丙酮酸羧化酶对酿酒酵母积累琥珀酸的作用探究

为探究丙酮酸羧化酶及微量元素Ca~(2+)和生物素对酿酒酵母积累琥珀酸的作用,敲除了琥珀酸脱氢酶编码基因(SDH2)并过量表达来自米根霉的丙酮酸羧化酶基因(Ro PYC)。琥珀酸产量由(0. 011±0. 002) g/L提高至(0. 841±0. 020) g/L,较表达前提高了75. 45%。随后,外源添加不同浓度Ca~(2+)和生物素考察其对琥珀酸发酵的影响,结果发现,Ca~(2+)的添加促进了菌体的生长但不利于琥珀酸的积累,而外源添加浓度为16、32、64、96μg/L生物素时,琥珀酸产量分别提高75. 04%、84. 26%、69. 28%、66. 79%。其中生物素质量浓度为32μg/L时,琥珀酸产量达到最大为(0. 964±0. 02) g/L,且丙酮酸羧化酶(pyruvate carboxylase,PC)酶活提高14. 42%,说明PC酶活的提高促进了酿酒酵母中琥珀酸的积累。该研究为解决酿酒酵母琥珀酸合成过程中前体碳代谢流不足问题提供了新的解决思路。     

生物素和VB1在谷氨酸棒杆菌过量合成缬氨酸中的作用

通过对谷氨酸棒杆菌突变株AATV341进行生物素和VB1添加量的实验，探讨了生物素和VB1添加量对该菌株菌体质量及缬氨酸产量的影响；同时根据对AATV341进行的胞内外某些特定氨基酸和有机酸检测，研究了这两种维生素对碳架代谢流的分布，尤其是对丙酮酸节点处的代谢流量分布的影响。结果表明，生物素添加量在40μg／L时，菌体质量和缬氨酸在胞外的累积明显提高，谷氨酸在胞外累积降低；VB1的适量添加，可提高缬氨酸在胞外的累积，降低发酵液中乙酸和草酰乙酸的含量。     

丙酮酸对Bacillomycin D合成的影响及调控

目的:本文主要研究外源添加丙酮酸和过表达丙酮酸激酶（pyk）、乙酰辅酶A合成酶（acs）对Bacillomycin D合成的影响及调控作用。方法:本研究以B.amyloliquefaciens fmbJ为出发菌株,首先通过HPLC检测了外源添加不同浓度的丙酮酸对Bacillomycin D产量的影响,并通过RT-PCR检测了丙酮酸在此过程中的调控作用。然后通过电转化的方法构建了丙酮酸激酶和乙酰辅酶A合成酶的诱导型过表达菌株B.amyloliquefaciens fmbJ-pyk、fmbJ-acs,并研究了二者在不同浓度IPTG诱导下的Bacillomycin D产量。结果:0.075%的丙酮酸可将Bacillomycin D产量提高至对照组的1.41倍,Bacillomycin D合成基因的上调也证实了这一提高作用。此外,丙酮酸能够上调comA、comP、comQ、sigH、sigM、degU、degQ、spo0A、codY等调控因子的表达,下调rapC和abrB的表达,并促进乙酰辅酶A合成酶的表达,最终共同促进Bacillomycin D的合成。在50 mg/L的IPTG的诱导下,fmbJ-acs和fmbJ-pyk的Bacillomycin D产量也分别提高至fmbJ的1.16和1.34倍。结论:本研究发现外源添加丙酮酸以及过表达丙酮酸激酶和乙酰辅酶A合成酶可以促进Bacillomycin D的合成,揭示了丙酮酸在Bacillomycin D合成中的调控作用,为推进Bacillomycin D高效生产提供了新的研究思路。     

乳酸发酵短杆菌中的赖氨酸代谢控制发酵

以本室选育的赖氨酸产生菌AL 0 39为出发菌株 ,以亚硝基胍诱变 ,筛选到一株氟丙酮酸敏感型突变株FP 0 94,其赖氨酸产量比出发菌株提高 37.5 %。进而研究了生物素、醋酸钙对该菌产赖氨酸的影响。     

