巨大芽孢杆菌（Bacillus megaterium）突变株Bn,B5的生物学特性及在Vc二步发酵中的伴生作用

对选出的巨大芽孢杆菌突变株Bn,B5进行了生物学特性及发酵条件的研究,发现它们具耐低pH和抗高浓2KGA特性.可促进氧化葡萄糖酸杆菌生长,使其延迟期缩短,产酸增加.适宜的通气量下,摇瓶糖酸转化率提高10%～14%;当发酵pH为6.2～6.6时,转化率提高20%～30%.     

VC二步发酵产酸菌氧化葡萄糖酸杆菌的选育

实验通过紫外线两轮诱变的方法诱变选育氧化葡萄糖酸杆菌（Gluconobacter oxydans）,以实现提高2-酮基-L-古龙酸（2-KLG）产量的目的,获得1株高产2-KLG的菌株G5。结果证明该突变菌株在pH6．5—6．7的发酵培养基中与蜡质芽孢杆菌（Bcillus cereus）混合发酵,G5的平均糖酸转化率提高了13．49％,酸量达到83．6mg／mL,发酵周期缩短了2—3h。经连续10代转接发酵实验,证明其产酸稳定性较好。结论：氧化葡萄糖酸杆菌（Gluconobacter oxydans）的突变体G5提高了糖酸转化率,缩短了发酵周期。     

丁酸梭杆菌发酵甘油制备1,3-丙二醇的研究

本研究采用丁酸梭芽孢杆菌(Clostridium butyricum)从甘油发酵制备1,3—丙二醇。该发酵需厌氧培养,发酵培养基为：甘油6％,葡萄糖1％,玉米浆2％,(NH4)2SO40．2％。发酵温度为34℃,pH为6．5～7。在最佳条件下,发酵50h可产1,3—丙二醇40．7g／L,甘油摩尔转化率达68％。     

芽孢乳杆菌发酵葡萄糖制备D(-)-乳酸的研究

在国内首次报道了发酵法制备D(-)-乳酸,采用芽孢乳杆菌(Sporolactobacillus sp．)从葡萄糖发酵制备D-乳酸。该发酵为微需氧,发酵培养基组成(g/L):葡萄糖60,酵母膏6,玉米浆5,NH4NO3l,麸皮2,NaH2PO4 4,CaCO340。发酵液的初始pH为7．0.在最佳条件下,37℃发酵72h可产40．7g/L的D-乳酸,葡萄糖转化率67．83％。通过离子配位色谱分析,产物的D-乳酸光学纯度为96．04％e．e．。     

酪酸梭状芽孢杆菌低成本培养基的优化

本文重点在于提高酪酸梭状芽孢杆菌活菌数量,降低发酵培养基成本。通过对发酵培养基中不同碳源、氮源、生长因子等进行单因素研究,得到最佳培养基组成：可溶性淀粉10/L,豆粕（中性蛋白酶水解3h）20g／L,玉米浆3g／L。用此培养基在37℃培养24h,采用高层半固体琼脂试管法对酪酸梭状芽孢杆菌进行活菌计数,活菌数可达8．2×10^8cfu／mL.培养基中添加K2HPO45g／L、MgSO4·7H2O0．2g／L、MnSO3·H2O0．2g/L培养32h时,酪酸梭状芽孢杆菌芽孢转化率可达95％。     

β-葡聚糖酶高产菌株BS9418F的选育及其发酵条件的研究

经60 Coγ射线辐照处理获得的诱变菌株芽孢杆菌BS9418F ,其产酶活力比出发菌株提高 30 %以上。该菌株以大麦粉 7%、玉米粉 3%、豆粕 3%及适量无机盐为培养基最佳配比 ,其最适培养条件为 :培养基初始 pH 7.0 ,摇瓶装量 5 0mL/ 30 0mL三角瓶 ,种龄 16～ 2 0h ,接种量 2 %～ 3% ,培养温度 36～ 37℃ ,发酵周期 40h。在优化条件下 ,摇瓶发酵产 β 葡聚糖酶活力高达 5 5 0 0u/mL以上 ,比出发菌株初始发酵水平提高了 4倍以上     

