SAM产生菌酿酒酵母HYS98分批发酵动力学研究及应用

在分批发酵过程特征分析的基础上,建立了分批发酵动力学模型并验证了其可靠性.根据所建立的动力学模型,提出了分阶段控制蔗糖和乙醇浓度的策略,据此策略在5 L反应器中发酵40 h,菌体浓度为41.76 g·L-1、SAM胞内含量为84.21 mg·(g DCW)-1、SAM浓度为3.51 g·L-1发酵产率为0.088 g·(L·h)-1,分别比原分批发酵高出13%、20%、36%和22%.     

氨基树脂固定化S-腺苷甲硫氨酸合成酶的研究

以氨基树脂为载体对S-腺苷甲硫氨酸(SAM)合成酶进行固定化,优化了酶的固定化条件并对固定化酶的性质进行了研究。优化的固定化条件为:戊二醛体积分数5%、SAM合成酶添加量20mg·g-1、固定化时间5h。所制备的固定化SAM合成酶的酶活力为476.8U·g-1,酶活力回收率为74.5%。与游离SAM合成酶相比,固定化SAM合成酶的稳定性大幅提高,在50℃孵育5h酶活力仍保留61.2%,而游离SAM合成酶则完全失活;在pH值为6.0~6.5、8.0~9.5的缓冲溶液中,固定化SAM合成酶也更加稳定;固定化SAM合成酶连续催化反应10批次,酶活力保留86.3%;固定化SAM合成酶在4℃储存30d,酶活力保留81.4%。固定化SAM合成酶米氏常数KATPm=0.14mmol·L-1,KLm-Met=0.28mmol·L-1。     

酿酒酵母生物转化蛋氨酸生产S-腺苷-L-蛋氨酸

摇瓶考察筛选酿酒酵母（zjus1)培养1d后补加蛋氨酸和葡萄糖生产S－腺苷－L－蛋氨酸（SAM），并考察了酵母提取物，补加蛋氨酸浓度，葡萄糖浓度及转化pH对酵母细胞的生长和SAM产量的影响，15LB.Braun罐间歇培养及转化过程实验表明，溶氧提高利于碳源利用、细胞生长和SAM产量提高，SAM水平可达800mg/L，酵母密度DCW=15g/L，蛋氨酸转化率约30％。     

高效液相色谱法分析胞内S-腺苷-L-甲硫氨酸含量及其合成酶活性的优化研究

对重组毕赤酵母胞内S-腺苷-L-甲硫氨酸(SAM)含量以及SAM合成酶活性的高效液相色谱(HPLC)测定方法进行了优化。通过调整流动相浓度和洗脱程序,建立了保留时间适中、分离效果好的SAM定量分析方法。结果表明,SAM浓度在0.05～0.5 g·L-1时,其峰面积与浓度线性关系良好,相关系数为0.99996,平均回收率...     

高生物利用率L-蛋氨酸技术商业化

<正>近日,韩国希杰集团在马来西亚的L-蛋氨酸工厂日前投料,开始商业化生产。该技术以植物为原材料,生产的DL-蛋氨酸与以石油为原材料的产品比起来,更易被动物消化吸收,其相对生物利用率高出20%~40%。随着L-蛋氨酸商业化生产的开始,希杰成为了世界唯一的能用生物发酵工艺生产包括L-赖氨酸、L-苏氨酸、L-色氨酸和L-蛋氨酸四种主要氨基酸的企     

乙酰-D-蛋氨酸消旋工艺的研究

在酶法拆分蛋氨酸的工艺中,经离子交换分离L-蛋氨酸后余下的N-乙酰基-D(L)-蛋氨酸溶液进行消旋反应,可将一部分N-乙酰基-D-蛋氨酸转变成L型乙酰基蛋氨酸后,可重新作为N-乙酰基D、L-蛋氨酸拆分底物使用。本文针对目前工厂实际生产中消旋产量和收率不太好的现状,利用正交试验法对影响消旋的各个因素作了分析,得出了较适宜的工艺条件,提高了产品的质量和收率。     

