不同冷冻保藏条件对Glarea lozoyensis生长及产纽莫康定B_0能力的影响

棘白菌素类化合物纽莫康定B_0生产菌株Glarea lozoyensis,在其连续传代和发酵过程中都观察到菌株的变异,给科研、生产带来极大困扰。因此,采用低温冷冻保藏对G.lozoyensis Q1进行长期保存。采用单因素实验法研究了低温保护剂类型、保藏温度以及低温保护剂浓度对G.lozoyensis Q1菌体形态和发酵性能的影响。以作用于全细胞的80 g/L甘油和作用于细胞壁外的200 g/L PEG-6000作为低温保护剂,在-80℃保藏3个月后,纽莫康定B_0产量达到1.6 g/L,与保藏前基本一致。并且,当菌体保藏过后菌丝形态能维持一定褶皱的实验组产量均能保持在1 g/L以上,当表面趋于光滑时产量急剧下降,仅为初始的25%左右。结果表明,以甘油及PEG-6000作为冷冻保护剂,在-80℃条件下可实现G.lozoyensis Q1长期保藏。     

纽莫康定Bo色谱纯化工艺的研究

本文介绍了纽莫康定Bo的抗菌机理及其发酵生产中出现的杂质情况,探讨了色谱填料对纽莫康定Bo粗品纯化效果的影响,并考察了纯化过程中的主要影响因素。以上样量、有机相浓度、洗脱流速、纯化温度为考察因素,纯化后纯度在98%以上的回收率为考察指标。采用正交实验法对纯化工艺进行优化,确立了最优的纯化工艺为:上样量为3mg/ml填料,有机溶剂浓度30%乙腈水溶液,洗脱流速1ml/min,纯化温度为20℃,柱床高度460mm。     

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。     

响应面法优化赖氨酸发酵培养基

运用Ｂｏｘ－Ｂｅｈｋｅｎ设计的响应面试验（Ｒｅｓｐｏｎｓｅ－ｓｕｒｆａｃｅ ｅｘｐｅｒｉｍｅｎｔ）对黄色短杆菌（Ｂｒｅｕｉｂａｃｔｅｒｉｕｍ ｆｌａｕｕｍ）ＦＢ４２进行赖氨酸发酵的培养基进行了优化。响应面法对７２小时分批发酵结果的优化分析表明：硫酸铵、豆饼水解液及玉米浆三种因素对赖氨醚发酵无明显的交互作用,在硫酸铵、豆饼水解液和玉米浆的浓度分别为５％、２％和３％时,发酵液中的产酸达到５４．０６ｇ／     

响应面法优化康定鼠尾草中迷迭香酸提取工艺

利用单因素实验和响应面法优化康定鼠尾草中迷迭香酸提取工艺。采用高效液相色谱法测定迷迭香酸含量,以提取率为参考指标。通过单因素实验筛选出料液比、提取时间和乙醇浓度三个主要因素,通过BoxBehnken设计方案,建立迷迭香酸提取得率的二次回归方程,得到优化组合条件。最佳提取条件为乙醇浓度40%,液固比15∶1,提取时间50 min。最佳提取条件下,迷迭香酸提取得率为12.30 mg/g。优化得到的康定鼠尾草中迷迭香酸的提取工艺合理,操作可行,质量稳定。     

一种Zalerion arboricola发酵产物的分离、纯化和结构鉴定

抗真菌药物卡泊芬净是以丝状真菌Zalerion arboricola的菌体代谢产物纽莫康定B0为中间体经化学合成得到.该菌在代谢产纽莫康定B0的同时还会产生多种具有相似结构的代谢物.为了从这些结构类似物中寻找潜在的抗真菌药物中间体,对Zalerion arboricola菌株HCCB30111的代谢产物进行了分离纯化和...     

