重组大肠杆菌全细胞催化合成莱鲍迪苷D

莱鲍迪苷D(Rebaudioside D,RD)是一种稀有具有高甜度的甜菊糖苷类化合物。本文实现了重组大肠杆菌全细胞催化莱鲍迪苷A(Rebaudioside A,RA)合成RD。以水稻c DNA为模板,扩增得到葡萄糖基转移酶基因eugt11,构建了重组菌株E.coli BL21(p ETDuet-eugt11),并成功表达了重组蛋白6His-EUGT11。通过Ni柱亲和层析纯化并在体外酶催化反应表征了其催化活性。将重组菌BL21(p ETDuet-eugt11)应用于催化合成RD研究。探讨了反应体系pH、温度、柠檬酸钠浓度、菌体密度、二价金属离子、二甲苯体积分数、UDPG添加浓度对反应效率的影响。单因素考察结果显示,在菌体密度0.16 g湿细胞/m L反应液,底物RA浓度为1.0 mmol/L,pH 8.0,60 mmol/L柠檬酸钠,1%二甲苯,0.1 mmol/L Zn Cl2,12.0 mmol/L UDPG,反应温度42℃,反应时间24 h的条件下,RD产量为123.6 mg/L(约0.1 mmol/L)。     

全细胞催化合成阿糖胞苷工艺的研究

以阿糖尿苷和胞嘧啶为原料,采用枯草芽胞杆菌Bacillus subtilis A302作为全细胞催化剂进行阿糖胞苷的生物合成,通过单因素与正交试验的考察,得到了全细胞催化合成阿糖胞苷的最佳反应条件:反应初始pH为7.0,反应温度为36℃,催化剂添加量为250 mg,摇床转速为190 rpm,该条件得到的阿糖胞苷收率达到65.3％.该方法操作简单,反应体系杂质较少,是合成阿糖胞苷的一种有效可行的方法.     

球孢白僵菌全细胞催化苯甲酸合成对羟基苯甲酸

[目的]为实现对羟基苯甲酸的生物合成.[方法]通过形态学和分子学鉴定得到一株球孢白僵菌B2660,利用B2660对苯甲酸进行全细胞催化,经高效液相色谱(HPLC)和GC-MS检测确定产物为对羟基苯甲酸,并通过正交试验对全细胞催化条件进行了优化.[结果]未优化前,球孢白僵菌B2660在48 h合成对羟基苯甲酸0.434g...     

重组大肠杆菌全细胞催化L-苏氨酸合成2,5-二甲基吡嗪

2,5-二甲基吡嗪(2,5-dimethylpyrazine,2,5-DMP)在食品香料与医药方面具有重要的经济价值,工业上普遍采用环境不友好且反应条件苛刻的化学合成法来生产.文中结合代谢工程和辅因子工程策略设计高效催化L-苏氨酸合成2,5-DMP的全细胞催化剂,实现微生物转化法合成2,5-DMP.本研究首先分析了不同...     

生物转化-从全细胞催化到代谢工程

与传统的化学合成方法相比,利用生物的手段转化生产活性化合物及其衍生物无疑具有更大的吸引力。随着用于生物转化微生物种类的增多,生物转化的应用领域不断得到扩大。生物转化的发展经历了野生型全细胞催化,基因工程微生物全细胞反应,以及利用系统分析和代谢工程进行全局性调控等几个阶段。以下对这一发展趋势及相关研究的最新进展作一简要综述。     