柠檬酸钠和生物素对白色链霉菌发酵产ε-聚赖氨酸的影响

本文研究了不同生长期在培养基中添加柠檬酸钠和生物素对白色链霉菌生长及产ε-PL的影响,结果表明添加不同浓度柠檬酸钠对菌体生长的影响不明显,但对白色链霉菌ε-PL合成有正向促进作用。0 h添加2 g/L的柠檬酸钠可获得最大的ε-PL产量0.92g/L。随着柠檬酸钠浓度的增加,ε-PL产量先增加后降低。在0 h添加2 g/L柠檬酸钠并在36 h添加300μg/L生物素,发酵72 h后菌体干重和ε-PL产量分别达到了7.86 g/L和1.10 g/L,是空白对照组的1.30倍和1.93倍,说明外源添加柠檬酸钠和生物素对白色链霉菌发酵生产ε-PL有促进作用。     

中间代谢产物对白色链霉菌ε-聚赖氨酸合成的影响

探讨白色链霉菌ε-聚赖氨酸(ε-poly-L-lysine,ε-PL)生物合成过程中,中间代谢产物柠檬酸、L-天冬氨酸和L-赖氨酸对ε-PL合成的影响。单因素实验结果表明,在摇瓶发酵开始(0 h)添加柠檬酸至终质量浓度为1.0g/L,培养到12 h分别添加终质量浓度为0.3 g/L的L-天冬氨酸和终质量浓度为1.0 g/L的L-赖氨酸,可分别提高ε-PL产量42.5%、28.7%和44.1%。正交试验显示,只有柠檬酸和L-赖氨酸对ε-PL合成影响显著(P0.05),而L-天冬氨酸影响不显著;在培养基中添加1.0 g/L的柠檬酸和L-赖氨酸ε-PL产量可提高60.1%。在破碎的无细胞体系中,只有添加L-赖氨酸可以检测到ε-PL的生成,进一步证实了L-赖氨酸是ε-PL合成的直接前体。因此,通过添加合适的中间代谢产物,可以有效提高ε-PL产量。     

生物素对L-缬氨酸发酵的影响

为研究生物素添加量对谷氨酸棒状杆菌（Corynebacterium glutamate）发酵生产L-缬氨酸的影响,以谷氨酸棒状杆菌XV0505（Leu－＋Ile－＋2-TAr＋α-ABr＋SGr）为供试菌株,考察不同生物素添加量条件下菌体量、耗糖、产酸以及副产物L-丙氨酸的情况,确定了生物素最适添加量为50 μg/L;利用膜偶联透析发酵方式有效解除了发酵生产过程中产生的反馈抑制现象,降低了副产物的产量,提高了L-缬氨酸的转化率及产量。与原单批次发酵的工艺相比,新工艺的最终L-缬氨酸总量达到106.1 g/L,产量提高了47.4%,糖酸转化率提高到34.5%。     

乳酸发酵短杆菌中碳架流向赖氨酸的导向与分析

对乳酸发酵短杆菌生物合成赖氨酸的代谢途径及其代谢流量从理论上进行了分析，计算了乳糖发酵短杆菌合成赖氨酸的最大理论转化率。对代谢途径及其遗传调控进行了分析，通过NTG诱变育成了一株带有FP^S、AEG^r、Ma^r和Leu^-4种遗传标记的赖氨酸高产菌株，使赖氨酸的产量提高到54.6g/L，并分析了这些标记对代谢流量改变的影响。     

以甜菜糖蜜为原料进行谷氨酸发酵。生物素本身已过量，为什么有的厂还要配加100μg／L以上的纯生物素？

以甜菜糖蜜为原料进行谷氨酸发酵时,发酵培养基预先配加100μg／L以上的纯生物素,是为了削弱每批原料中生物素含量变化不易掌握的影响,使每批发酵培养基中的生物素都处于生物素已知的大过量状态,再进行人为的强制控制,易于稳产、高产;与此同时,高浓度的生物素还能激活丙酮酸羧化酶,强化CO2固定反应,有利于提高转化率。这样,通过高生物素与大接种量相结合,促进菌体迅速繁殖,而在对数生长早期（5h左右）及时加入相对高一些的生长抑制剂吐温60（约0．2％）,拮抗脂肪酸的生物合成,导致形成磷脂合成不足的不完全的细胞膜．经再度倍增,细胞伸长、膨大、完成谷氨酸非积累型细胞向谷氨酸积累型细胞的转变。     

