浓度。得到最佳发酵培养基为:马铃薯淀粉3.9 g/L、脱脂奶粉2.2 g/L、无水氯化钙1.3 g/L、葡萄糖1 g/L,豆饼粉1.5g/L,七水硫酸镁2.5 g/L,EDTA-Fe3+3 mL/L,微量元素(TE)0.5 mL/L,VB121 mL/L。在此最优条件下发酵埃博霉素B的产量为29.95 mg/L,与模型预测值接近,发酵产量比优化前提高了1.1倍。     

多孔陶瓷吸附固定纤维堆囊菌发酵制备埃博霉素

由纤维堆囊菌产生的埃博霉素具有极大药用价值,但因纤维堆囊菌液体环境中聚团非均匀生长限制了埃博霉素的大规模生产。借助多孔陶瓷的多孔结构,可为粘细菌提供固体附着生长面,提高埃博霉素产量。利用造孔剂法制备硅藻土基多孔陶瓷,在优化制备及改性条件后,当30目木屑造孔剂用量为2.5%(质量分数),7 MPa下制得的硅藻土基多孔陶瓷性能良好,孔径集中在5μm,比表面积为23.55 m2/g,孔隙率为32%,机械强度为10.2 MPa;经1.5 mol/L FeCl3改性的多孔陶瓷对纤维堆囊菌的吸附量达36.8 mg/g。在优化固定化发酵条件后,当在300 ml三角瓶中装液量为45 ml,固液比3:5,接种量10%,温度30℃,转速220 r/min,最初pH 7.5,发酵时间为8 d时,埃博霉素的产量达90.2 mg/L,与游离发酵相比提高了近4倍。     

埃博霉素B小试发酵罐发酵条件研究

研究了纤维堆囊菌(Sorangium cellulosum)So F5-76在5 L发酵罐水平上发酵生产埃博霉素B的基本工艺参数,具体考察了接种量、搅拌转速、通气量、添加消泡剂及补糖等5个工艺参数对埃博霉素B发酵产量的影响。最后确定发酵罐基本发酵条件为接种量9%,搅拌转速180 r/min,空气流量3.5 L/min,消泡剂种类选择Antifoam B聚醚类消泡剂,补糖控制在发酵液糖浓度为0.2 g/L,在此条件下埃博霉素B的产量可达25.6 mg/L。     

纤维堆囊菌SoF5-76产埃博霉素B分批发酵动力学研究

埃博霉素发酵生产体系复杂,且生产菌株较难操作,导致发酵参数测定困难,迄今为止未见关于埃博霉素发酵动力学方面的报道。利用5 L发酵罐,对纤维堆囊菌(Sorangium cellulosum)产埃博霉素B(Epothilone B)的分批发酵动力学进行了研究,整个发酵过程中维持温度30℃、p H7.4。发酵结束时,菌体干重达3.00 g/L,埃博霉素B产量达18.20 mg/L,葡萄糖含量为0.049g/L。基于Logistic方程和Luedking-Piret方程,利用MATLAB软件对其进行非线性拟合,构建了菌体生长、埃博霉素B合成和葡萄糖消耗的动力学模型。结果表明,该组模型能较好的拟合发酵过程。     

埃博霉素（Epothilones）的PKS/NRPS杂合基因簇

埃博霉素是由粘细菌纤维堆囊菌产生的一类具有促微管聚合活性的大环内酯类化合物。埃博霉素生物合成的多酶复合体是一个由多个功能模块组成,同时含有多聚酮合酶（PKS）和非核糖体肽合成酶（NRPS）的大操纵子。根据同位素标记试验结果和合成酶全基因簇功能的推测,埃博霉素的生物合成包括聚酮链的引发、链合成的起始和噻唑环的形成、链的延伸和转移、链合成的终止释放和环化、及产物的后修饰5个阶段。埃博霉素的PKS/NRPS杂合基因簇是开展组合生物合成研究的良好材料。     