重组枯草芽孢杆菌全细胞催化合成2-O-α-D-甘油葡糖苷

2-O-α-D-甘油葡糖苷是一种在食品、化妆品、保健品及医药领域有着重大应用前景的高附加值产品,但国内仍未实现2-O-α-D-甘油葡糖苷的工业化生产,且鲜有关于2-O-α-D-甘油葡糖苷合成的相关报道。文中旨在开发一种利用食品安全级重组枯草芽孢杆菌全细胞催化合成2-O-α-D-甘油葡糖苷的方法,通过构建一株异源表达肠膜明串珠菌蔗糖磷酸化酶(Sucrose phosphorylase,SPase)的重组枯草芽孢杆菌Bacillus subtilis 168/pMA5-gtfA,并将其用作全细胞催化剂合成2-O-α-D-甘油葡糖苷,通过优化培养温度、时间及全细胞转化条件,提高其转化合成2-O-α-D-甘油葡糖苷的产量。结果表明,重组枯草芽孢杆菌B. subtilis 168/pMA5-gtfA在30℃下培养20 h,菌体裂解物酶活力最大达1.43 U/mL,并且在1 mol/L蔗糖、2.5 mol/L甘油、pH 7.0、菌体OD600为40、30℃下全细胞转化反应48h,共生成2-O-α-D-甘油葡糖苷189.3g/L,平均转化速率为15.6mmol/(L·h),蔗糖转化率约为75.1%,...     

蔗糖磷酸化酶全细胞催化AA-2G的条件优化

目的:使用表达蔗糖磷酸化酶(EC 2.4.1.7,Sucrose phosphorylase,SPase)的大肠杆菌重组工程菌E.coli BL21/pET-spase,作为全细胞催化剂,合成2-O-D-吡喃葡糖基-L-抗坏血酸(Ascorbic acid 2-glucoside,AA-2G)。通过反应条件的优化研究,提高AA-2G的收率。方法:分别考察菌体量、缓冲液pH、蔗糖浓度、维生素C浓度、反应时间和温度对AA-2G合成反应的影响,再组合上述最佳条件进行反应。AA-2G的产量使用高效液相色谱法进行定量。结果:最佳反应条件为:菌体量15 mg/mL,缓冲液pH 4.5,蔗糖浓度100 g/L,维生素C浓度175 g/L,反应时间20 h,温度37℃。在此条件下,AA-2G产量达到了35.7 g/L。结论:以蔗糖为底物,使用SPase合成AA-2G的研究报道较少。本研究通过优化此方法的反应条件,让AA-2G的产量得到了大幅提高。同时本研究中成功地采用了大肠杆菌工程菌作为全细胞催化剂,这比传统的使用粗酶液的方法更省时省力,有良好的应用潜力。     

杰氏棒杆菌L-天冬氨酸α脱羧酶半理性改造及全细胞催化合成β-丙氨酸

β-丙氨酸是多个药物合成的重要砌块,可以通过天冬氨酸α脱羧酶（Pan D）催化L-天冬氨酸脱羧来合成,但普遍在用的Pan D酶活性不高是制约全细胞催化合成β-丙氨酸的瓶颈。因此,本研究通过酶的挖掘,选择将杰氏棒杆菌来源（Corynebacterium jeikeium）Pan D在Escherichia coli中异源表达。对杰氏棒杆菌来源Pan D进行Alaph Fold2建模和分子对接,采用Rosetta虚拟突变确定突变热点,结合薄层层析初筛和纯化后复筛,最终筛选到突变体L39A,其比酶活为13.45 U/mg,相比野生型酶的比酶活（9.6 U/mg）提升了1.4倍。酶学性质表征数据表明,野生型酶和L39A突变体最适p H均为6.5,且在p H 6.0-7.0之间酶活性稳定;两者最适温度为55℃,但L39A热稳定性较野生型提高;突变体酶的催化效率比野生型提升了1.4倍。对突变体进行结构解析发现,39位取代为侧链基团更小的丙氨酸,亲水性增强,增加了关键催化氨基酸58位酪氨酸与其他氨基酸的相互作用,使活性中心周围的区域稳定性提高,从而提高了催化活性。全细胞催化数据表明,在OD600=4...     