Box-Behnken响应面法优化司替霉素B产生菌发酵条件

【背景】粗糙链霉菌(Streptomyces scabrisporus) HBERC-53204是本中心自主分离的一株链霉菌,经鉴定,其产生一种活性化合物司替霉素B (steffimycin B,SMB),对多种动植物重要病原菌具有良好生物活性。【目的】提高SMB发酵水平,拓宽放线菌活性天然产物在农牧业领域的研究及应用。【方法】以本实验室筛选出的一株产SMB的粗糙链霉菌HBERC-53204为研究对象,运用单因素试验筛选培养基的主效碳源、氮源、无机盐及各营养成分最适浓度,并基于单因素试验结果,通过Plackett-Burman(PB)试验设计筛选出显著影响因素,再结合最陡爬坡试验、Box-Behnken (BB)响应面法拟合显著因子与产量的非线性方程求解,进一步优化菌株产SMB的最佳发酵培养基配方。【结果】优化后最佳培养基配方为：葡萄糖36.22 g/L,蛋白胨8.00 g/L,酵母粉8.51 g/L,酸水解酪蛋白1.50 g/L,MgSO₄ 0.68 g/L,KNO₃ 1.00 g/L。经摇瓶验证,优化后SMB效价达到477.26 mg/L...     

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%.     

响应面-满意度函数优化重组大肠杆菌产NMN转移酶发酵条件

重组大肠杆菌Escherichaia coli能高效表达NMN转移酶,以此为出发菌株,以菌体生长量OD600和NMN转移酶的活力为响应值,对重组大肠杆菌产NMN转移酶的发酵条件进行优化.首先以Plackett-Burman实验设计优化筛选出3个主要影响因子:胰蛋白胨、甘油、MgSO4;随后以Box-Behnken中心组合设计建立上述3个因子对OD600和NMN转移酶活力水平的数学模型;最后通过满意度函数获得最佳发酵条件为:酵母粉30 g/L,胰蛋白胨10.5 g/L,甘油3.49 mL/L,MgSO40.45 g/L,K2 HPO440.5 g/L,KH2 PO46.0 g/L,NH4 Cl 1.5 g/L,NaCl 0.6 g/L,接种量1.5%,诱导时间12 h.在该优化条件下,菌体生长和产酶水平均获得了显著的提升.重组NMN转移酶的活力水平从8.85 U/mg提高到15.48 U/mg,菌体生长量OD600从4.85提高到6.01,提高幅度分别为74.92%和23.92%.     

响应面法优化叶酸高产菌发酵培养基及发酵条件

采用响应面法对叶酸产生菌产朊假丝酵母Y2.12的发酵发酵培养基及发酵条件进行优化.首先,采用Plackett-Burman方法对影响发酵各因素的效应进行评价,筛选出有显著效应的3个因素：碳源乳糖、pH值和摇床转速;并通过Box-Benhnken设计和响应面分析法对这3个主要影响因素进行分析.结果表明,优化后这3个因素的最佳值为摇床转速196r/min、pH7和碳源乳糖含量为6.25%.采用优化后的发酵培养基及发酵条件进行摇瓶发酵,叶酸的含量可达23.14±0.13mg/L,取得了很好的优化效果.     

Effects of amino acid and trace element supplementation on pneumocandin production by Glarea lozoyensis: impact on titer, analogue levels, and the identification of new analogues of pneumocandin B0

Addition of the amino acids threonine, serine, proline, and arginine to fermentations of the fungus Glarea lozoyensis influenced both the pneumocandin titer and the spectrum of analogues produced. Addition of threonine or serine altered the levels of the “serine analogues” of pneumocandins B₀ and B₅ and allowed for their isolation and identification. Proline supplementation resulted in a dose-dependent increase in the levels of pneumocandins B₀ and E₀, whereas pneumocandins C₀ and D₀ decreased as a function of proline level. Moreover, proline supplementation resulted in an overall increase in the synthesis of both trans-3- and trans-4-hydroxyproline while maintaining a low trans-4-hydroxyproline to trans-3-hydroxyproline ratio compared to the unsupplemented culture. Pneumocandin production and the synthesis of hydroxyprolines was also affected by addition of the proline-related amino acid arginine but not by the addition of glutamine or ornithine. Zinc, cobalt, copper, and nickel, trace elements that are known to inhibit α-ketoglutarate-dependent dioxygenases, affected the pneumocandin B₀ titer and altered the levels of pneumocandins B₁, B₂, B₅, B₆, and E₀, analogues that possess altered proline, ornithine, and tyrosine hydroxylation patterns. Journal of Industrial Microbiology & Biotechnology (2001) 26, 216–221. Received 05 November 2000/ Accepted in revised form 27 January 2001     