维生素对大肠杆菌Escherichia coli.JN8产L-色氨酸的影响

研究了9种维生素对大肠杆菌Escherichia coli.JN8生长及其发酵产L-色氨酸产量的影响,并对发酵培养基中的维生素浓度进行了优化。结果表明,低浓度肌醇和叶酸可显著促进大肠杆菌的生长及L-色氨酸的合成,叶酸和肌醇的最适添加量分别为0.000 2 mg/L和0.2 mg/L;较高浓度的生物素可以明显促进菌体生长及色氨酸的合成,其最适添加量为0.12 mg/L;较低浓度VB6的添加严重抑制色氨酸的合成,当其质量浓度增至0.8 mg/L时,菌体浓度和色氨酸的产量均较对照有显著的提高,分别提高11.8%和27.2%。添加VB5可略微提高色氨酸的产量,最优质量浓度为0.4 mg/L。添加VB1、VB2、VB3和VC虽然对菌体的生长均有较强的促进作用,但对于色氨酸合成却有不同程度的抑制作用。     

赖氨酸强化面包的研究

论述了用赖氨酸强化面包时,对面团质量的影响及烘焙过程中赖氨酸的损失情况以及如何合理强化等问题,并进行了初步的研究探讨。     

Ninhydrin-Reactive Lysine in Food Proteins

A lithium acetate-dimethyl sulfoxide ninhydrin reagent was shown to be useful for reaction of amino groups in a series of structurally diverse food proteins and flours of variable lysine content. The proteins were bovine serum albumin, casein, hemoglobin, lactalbumin, lysozyme, soybean protein, soybean trypsin inhibitor, trypsin, and wheat gluten. The flours were barley, high lysine corn, high protein rice, lima bean, nonfat dry milk, rice, soy, and wheat. The ninhydrin-reactive lysine content of these proteins varied from 63-109% of the theoretical values. This variation needs to be taken into account in possible applications of the ninhydrin reaction to food proteins.     

赖氨酸发酵动力学研究——兼流加方式优化

在40L中试发酵罐上用优选菌株进行了L-赖氨酸分批流加发酵,用实验数据对文献报道的分批发酵动力学模型进行了验证。结果发现所有模型都不能很好的描述赖氨酸分批发酵过程动力学。最后探讨了赖氨酸分批发酵的动力学机理,给出了流加优化方案。     

Influence of Soluble Lysine Maillard Reaction Products on Escherichia coli Amino Acid Lysine Auxotroph Growth-Based Assay

ABSTRACT: Lysine Maillard reaction products (LMRP) were generated by autoclaving different concentrations and ratios glucose and lysine for 3 h. Effects of LMRP on the Escherichia coli lysine auxotroph (lys⁻) were examined in model systems. The slope and y-axis intercept of the standard curve in the absence of LMRP were significantly ( p < 0.05) greater than in the presence of LMRP. Our study shows that LMRP can decrease the growth extent of lys⁻. The E. coli available lysine assay may provide the food industry with a practical approach to rapid and accurate estimation of lysine for least cost formulation in foods.     

Chapter 6: Maize with Increased Lysine (Lysine Maize—LY038)