全细胞催化生产N-乙酰神经氨酸的条件优化

【目的】N-乙酰神经氨酸有多种生物学功能,其在治疗流感、神经性疾病、炎症和肿瘤等方面具有重要的医药价值。N-乙酰神经氨酸现有的生产方法产量低、成本高,难以满足医药工业大规模的需求,因而急需建立一种经济高效的生产方法。【方法】在前期研究中,构建了一株产N-乙酰神经氨酸的代谢工程菌(Escherichia coliΔnanTEK/pNA),本研究通过单因素试验对工程菌生物转化生产N-乙酰神经氨酸的过程进行优化,包括工程菌细胞培养条件(培养温度和时间)和全细胞生物转化的各种条件(转化温度、时间、表面活性剂等)。【结果】经过条件优化后,建立了全细胞催化法生产N-乙酰神经氨酸的工艺流程,使N-乙酰神经氨酸的产量提高到294.39 mmol/L。【结论】该方法具有操作简便、高效和经济的优势,为N-乙酰神经氨酸的规模化生产奠定了基础。     

重组大肠杆菌全细胞催化高效合成胆绿素北大核心CSCD

胆绿素作为一种重要的保护细胞的抗氧化剂,其传统生产方法主要由胆红素的化学氧化产生,但过程复杂、纯度不高。本研究提出了一种高效、绿色、安全的生产胆绿素的方法。通过比较,筛选得到了破伤风梭状芽孢杆菌(Clostridium tetani)来源的血红素加氧酶(heme oxygenase,HO)基因,并成功构建具备转化血红素合成胆绿素能力的重组大肠杆菌(Escherichiacoli)BL21/pETDuet-hoCt。在pH 7.0、35℃、100 mg/L底物浓度条件下胆绿素产量为32.9 mg/L。为提高还原力,构建了基于谷氨酸脱氢酶(glutamate dehydrogenase,GdhA)的NADPH辅酶再生系统,获得重组菌E.coli BL21/pETDuet-gdhAEc-hoCt,胆绿素产量为71.5 mg/L。此外,通过引入膜表面展示系统,构建重组菌E.coli BL21/pETDuet-gdhAEc-blc/hoCt,缩短转化时间的同时,胆绿素产量进一步得到提高,达到76.3 mg/L,是目前生物法合成胆绿素的最高研究报道。本研究为胆绿素的绿色生产奠定了良好的基础。     

中心复合实验设计法优化重组毕赤酵母表达猪胰岛素前体的培养基和诱导pH

毕赤酵母中猪胰岛素前体(Porcine Insulin Precursor,PIP)的表达受到培养基组成及培养环境的影响。本文首先通过部分因子法设计实验,筛选出影响PIP表达的显著因子。实验结果表明,在试验范围内,甲醇补料量和硫酸铵浓度为正效应因子,诱导pH为负效应因子。在此基础上,经过快速登高法逼近显著因子的最优值后,再在此值附近利用中心复合法设计实验,最终得到了PIP表达的最佳条件。经过多批次实验验证,在此条件下PIP的表达量为120.4mg/L,比优化前的值41.5mg/L提高了将近两倍。     

Optimization of culture media for large‐scale lutein production by heterotrophic Chlorella vulgaris

Lutein is a carotenoid with a purported role in protecting eyes from oxidative stress, particularly the high‐energy photons of blue light. Statistical optimization was performed to growth media that supports a higher production of lutein by heterotrophically cultivated Chlorella vulgaris. The effect of media composition of C. vulgaris on lutein was examined using fractional factorial design (FFD) and central composite design (CCD). The results indicated that the presence of magnesium sulfate, EDTA‐2Na, and trace metal solution significantly affected lutein production. The optimum concentrations for lutein production were found to be 0.34 g/L, 0.06 g/L, and 0.4 mL/L for MgSO₄·7H₂O, EDTA‐2Na, and trace metal solution, respectively. These values were validated using a 5‐L jar fermenter. Lutein concentration was increased by almost 80% (139.64 ± 12.88 mg/L to 252.75 ± 12.92 mg/L) after 4 days. Moreover, the lutein concentration was not reduced as the cultivation was scaled up to 25,000 L (260.55 ± 3.23 mg/L) and 240,000 L (263.13 ± 2.72 mg/L). These observations suggest C. vulgaris as a potential lutein source. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:736–743, 2014     