Protoplast mutant selection of Glarea Lozoyensis and statistical optimization of medium for pneumocandin B0 yield-up

A combination of microbial strain improvement and statistical optimization is investigated to maximize pneumocandin B₀ production from Glarea lozoyensis ATCC 74030. Atmospheric and room temperature plasma (ARTP) was used to enhance G. lozoyensis ATCC 74030 in pneumocandin B₀ yield. Mutant strain G. lozoyensis Q1 exhibited 1.39-fold increase in pneumocandin B₀ production to 1134 mg/L when compared with the parent strain (810 mg/L). Further, the optimized medium provided another 1.65-fold in final pneumocandin B₀ concentration to 1873 mg/L compared to the original medium. The results of this study indicated the combined application of a classical mutation and medium optimization can improve effectively pneumocandin B₀ production from G. lozoyensis and could be a tool to improve other secondary metabolites production by fungal strains.     

Novel osmotic stress control strategy for improved pneumocandin B0 production in Glarea lozoyensis combined with a mechanistic analysis at the transcriptome level

Applied Microbiology and Biotechnology - Pneumocandin B0, the precursor of the antifungal drug caspofungin, is a secondary metabolite of the fungus Glarea lozoyensis. In this study, we investigated...     

Residual fructose and osmolality affect the levels of pneumocandins B0 and C0 produced by Glarea lozoyensis

A high total pneumocandin titer (B₀ + C₀) with a low percentage of the structural isomer pneumocandin C₀ was achieved by carrying out fermentations of Glarea lozoyensis at a high residual fructose concentration (125 g/l initial). When the fermentation was carried out at a low residual fructose concentration (40 g/l initial), pneumocandin production increased by 34%. However, a disproportionate increase in the level of pneumocandin C₀ synthesized (250% increase vs 30% increase for pneumocandin B₀) was observed. Midcycle addition of 150 mM NaCl or 116 mM Na₂SO₄ to low residual fructose fermentations returned the titer and isomer levels to those seen for the high residual fructose fermentation. The increase in pneumocandin C₀ synthesis under low residual fructose conditions appears to be linked to the increase in the synthesis of trans-4 hydroxyproline, with the synthesis of trans-3 hydroxyproline remaining unaffected. This suggests that the formation of pneumocandin C₀ is the result of a misincorporation of trans-4 hydroxyproline instead of trans-3 hydroxyproline by the pneumocandin peptide synthetase, and that the amount of trans-4 hydroxyproline formed dictates the frequency of this misincorporation. Received: 16 February 2000 / Revised: 17 May 2000 / Accepted: 8 June 2000     

头孢菌素C产生菌的诱变育种及培养基优化

通过对顶头孢霉（Cephalosporium acremonium）FC-01进行诱变选育及特定种子培养基的优化,提高了头孢菌素C的发酵产量。分别采用紫外-氯化锂和钴-60（60Co）γ射线对FC-01进行诱变选育,筛选到高产菌株FC-1-4和FC-4-2,产量较出发菌株分别提高了26%和54.5%。运用Plackett-Burman设计方法和响应面法对种子培养基进行优化,头孢菌素C发酵效价较对照分别提高了34.7%和13.2%,优化后的种子培养基主要成分为玉米浆3.70%、葡萄糖2.62%和硫酸镁0.15%,得到的菌株及相应的种子培养条件已成功应用在160M³工业发酵罐生产中,具有重要的工业生产能力。     