食品及饲料添加剂

农业部批准使用的 饲料添加剂品种目录 饲料级氨基酸(7种)L-赖氨酸盐酸盐、DL-蛋氨酸、DL-羟基蛋氨酸、DL-羟基蛋氨酸钙、N-羟甲基蛋氨酸、L-色氨酸、L-苏氨酸。 饲料级维生素(21种)β-胡罗卜素、维生素A、维生素A乙酸酯、维生素A棕榈酸酯、维生     

全自动合成L-[S-11C-甲基]-蛋氨酸及其质量控制

目的研究新型脑肿瘤显像剂L-[S-11C-甲基]-蛋氨酸的合成方法。方法利用回旋加速器轰击产生11C-CO2,甲醇化后获得活性很强的甲基化前体11C-CH3I,该前体再与L-高胱氨酸硫内酯在常温下反应,获得11C标记的蛋氨酸(11C-MET)。结果11C-MET合成时间约3分钟,合成效率约为80%,放射化学纯>99%,质量控制指标合格。结论11C-MET合成速度快,合成效率高,适用于临床PET显像。     

酶法拆分N-乙酰-D,L-蛋氨酸转化条件优化

重组大肠杆菌BL21/pET22b-argE表达的N-乙酰鸟氨酸脱酰基酶(NAOase)可用于脂肪族氨基酸的手性拆分.水解N-乙酰-D,L-氨基酸中的L-型底物,N-乙酰-D,L-蛋氨酸为其最合适的底物.为了确定NAOase拆分N-乙酰-D,L-蛋氨酸合适的转化条件,考察了反应温度、pH值、Co2+浓度、转化时间、底物浓度和加酶量对产物的影响.结果表明,合适的反应条件为37℃,pH值7.0,Co2+ 1 mmol/L,转化时间20 min,底物浓度150 mmol/L,菌泥5 g/L.在上述反应条件下,N-乙酰-D,L-蛋氨酸的转化率可达81.4%.     

蛋氨酸开发利用前景广阔

蛋氨酸 (Methionine)又名甲硫氨酸、甲硫基丁氨酸 ,化学名称为 2 -氨基 - 4-甲硫基丁酸 ,是唯一含硫醚的氨基酸。外观为白色片状结晶或粉末 ,有特殊气味 ,味微甜 ,无旋光性。熔点 2 81℃ (分解 ) ,相对密度 (d4 2 0 ) 1 .340 ,溶于水、稀酸和稀碱 ,微溶于醇 ,不溶于醚。 1 %蛋氨酸水溶液的 p H值为 5.6- 6.1 ,对热和空气稳定 ,对强酸不稳定 ,可导致脱甲基作用。蛋氨酸的化学形式有 L-蛋氨酸、D-蛋氨酸、DL-蛋氨酸、蛋氨酸羟基类似物及其它类似物 N-丙酰蛋氨酸、N-丁酰蛋氨酸、N-棕酰蛋氨酸等。蛋氨酸羟基类似物包括羟基蛋氨酸 MHA、羟…     

Nonlinear model-based dissolved oxygen control in a biological wastewater treatment process

The dissolved oxygen (DO) concentration has been an important process parameter in the biological wastewater treatment process (WWTP). In this paper, we propose a nonlinear control scheme to maintain the dissolved oxygen level of an activated sludge system. Without any linearization or model reduction, it can directly incorporate the nonlinear DO process model with on-line estimation of the respiration rate (R) and the oxygen transfer rate (K_La). Simulation results show that it outperforms a control performance of the PID controller. Since it incorporates the process disturbance and nonlinearity in the controller design, the suggested method can efficiently deal with the operating condition changes that occur frequently in the wastewater treatment process.     