裂褶菌F17锰过氧化物酶酶活力影响因素的响应面优化(英文)

锰过氧化物酶是真菌分泌的一种糖基化的含有血红素辅基的胞外蛋白,在染料降解和脱色过程中起着重要作用。本实验利用本实验室保存的的白腐真菌裂褶菌Schizophyllum sp.F17产锰过氧化物酶(MnP),研究MnP的酶学性质,并对酶活条件进行优化。实验通过超滤浓缩、DEAE-纤维素、DE52离子交换层析和Sephadex G-75凝胶过滤等步骤,分离纯化得到电泳纯的锰过氧化物酶。该酶蛋白含量为23μg/mL,分子量大小为49.2kDa,在0.1mmol/L H2O2中半衰期为5~6min。Mn2+、H2O2以及酶的用量可以影响MnP酶促反应的效率,在单因子分析法的基础上,通过全因子中心组合设计响应面分析表明:H2O2以及H2O2与酶用量之间的交互作用对酶促反应的作用是最显著的。在优化条件下,酶对偶氮染料金橙G、刚果红显示出较强的脱色能力。     

固定化全细胞催化可再生油脂合成生物柴油的稳定性

酶法合成生物柴油具有反应条件温和、醇用量小、无污染物排放、产物易分离回收等优点,越来越得到关注。全细胞催化剂,无需酶的提取和纯化,减少了酶活损失,有望大幅降低生产成本;Rhizopus oryzae IFO4697全细胞可以有效催化植物油脂合成生物柴油,进一步提高全细胞在催化植物油脂甲醇解制备生物柴油过程中的稳定性,对于工业放大具有重要意义。本实验对固定化全细胞Rhizopus oryzae IFO4697催化植物油脂合成生物柴油的稳定性进行了系统地研究,结果表明:反应体系水含量对于全细胞催化剂的反应活性和催化稳定性有重要影响,5%～15%含水量适宜;研究范围内,载体粒度及干燥方式对稳定性影响不显著;经过戊二醛交联后,全细胞催化油脂甲醇解反应的稳定性显著提高,1200h反应后,仍然可以保持75%的生物柴油得率;真空抽滤直接回用的方式有利于稳定性的保持。在优化条件下,回用20个批次,生物柴油得率可维持在80%。     

Response surface methodology for optimizing the fermentation medium of Clostridium butyricum

AIMS: Strains of Clostridium butyricum have been increasingly used as probiotics for both animals and humans. The aim of this study was to develop a growth medium for cultivating C. butyricum ZJUCB using a statistical methodology. METHODS AND RESULTS: Response surface methodology (RSM) was used to evaluate the effects of variables, namely the concentrations of the glucose, pectin, soyabean cake extract, casein, corn steep flour, ammonium sulphate, sodium bicarbonate and the medium initial pH. A fractional factorial design was applied to study the main factors that affected the growth of a probiotic strain of C. butyricum currently preserved in our lab and the central composite experimental design was adopted to derive a statistical model for optimizing the composition of the fermentation medium. The experimental results showed that the optimum fermentation medium for the growth of C. butyricum was composed of 2% glucose (w/v), 0.5% pectin (w/v), 0.2% casein (w/v), 3.98% soyabean cake extract, 0.1% (NH4)2SO4 (w/v), 0.124% NaHCO3 (w/v), 0.37% corn steep flour (w/v), 0.02% MnSO4 H2O (w/v), 0.02% MgSO4 7H2O (w/v) and 0.002% CaCl2 (w/v) at pH 7.5. CONCLUSIONS: After incubating 24 h in the optimum fermentation medium, the populations of the viable organisms were estimated to be 10(9) CFU ml(-1). In the present study, we report the optimization of a growth medium that produced increased yields using statistical approach. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of bacteria as a probiotic is showing increasing potential. The development of a growth medium that has a high yield is an obvious need, and the approach to optimizing a growth medium is innovative.     