响应面法优化类芽胞杆菌HB172198产褐藻胶裂解酶发酵培养基

为了提高类芽胞杆菌新种HB172198产褐藻胶裂解酶活力,本研究采用响应面法对该菌株液体发酵培养基进行了优化实验。在单因素实验和Plackett-Burman试验筛选出海藻酸钠、胰蛋白胨、NaCl、MgSO4·7H2O等4个显著影响产酶因素的基础上,通过Box-Behnken设计及响应面法进行回归分析,得出产褐藻胶裂解酶最佳发酵培养基,其成分为:海藻酸钠7.50 g/L、胰蛋白胨13.57 g/L、NaCl 29.75 g/L、MgSO4·7H2O 0.08 g/L。优化条件下该菌株最大酶活性达14.60 U/mL,是优化前的1.87倍。本研究为菌株HB172198产褐藻胶裂解酶的大规模生产和工业应用提供了重要的理论依据。     

响应面模型分析在发酵产酶条件优化中的应用

<正>微生物蛋白酶是一种重要的工业用酶,其产量占工业酶总产量的40%,广泛应用于食品、纺织、医药、化工、环保等行业。其中高温蛋白酶具有热稳定性强的特点,适合工业化应用中的高温催化过程,具有广阔的应用前景,但仍存在产量较低、成本较高等缺点,如何提高菌株产酶能力一直是微生物高温蛋白酶应用研究中的热点问题。一般而言,高密度细胞培养技术的应用可显著提高产酶菌的酶产量[1],但与常见产酶工程菌株受单一诱     

利用响应面法优化红谷霉素发酵培养基*

在摇瓶条件下,对链霉菌702发酵生产过程中的主要培养基组成对产红谷霉素影响进行的研究。试验采用响应面法优化摇瓶发酵培养基,利用全因子实验设计筛选出对链霉菌702产红谷霉素重要影响因子黄豆饼粉和工业蛋白胨,应用最陡爬坡实验法接近重要因子的最优水平,然后应用中心复合设计确定重要因子的最优水平。优化后的培养基组成为：玉米淀粉20g,玉米粉20g,葡萄糖20g,磷酸二氢钾0．3g,蛋白胨9g,黄豆饼粉23g,硝酸钾2．5g,硫酸铵2．5g,豆油5mL,氯化钠3g,碳酸钙6g,定容至1L。实验结果表明,采用优化后的培养基,其发酵液红谷霉素效价达到1,500μg／mL,比优化前提高了3.08倍。     

重组谷氨酸棒杆菌发酵L-苯丙氨酸培养基的优化

【目的】提高重组谷氨酸棒杆菌发酵L-苯丙氨酸(L-phenylalanine,L-Phe)的产量。【方法】使用正交试验设计以及响应面优化法分别对种子培养基及发酵培养基进行优化,确定了重组谷氨酸棒杆菌发酵L-Phe的最佳种子培养基及最佳发酵培养基。【结果】重组谷氨酸棒杆菌发酵L-Phe最佳种子培养基(g/L):葡萄糖25.0,玉米浆25.0,硫酸铵15.0,硫酸镁1.0,磷酸二氢钾2.0,尿素2.0,p H 6.8-7.0;最佳发酵培养基(g/L):葡萄糖110.0,玉米浆7.0,硫酸铵25.0,硫酸镁1.0,磷酸二氢钾1.0,柠檬酸钠2.0,谷氨酸1.0,碳酸钙25.0,p H 6.8-7.0;在最佳培养基条件下L-Phe产量最高达到9.14 g/L,较优化前的7.46 g/L提高了22.5%。【结论】通过正交试验和响应面分析对重组谷氨酸棒杆菌发酵L-Phe培养基进行优化,明显提高了L-Phe的产量,并确定了葡萄糖、玉米浆和硫酸铵为发酵培养基中影响L-Phe产量的3个关键因子。研究结果为L-Phe的发酵放大提供了依据。     

Use of response surface methodology to optimize culture medium for production of lipase with Candida sp. 99-125

Response surface methodology (RSM) was employed to optimize culture medium for production of lipase with Candida sp. 99-125. In the first step, a Plackett–Burmen design was used to evaluate the effects of different components in the culture medium. Soybean oil, soybean powder and K₂HPO₄ have significant influences on the lipase production. The concentrations of three factors were optimized subsequently using central composite designs and response surface analysis. The optimized condition allowed the production of lipase to be increased from 5000 to 6230 IU/ml in shake flask system. The lipase fermentation in 5 l fermenter reached 9600 IU/ml.