响应面分析法优化耐高温蛋白酶发酵培养基

采用响应面分析方法对产耐高温蛋白酶的苏云金芽孢杆菌(Bacillus thuringien- sis)FZ62发酵培养基进行优化．首先,进行发酵培养基碳源、氮源及初始pH值的单因素筛选,优化结果表明最适氮源为酵母粉、碳源是葡萄糖,初始pH值范围6-8．在此基础经响应面法优化,发酵培养基最佳组合为:酵母粉2．04%,葡萄糖为0．10%,初始pH7．07．经以上优化后发酵水平比初始设计提高了3．22倍．     

生防菌Ⅲ-61抗真菌活性产物的摇瓶发酵

对生防链霉菌Ⅲ-61产生抗真菌活性物质的摇瓶发酵工艺进行了研究。利用正交试验设计优化了发酵培养基组分,其最适配方为黄豆粉1．5％,蛋白胨0．3％,蔗糖1．0％,淀粉1．3％,磷酸二氢钾0．02％,硫酸镁0．025％,氯化钠0．5％,配咸水溶液,调pH至7～7．4,加碳酸钙1％。通过单因素试验,筛选获得了最优培养条件组合：液体种龄24h,接种量5％～10％,500mL摇瓶培养基装量为80mL,摇床转速240r／min,培养温度31℃,发酵周期96～120h。此优化的发酵培养基与发酵条件的组合昕得菌株Ⅲ-61发酵液对主要靶标黄瓜灰霉病菌的抑菌圈直径达49．5mm,较优化前提高了45．59％。     

苏云金杆菌33菌株最佳培养基和发酵条件研究

应用正交试验L27(313)对鳞翅目昆虫高毒效的苏云金杆菌33菌株的发酵培养基进行研究,得到了适合其发酵培养的优化复合培养基配方—0.5％玉米粉 2．00％黄豆粉 0．15％酵母粉 0．25％鱼粉 0．075％蛋白胨 0．25％磷酸二氢钾 0．05％碳酸钙 0．035％硫酸镁。另外实验还证实适当增加通气量、发酵初始酸碱度控制在pH7．5、发酵温度30℃、转速180r／min、培养40h更适合该菌株液体深层发酵。     

一株凝结芽孢杆菌产生糖脂的工艺研究

对分离出的一株凝结芽孢杆菌生产糖脂的摇瓶发酵工艺进行了优化和 1 0L罐发酵实验。适宜发酵条件为 :培养基由 6 %豆油、 3 5g/LNaNO3、 0 75g/L酵母膏以及一定量的无机盐组成 ,发酵温度 3 0℃ ,初始pH8 5 ,搅拌转数 1 5 0～ 2 4 0r/min ,发酵周期 96h。糖脂产量达到 7 0 73g/L。     

蛭弧菌J26转化山梨糖生产2—酮基—L—古龙酸发酵条件的研究

对新分离得到的能产生维生素C前体2-酮基-L-古龙酸（2-KGA）的优良菌株蛭弧菌J26,进行了一系列摇瓶发酵条件试验,通过添加一些其它有机碳源或其它氮源,可使蛭弧菌J26摇瓶发酵产生2－KGA由50-60mg／ml上升到70mg／ml以上,采用SMA探针技术的流加发酵摇瓶试验产生2－KGA可以达到83．9－91．7mg／ml。本文还对发酵时种子液的接种量、发酵初始精浓度、初始pH值等对产酸的影响,进行了比较研究,对摇瓶发酵全过程的单一菌体形态特征进行了电子显微镜观察。     

Selective, high conversion of D-glucose to 5-keto-D-gluoconate by Gluconobacter suboxydans

Selective, high-yield production of 5-keto-D-gluconate (5KGA) from D-glucose by Gluconobacter was achieved without genetic modification. 5KGA production by Gluconobacter suffers byproduct formation of 2-keto-D-gluconate (2KGA). By controlling the medium pH strictly in a range of pH 3.5-4.0, 5KGA was accumulated with 87% conversion yield from D-glucose. The pH dependency of 5KGA formation appeared to be related to that of gluconate oxidizing activity.     