E.coli M15 (pQTPL)高效发酵生产酪氨酸酚裂解酶的控制策略

在摇瓶和4 L发酵罐上研究了营养和环境条件对重组菌E. coli M15 (pQTPL)分批发酵生产酪氨酸酚裂解酶(TPL)的影响. 在培养基中添加20 g/L葡萄糖和1.0 g/L玉米浆使TPL酶活提高到63.1 U/g(干重). 在此基础上,维持发酵液中溶氧水平为30%,可使菌体浓度在8 h达到4.78 g/L,酶活为54.6 U/g,比对照组(不控制溶氧)分别提高了21%和31.6%. 采用溶氧反馈调节-限制性补料策略,可使菌体浓度提高到31.5 g/L. 采用两阶段温度和pH控制策略,在发酵前8 h控制pH 7.0、温度37℃,8 h 至发酵结束之间控制pH为8.0、温度为30℃,可使重组菌的TPL酶活达到154.4 U/g,并使TPL在细胞中过量表达,实现了高菌体浓度和高TPL酶活的统一.     

Productivity enhancement of S‐adenosylmethionine in Saccharomyces cerevisiae using n‐hexadecane as oxygen vector

BACKGROUND: Saccharomyces cerevisiae is one of the main microorganisms that can produce S‐adenosylmethionine (SAM) from L‐methionine and ATP with high productivity. To satisfy the ATP requirement for SAM synthesis, sufficient oxygen should be supplied to the medium to improve aerobic metabolism in S. cerevisiae. In this study, n‐hexadecane used as oxygen vector for enhancement of SAM production by this yeast was investigated. RESULTS: N‐hexadecane was most favorable for cell growth and SAM synthesis in S. cerevisiae when added at the time of inoculation. It could increase glucose consumption, reduce ethanol accumulation, and ultimately improve biomass and SAM productivity in a fermentation process. In a bioreactor, the highest yield of SAM (2.27 g L^?1) was achieved in the presence of 4% (v/v) n‐hexadecane after 24 h of inoculation, which was 23.37% higher than the control (1.84 g L^?1). CONCLUSION: The addition of n‐hexadecane to cultures of S. cerevisiae significantly enhanced SAM production without increasing energy consumption, and has the potential for use in large‐scale fermentation processes to increase oxygen supply. Copyright © 2012 Society of Chemical Industry     

A transient mathematical model for maximum respiration activity and oxygen diffusion coefficient estimation in non-steady-state biofilms

A transient mathematical model was established in order to evaluate oxygen diffusivity in non-steady-state biofilms. A submerged fixed bed biofilm system with efficient medium recirculation was investigated for p-toluenesulphonic acid degradation by Comamonas testosteroni T-2 in a multi-species biofilm. Static mixer elements (Sulzer Chemtech Ltd, Switzerland) were used as a support matrix for biofilm formation. Biofilm respiration was estimated using the dynamic gassing-out oxygen uptake method. Based on the dissolved oxygen concentration profiles, the oxygen diffusion coefficient and the maximum respiration activity were calculated. The values of the dissolved oxygen diffusion coefficient varied with biolfilm development and values reported here (2×10⁻¹⁰–1.2×10⁻⁹ m² s⁻¹) are in good agreement with literature data. Calculated oxygen consumption rates fit well with values obtained in respirometry tests with washed out biofilms. The knowledge of diffusivity changes in biofilms is particularly important for removal capacity estimation and appropriate reactor design. © 1998 Society of Chemical Industry     

应用溶氧反馈控制高密度培养重组大肠杆菌过程中乙酸的产生

在培养重组大肠杆菌的过程中,乙酸的产生会抑制细菌的生长,也不利于重组蛋白的表达.研究了补料培养重组大肠杆菌过程中,应用脉冲补料方式时溶氧信号的响应特征来辩识是否产生乙酸.当大肠杆菌以0.1 h-1的比生长速率生长时,补料过程中发酵液的乙酸含量很低,溶氧pO2的响应信号随脉冲补料而振荡.而在诱导表达重组人表皮生长因子时,大肠杆菌的呼吸能力下降,过量补入的葡萄糖使重组菌产生大量的乙酸,同时pO2的响应信号失去振荡的特征.根据pO2响应信号的特征反馈控制葡萄糖的流加速率,有效地避免了重组蛋白表达过程中乙酸的产生,实现了重组大肠杆菌的高密度培养,菌体干重达到48 g·L-1,重组人表皮生长因子的表达水平提高了45%.     