ABSTRACT: Data and information provided in this case study relate to a crop derived by modern biotechnology, in which a specific nutrient (lysine) has been increased in maize grain.Lysine maize is a feed ingredient with enhanced nutritional characteristics for poultry and swine and provides an alternative to adding supplemental lysine to diets for these animals. Lysine maize is in an advanced state of development; therefore, extensive unpublished data and information are presented to demonstrate that (1) Lysine maize,and the feeds and foods derived from it,are as safe as those derived from conventional maize,and (2) the increased lysine in Lysine maize grain produces the intended nutritional benefit for broiler chickens when compared to a diet containing conventional maize grain and a crystalline lysine supplement.These conclusions are based on a detailed molecular characterization of Lysine maize,a safety assessment of the introduced protein,a safety and nutritional assessment of the LY038 crop,and a comparison of the agronomic and phenotypic properties of maize hybrids with and without the Lysine maize trait.Although Lysine maize is a specialty crop for use in animal feed,its safety for both animals and humans must be demonstrated. Free lysine is significantly increased in Lysine maize by the introduction of the dapA gene (cordapA) from Corynebacterium glutamicum that encodes a form of dihydrodipicolinate synthase (cDHDPS) that is insensitive to lysine feedback inhibition.Analysis of lysine anabolic and catabolic pathways in maize identified 6 metabolites that might change as a consequence of the introduction of cDHDPS insensitive to lysine‐feedback inhibition.The results of compositional analysis demonstrated that Lysine maize grain is comparable to conventional maize, with the exception of the intended increase in lysine and a corresponding increase in 2 products of lysine catabolism,saccha‐ropine and ‐aminoadipic acid. Therefore, the safety and/or nutritional implication of these 3 compounds under the conditions of use were the focus of additional assessments and found to not present either a safety or nutritional problem.     

Lysine availability in protein-alginate ester gels

The reduction in available lysine through the interaction of ε-amino groups with propylene glycol alginate to form thermostable gels was investigated using three methods—FDNB, formol titration and dyebinding. Formol titration is preferred for reliability and rapidity. The available lysine content was reduced by at most 18% for disordered proteins (gelatin, caseinate) and from 11 to 18% for structural proteins (whey, soya, egg albumen). The gels are readily attacked by the main digestive proteolytic enzymes, pepsin and trypsin.     

Measurement of Available Lysine Using the Guanidination Reaction

Available lysine with its free ε-amino group can be chemically converted to homoarginine by means of a guanidination reaction using methylisourea (MIU). Various factors that can affect this reaction were investigated. For milk protein optimum conditions were found to be pH 10.8 for 96 hr at 20°C with a MIU concentration of 0.5M. Other protein sources had a different optimum pH for the guandination reaction. Addition of various carbohydrate sources and amounts did not interfere with the quantitation of available lysine.     

赖氨酸螯合铁在母猪日粮中的应用

试验分4组。用赖氨酸螯合铁和硫酸亚铁分别进行饲喂妊娠，哺乳期母猪的试验，并同时用葡聚糖铁给初生仔猪作补铁的试验。结果表明：饲喂赖氨酸螯合铁组的母猪所生仔猪，比对照组母猪所生仔猪断奶成活率高9．2个百分点（P＜0．05），平均断奶重高18．8％（P＜0．01）。比饲喂硫酸亚铁组母猪所生仔猪断奶成活率高3．1个百分点（P＞0．05），平均断奶重高9．9％（P＜0．05）。而与给3日龄仔猪注射葡聚糖铁组相比，成活率和断奶重分别提高0．2个百分点和2．3％（P＞0．05）。     

Investigations on the Guanidination of Lysine in Proteins

The present study was conducted to compare the efficacy of five different reaction conditions on the guanidination of lysine in casein and to establish optimum lysine: O -methylisourea (OMIU) for maximum guanidination of lysine in casein and soya bean meal. The results indicate that the presence of glycine–NaOH buffer is not required for guanidination of proteins at pH 10·5. A OMIU concentration of 0·4 M was found to be as effective as 0·6 M for guanidination. Both OMIU–hydrogen sulphate and free OMIU were equally effective reagents in terms of conversion of lysine to homoarginine. The use of OMIU–hydrogen sulphate for guanidination and the use of ethanol to recover guanidinated protein, however, resulted in the formation of crystalline sodium sulphate, a known purgative agent, in the guanidinated material, and therefore are not recommended if the guanidinated protein is to be used in animal trials. The molar ratio of lysine: OMIU required for efficient lysine conversion to homoarginine varied for different protein sources. Ratios required for maximum conversion for casein and soya bean meal were determined to be 1:10 and 1:16, respectively. A simple procedure developed for the large-scale guanidination (5–10 kg batches) of proteins is also described. The results showed that guanidination of proteins can be easily scaled up from 20 g to 5–10 kg and that large-scale guanidination is feasible and efficient.