菌蜕搭载的埃博霉素促进Caspase-3介导的HeLa细胞凋亡

[目的]初步探讨菌蜕搭载的埃博霉素抑制HeLa细胞增殖的机制。[方法]用半抑制浓度的游离或菌蜕搭载的埃博霉素B分别处理HeLa细胞4~48 h,应用凋亡检测试剂盒分析细胞凋亡情况,应用Caspase-3分析试剂盒检测Caspase-3的活性,并用透射电镜观察细胞的形态变化。[结果]菌蜕搭载的埃博霉素B能更快地诱导HeLa细胞发生时间依赖的凋亡和坏死,处理24 h时早期凋亡细胞及晚期凋亡和坏死细胞的比例分别达32.72%和18.36%,处理48 h时比例分别达23.14%和51.52%。该药物还能诱导更强的Caspase-3激活,处理24 h时其活性约为游离埃博霉素B的1.5倍。Caspase-3活性的升高程度与细胞的早期凋亡程度相平行。该药物处理的HeLa细胞显示典型的凋亡特征。[结论]菌蜕搭载的埃博霉素B能更有效地促进Caspase-3介导的HeLa细胞凋亡。     

吸水链霉菌NND-52-C菌株产阿扎霉素B 发酵条件的优化

通过碳源、氮源的单因子实验和正交实验,确定了吸水链霉菌NND-52-C菌株产阿扎霉素B最佳的液体发酵培养基组分(%,质量分数)甘油4.5,玉米粉3,黄豆粉1,蛋白胨1,NaNO30.5,NaCl 0.2,CaCO3 0.3,MgSO4·7H2 O0.05,FeSO4 0.05,PH 7.5;阿扎霉素B最佳的发酵条件发酵前期温度30℃,起始pH 7.5;发酵后期温度28℃,pH6.8;接种量10%,装液量30 mL/250mL摇瓶,发酵时间5 d,最终阿扎霉素B的产量达到l 434 mg/L,比原配方以及原发酵条件提高了76%.     

刺芹侧耳生长条件和栽培特性的研究

对刺芹侧耳[Pleurotus eryngii(DC.ex Fr.)Quel]生长条件与栽培特性进行了分析.结果表明:刺芹侧耳菌丝生长的适宜pH值为pH 5～pH 7,最适pH值为pH 6;黑暗环境有利于菌丝生长,光照对菌丝生长具有抑制作用;菌丝生长适宜的碳源为可溶性淀粉、葡萄糖和蔗糖,适宜的氮源为蛋白胨和钼酸铵,葡萄糖和蛋白胨的适宜浓度分别为50和1 g·L-1;在马铃薯黄豆粉培养基、黄豆粉玉米粉培养基和马铃薯蛋白胨培养基中可较好地形成菌丝球.采用液体菌种栽培,菌丝的生长速度比使用玉米固体菌种快,其中用马铃薯黄豆粉液体菌种栽培,菌丝生长最快;使用马铃薯蛋白胨液体菌种在木屑棉籽壳混合培养料中栽培,可获得菌柄长菌盖大的子实体,而且产量高.     

高产申嗪霉素和吩嗪-1-酰胺的水稻根际铜绿假单胞菌PA1201分离、鉴定与应用潜力

【目的】从水稻根际筛选能高效抑制水稻病原菌的细菌,分析和鉴定其形态和生化特征,为开发新型绿色农药奠定基础。【方法】从水稻根际分离能以1-氨基环丙烷-1-羧酸(ACC)为唯一碳源的菌株,根据菌株形态和生化特性、16S r DNA序列比对和磷脂脂肪酸图谱,对该菌株进行鉴定。通过氯仿萃取抽提、高效液相色谱分析,确定菌株PA1201在PPM培养基和黄豆粉培养基中申嗪霉素和吩嗪-1-酰胺的产量。【结果】菌株PA1201能有效抑制水稻纹枯病菌和水稻白叶枯病菌的生长,属于铜绿假单胞菌(Pseudomonas aeruginosa sp.PA1201);PA1201产生两种抑菌代谢产物申嗪霉素和吩嗪-1-酰胺,在PPM和黄豆粉培养基中申嗪霉素的产量可达81.7 mg/L和926.9 mg/L,吩嗪-1-酰胺的产量亦可达18.1 mg/L和489.5 mg/L;PA1201产生大量胞外蛋白酶,对人肺腺癌细胞系A549和黑腹果蝇具有一定毒性。【结论】PA1201的申嗪霉素产量比现有生产菌株的出发菌株M18高3-4倍,还能产生另一种抑菌活性更高的衍生物吩嗪-1-酰胺,具有进一步开发的潜力。     

Selective production of epothilone B by heterologous expression of propionyl-CoA synthetase in Sorangium cellulosum

The metabolic engineering of epothilones, as secondary metabolites, was investigated using Sorangium cellulosum to achieve the selective production of epothilone B, a potent anticancer agent. Thus, the propionyl-CoA synthetase gene (prpE) from Ralstonia solanacearum was heterologously expressed in S. cellulosum to increase the production of epothilone B. Propionyl-CoA synthetase converts propionate into propionyl-CoA, a potent precursor of epothilone B. The recombinant S. cellulosum containing the prpE gene exhibited a significant increase in the resolution of epothilones B/A, with an epothilone B to A ratio of 127 to 1, which was 100 times higher than that of the wild-type cells, demonstrating its potential use for the selective production of epothilone B.     