Production of S‐lactoylglutathione by high activity whole cell biocatalysts prepared by permeabilization of recombinant Saccharomyces cerevisiae with alcohols

The permeabilization of yeast cells with methanol, ethanol, and isopropyl alcohol under various conditions was studied to develop the preparation method of high activity whole cell biocatalysts. Recombinant Saccharomyces cerevisiae, which intracellularly overexpresses glyoxalase I and catalyzes the conversion of methylglyoxal to S‐lactoylglutathione in the presence of glutathione, was used as the model system. The permeabilization treatments with alcohols significantly enhanced the activities of yeast cells. Especially, the initial S‐lactoylglutathione production rates of cells permeabilized with 40% ethanol and isopropyl alcohol solutions for 10 min at 4°C were high and were 364 and 582 times larger than those of untreated cells, respectively. These permeabilized yeast cells retained high activities during repeated batch reactions. Even in third batch reaction, they showed approximately 70–80% of the activity in the first batch. The plasma membrane of S. cerevisiae cells was damaged by the treatment with alcohol solutions in such a way that leakage of glyoxalase I from the cells is rather small and that both substrate and product show very high permeability. The initial S‐lactoylglutathione production rates of these permeabilized cells were 1.5–2.5 times larger than those of glyoxalase I in cell extracts prepared by ethyl acetate method from the same amount of cells. These results demonstrate that the recombinant S. cerevisiae cells permeabilized with alcohol solutions under the optimum condition are very effective whole cell biocatalysts. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 54–60, 1999.     

Optimization of a culture medium for xylanase production by Bacillus sp. using statistical experimental designs

The concentrations of oat spelt xylan, casein hydrolysate and NH4Cl in the culture medium for production of xylanase from Bacillus sp. I-1018 were optimized by means of response surface methods. The path of steepest ascent was used to approach the optimal region of the medium composition. The optimum composition of the nutrient medium was then easily determined by using a central composite design and was found to be 3.16g/l of xylan, 1.94g/l casein hydrolysate, 0.8g/l of NH4Cl. The xylanase production was increased by 135% when the strain was grown in the optimized medium compared to initial medium.     

Optimization of a medium for enhancing nicotine biodegradation by Ochrobactrum intermedium DN2

AIMS: To optimize a medium for nicotine degradation by Ochrobactrum intermedium DN2 in presence of yeast extract, glucose and Tween 80 using response surface methodology (RSM). METHODS AND RESULTS: In this study, the effects of yeast extract, glucose and Tween 80 on nicotine degradation were investigated in flasks using a novel nicotine-degrading bacterium, O. intermedium DN2. A full factorial central composite design was applied in the design of experiments and in the analysis of the experimental data. The results showed that the most significant variable influencing nicotine degradation was yeast extract, followed by glucose, and then Tween 80. Moreover these three factors interacted with each other and combined to produce positive effects on nicotine degradation. The experimental data also allowed the development of an empirical model (P < 0.0001) describing the inter-relationship between independent and dependent variables. By solving the regression equation, the optimal values of the variables were determined as: yeast extracts 0.094%, glucose 0.101% and Tween 80 0.080%. Using the medium obtained, about 1,220 mg l(-1) of nicotine was degraded (95.55%) within 10 h at the specific biodegradation of 116.59 mg l(-1) h(-1) in 30-l bioreactor containing 25-l tobacco extract. CONCLUSIONS: An optimal medium of nicotine degradation by the strain DN2 was obtained. SIGNIFICANCE AND IMPACT OF THE STUDY: RSM proved to be reliable in developing the model, optimizing factors and analysing interaction effects. The results provide better understanding on the interactions between yeast extract, glucose and Tween 80 for nicotine biodegradation.