溶氧对Candida glycerinogenes产甘油发酵代谢流分布的影响

分析了耐高渗高产甘油工业菌株产甘油假丝酵母(Candida glycerinogenes)的摇瓶发酵过程,发现溶氧水平是影响C.glycerinogenes 生长、代谢产物生成的重要因素。在此基础上,以5 L发酵罐研究了不同恒溶氧水平甘油的合成过程,通过代谢流分析表明,低溶氧环境下乙醇碳流量增加,导致糖耗快而甘油合成相对较少;碳流分布表明HMP途径在甘油过量合成中起着重要的作用。分析C.glycerinogenes发酵过程中能量代谢和物质代谢（氧化磷酸化和底物水平磷酸化）的变化发现高溶氧环境下ATP合成水平高,一定程度抑制底物水平磷酸化从而使碳流更多地流向了甘油合成;C.glycerinogenes胞内氧化还原平衡(NADH/NAD+比率)的研究进一步解释了产甘油假丝酵母中甘油合成和乙醇生成的竞争关系。     

LIQUID-FILM TIME CONSTANT ASSESSMENT FOR kLa MEASUREMENTS IN RESPIRING FERMENTATION BROTHS

To determine the liquid-film time constant (τ_F) of the dissolved oxygen electrode for k_La measurements with the dynamic method in respiring fermentation broths, an approach using the steady-state readings of the electrode in the broths containing respiring and non-respiring cells, respectively, has been established. The results showed that τ_F in microbial suspensions with high volumetric oxygen uptake rates could be very large under the conditions of low aeration and agitation, indicating the slower response of the oxygen electrode caused by the more difficult penetration of oxygen molecules through the thicker films with active cell respiration. The significance and adequacy of the τ_F corrections for k_La measurements were evaluated experimentally.     

Fermentation characteristics of a low‐oxygen inducible hmp promoter system in Bacillus subtilis LAB1886

In an attempt to find a low‐cost promoter system which could be used for producing recombinant proteins, a low oxygen inducible hmp promoter system in Bacillus subtilis (LAB1886) was characterized by studying the effects of cell density (OD₆₀₀), glucose concentration, pH, dissolved oxygen (DO) level, nitrate and nitrite concentration on cell growth and β‐galactosidase expression. The optimal cultivation strategy was found to be to grow the cells in the presence of nitrate to OD₆₀₀ = 0.2 at a high DO level (80%), and then to induce the hmp promoter by lowering the DO level to 1–2%. As a result, the specific β‐galactosidase activity increased continuously to the maximal value of 1750 Miller Units. Nitrite, as the apparent inducer of the hmp promoter, was produced from nitrate reduction, and had profound effects on cell growth and β‐galactosidase expression. Copyright © 2006 Society of Chemical Industry     

重组戊型肝炎疫苗抗原工程菌发酵工艺的优化

目的优化重组戊型肝炎疫苗抗原工程菌的发酵工艺,为其规模化生产奠定基础。方法以目的蛋白的表达量及菌体浓度作为综合评价指标,对重组戊型肝炎疫苗抗原工程菌的摇瓶发酵温度、初始pH值、溶解氧、IPTG浓度及诱导时间进行优化;以摇瓶发酵结果为基础,进一步对发酵罐培养工艺中诱导时机、诱导时间及补料培养基配方进行优化;以确定的罐发酵工艺条件连续发酵5批,验证该发酵工艺。结果最适摇瓶发酵工艺条件为:发酵温度为37℃,初始pH值为7.0,高溶氧,IPTG诱导浓度为0.05 mmol/L,诱导时间为4 h;最适罐发酵工艺条件为:菌体A600值约为40时开始诱导,诱导时间为4 h,补料培养基配方为葡萄糖25%,MgSO4.7H2O 0.5%。按照确定的工艺连续发酵5批,最终菌体A600均达50.0以上,目的蛋白的表达量均高于15%。结论优化的重组戊型肝炎疫苗抗原工程菌的发酵工艺稳定性良好,已达到中试生产规模的要求。