Effects of alginates and sodium carrageenates mixed with soy proteins on the coefficient of protein efficiency

Male 21 d-old Wistar rats, were fed for 4 wk with diets containing casein or soybean proteins (10%) with 0.5, 1, 2 or 3% sodium alginate or sodium carrageenan or without any alginate or carrageenan. Daily protein intake and weight gain of casein-fed rats were not significantly different (P less than 0.05) from those of rats fed soybean meal with alginate, whatever the dose received. Rats fed 3% carrageenan in soybean meal had significantly higher feed intake than that of rats fed casein. At the levels studied, alginate had no effect on the Protein Efficiency Ratio (PER), but carrageenan did. The addition of increased quantities of carrageenan to soybean meal followed by heating the mixture led to a progressive and significant decrease in PER at all levels of carrageenan compared to casein feeding. The addition of 3% carrageenan to heated soybean meal, corresponding to 0.62% of meal diet, led to a significant decrease in PER. These results confirm the precipitating role of carrageenans on proteins.     

Optimization of medium composition for actinomycin X2 production by <Emphasis Type="Italic">Streptomyces </Emphasis><Emphasis Type="Italic">spp</Emphasis> JAU4234 using response surface methodology

The effects of cultivation medium compositions including soybean meal, peptone, soybean oil and cornstarch for actinomycin X2 production by Streptomyces spp JAU4234 were accessed by using response surface methodology. The 2(4) full factorial designs and the paths of steepest ascent were effective in searching for the major factors of actinomycin X2 production. In this study, cornstarch and soybean oil showed negative effect on actinomycin X2 production based on the first-order regression coefficients derived from MINITAB software. Subsequently, a central composite design for optimization was further investigated. Preliminary studies showed that soybean meal and peptone were believed to be the major factors for actinomycin X2 production. Estimated optimum compositions for the production of actionmycin X2 were as follows (g/l): soybean meal 21.65 and peptone 9.41, and result in a maximum actionmycin X2 production of 617.4 mg/l. This value was closed to the 612 mg/l actionmycin X2 production from actual experimental observations. The yield of actionmycin X2 was increased by 36.9% by culturing the strain Streptomyces spp JAU4234 in the nutritionally optimized fermentation medium.     

手性拆分环氧氯丙烷菌株的筛选、鉴定及产酶条件研究

从土壤中筛选到5株环氧化物水解酶生产菌,并通过ITS序列鉴定了其中的C375菌,结果为黑曲霉（Aspergillus nigerZJB-09103）。考察了培养基不同碳源、氮源、金属离子和pH等对产酶的影响,得到了较佳的培养基条件：淀粉16g／L,豆饼粉3g／L,蛋白胨3g／L,KH2PO4 0．4g／L,K2HPO4 0．8g／L,MgSO4 0．2g／L,ZnSO4 0．03g／L,pH6．5。采用优化后的培养基条件,酶活力达到156．1U／L,比优化前初始发酵培养条件下的酶活提高了252％,当环氧化物水解酶催化时间为10h时,（s）-环氧氯丙烷的对映体过量值（e．e．）可达99．0％。产率为18．6％。     

响应面法优化波赛链霉菌JMC 06001发酵培养基

采用响应面法对产生抑菌活性物质的波赛链霉菌（Streptomyces peucetius）菌株JMC 06001的发酵培养基进行优化。首先采用Minnimum Run Equireplicated Res IV设计对初始发酵培养基的8个营养因素进行筛选,获得影响产生抑菌活性物质的3个主要影响因素：葡萄糖、大豆粉和NaCI;然后用最陡爬坡实验快速逼近最大响应区域;最后,结合Box-Behnken设计及响应面分析,确定主要影响因素的最佳浓度,得出该菌株产抑菌活性物质的最优发酵培养基配方为：葡萄糖1．2％,麦芽糖0．7％,蛋白胨0．9％,大豆粉1．4％,NaCl3．7％,CaCO3 0．1％,复合盐A液2．0％,复合盐B液0．1％,起始pH值7．0。用优化后的培养基发酵所得发酵液对敏感指示菌藤黄八叠球菌的抑菌圈直径达31．5mm,与预测值的相对偏差仅为1．59％,与用初始发酵培养基发酵所得发酵液的抑菌效果（抑菌圈直径26．5mm）相比提高了18．9％。     

Oxidation of terfenadine by Streptomyces platensis: Influence of culture medium on metabolite formation

The biotransformation of terfenadine into a primary alcohol, hydroxyterfenadine, followed by its oxidation to an acid, fexofenadine, was investigated using Streptomyces platensis cells. Time-courses of metabolite formation were established, and the results underlined the modulation of the alcohol to acid formation ratio according to culture conditions. Optimization of the hydroxylation step (pH, temperature, culture medium composition) led to the preparation of hydroxyterfenadine with a good yield (51%) using cells grown in culture medium without soybean peptone. In contrast, when incubations were performed with cells cultured in a medium containing soybean peptone, the alcohol to acid formation ratio decreased. The efficiency of the conversion to fexofenadine was shown to depend on the age of the cells, thus suggesting the induction of an oxidative activity. Both the hydroxylation reaction and the following two-oxidation steps leading to the acid seemed to depend on oxygen.     

Oxidation of terfenadine by Streptomyces platensis: Influence of culture medium on metabolite formation

The biotransformation of terfenadine into a primary alcohol, hydroxyterfenadine, followed by its oxidation to an acid, fexofenadine, was investigated using Streptomyces platensis cells. Time-courses of metabolite formation were established, and the results underlined the modulation of the alcohol to acid formation ratio according to culture conditions. Optimization of the hydroxylation step (pH, temperature, culture medium composition) led to the preparation of hydroxyterfenadine with a good yield (51%) using cells grown in culture medium without soybean peptone. In contrast, when incubations were performed with cells cultured in a medium containing soybean peptone, the alcohol to acid formation ratio decreased. The efficiency of the conversion to fexofenadine was shown to depend on the age of the cells, thus suggesting the induction of an oxidative activity. Both the hydroxylation reaction and the following two-oxidation steps leading to the acid seemed to depend on oxygen.     

Growth of Bifidobacterium bifidum in whey-based media

Bifidobacteria play an important role in human health including the enhancement of resistance against infection in infants. To develop an inexpensive whey-based medium for Bifidobaterium bifidum, potential growth promoters — yeast extract, casein, bovine casein digest, tryptone, peptone and glucosamine — singly or in combinations, were evaluated for their bifidus growth-promoting activity. The effect of environmental conditions on growth in cheese whey was also evaluated. A whey-based medium for B. bifidum was formulated. Cheese whey supplemented with N-acetylglucosamine (1 mg/ml) and yeast extract (10 mg/ml) in the presence of sodium thioglycolate (0.1%) at pH 6.8 promoted the growth of B. bifidum at 37°C. Journal of Industrial Microbiology & Biotechnology (2000) 25, 177–179. Received 20 May 2000/ Accepted in revised form 20 July 2000     

Factors affecting lipase production in Syncephalastrum racemosum

Syncephalastrum racemosum grown as a static culture showed maximum lipase production at 30°C in 2d at pH 8.0. When the medium was supplemented with fructose, maximum production of lipase per unit of growth was achieved, followed by raffinose, sucrose, ribose, galactose, maltose, lactose, mannitol and glucose. Amongst the nitrogen sources tested, corn steep liquor at 8% (v/v) produced maximum enzyme; there was evidence of catabolite repression by glucose when groundnut protein, soybean meal, milk casein or wheat bran were the sources of nitrogen. Calcium, potassium and sodium citrates, each at 0.1% (w/v), increased the yield of lipase.     

细菌素发酵培养基的优化及动力学初步分析

用响应面方法对Lactococcus lactis生产细菌素乳链菌肽的培养基进行了优化。首先用部分重复因子实验对培养基组份蔗糖,大豆蛋白胨,酵母粉,KH₂PO₄,NaCl,MgSO₄·7H2O对乳链菌肽的影响进行评价,并找出主要影响因子为大豆蛋白胨和磷酸二氢钾,前者为负影响,后者为正效应,其它组份对乳链菌肽产量的影响不显著。第二步用最陡爬坡路径逼近最大响应区域。最后用中心组合设计及响应面分析确定主要影响因子的最佳浓度。菌株在优化培养基中的乳链菌肽产量增加1倍为2150(IU/mL)。动力学分析表明,菌株生长与细菌素的产生为部分耦联型,进入对数中期菌体比生长速率和细菌素比产率在优化培养基中均大于优化前培养基。