以掷孢酵母作为伴生菌产生VC前体KGA的研究

以掷孢酵母作为伴生菌与氧化葡萄糖酸杆菌组成新混菌体系,通过测定生长代谢曲线,对其产酸性能和特点进行了研究。结果表明：相同条件下,新菌系产酸能力高于现有菌系,酸量增加5-7mg/ml,发酵周期缩短6-8h,酸转化率提高3-4%,最高产酸点pH值下降约0.5个单位,表现出较大的产酸潜力和可修饰性。     

Selective,High Conversion of D-Glucose to 5-Keto-D-gluoconate by Gluconobacter suboxydans

Selective, high-yield production of 5-keto-D-gluconate (5KGA) from D-glucose by Gluconobacter was achieved without genetic modification. 5KGA production by Gluconobacter suffers byproduct formation of 2-keto-D-gluconate (2KGA). By controlling the medium pH strictly in a range of pH 3.5–4.0, 5KGA was accumulated with 87% conversion yield from D-glucose. The pH dependency of 5KGA formation appeared to be related to that of gluconate oxidizing activity.     

Membrane-bound, 2-keto-D-gluconate-yielding D-gluconate dehydrogenase from "Gluconobacter dioxyacetonicus" IFO 3271: molecular properties and gene disruption

Most Gluconobacter species produce and accumulate 2-keto-d-gluconate (2KGA) and 5KGA simultaneously from d-glucose via GA in culture medium. 2KGA is produced by membrane-bound flavin adenine dinucleotide-containing GA 2-dehydrogenase (FAD-GADH). FAD-GADH was purified from "Gluconobacter dioxyacetonicus" IFO 3271, and N-terminal sequences of the three subunits were analyzed. PCR primers were designed from the N-terminal sequences, and part of the FAD-GADH genes was cloned as a PCR product. Using this PCR product, gene fragments containing whole FAD-GADH genes were obtained, and finally the nucleotide sequence of 9,696 bp was determined. The cloned sequence had three open reading frames (ORFs), gndS, gndL, and gndC, corresponding to small, large, and cytochrome c subunits of FAD-GADH, respectively. Seven other ORFs were also found, one of which showed identity to glucono-delta-lactonase, which might be involved directly in 2KGA production. Three mutant strains defective in either gndL or sldA (the gene responsible for 5KGA production) or both were constructed. Ferricyanide-reductase activity with GA in the membrane fraction of the gndL-defective strain decreased by about 60% of that of the wild-type strain, while in the sldA-defective strain, activity with GA did not decrease and activities with glycerol, d-arabitol, and d-sorbitol disappeared. Unexpectedly, the strain defective in both gndL and sldA (double mutant) still showed activity with GA. Moreover, 2KGA production was still observed in gndL and double mutant strains. 5KGA production was not observed at all in sldA and double mutant strains. Thus, it seems that "G. dioxyacetonicus" IFO 3271 has another membrane-bound enzyme that reacts with GA, producing 2KGA.     

Production of 5-keto-d-gluconate by acetic acid bacteria is catalyzed by pyrroloquinoline quinone (PQQ)-dependent membrane-bound d-gluconate dehydrogenase

Gluconobacter suboxydans IFO 12528 was selected as the best strain for 5-keto-d-gluconate (5KGA) production by oxidative fermentation. 5KGA was markedly accumulated by the strain during cultivation in a medium containing d-glucose and/or d-gluconate. The resting cells and the membrane fraction also catalyzed 5KGA formation with a minimal formation of 2-keto-d-gluconate (2KGA), an alternative keto-d-gluconate from d-gluconate. The membrane fraction of the organism was confirmed to contain a membrane-bound d-gluconate dehydrogenase (GADH) catalyzing d-gluconate oxidation to 5KGA of which optimum pH and temperature were found at pH 4 and 15°C, respectively. After treating the membrane fraction with EDTA allowing conversion from holo-GADH to the apoenzyme, 5KGA-forming GADH was confirmed to be a pyrroloquinoline quinone (PQQ)-dependent enzyme by the fact that the enzyme activity was restored by the addition of CaCl₂ and PQQ. The 5KGA-forming GADH was totally distinct from 2KGA-forming GADH in which a covalently bound FAD functions as coenzyme. 5KGA-forming GADH was well solubilized from the membrane fraction with n-octyl-β-d-thioglucoside and 5KGA formation was favourably catalyzed at relatively lower temperature, while 2KGA-forming enzyme was solubilized with Triton X-100 and relatively higher temperatures was optimum for 2KGA formation. These results are completely discrepant from the conclusion proposed by Klasen et al. [R. Klasen, S. Bringer-Mayer, H. Sahm, J. Bacteriol., 177, 1995, 2637] claiming that 5KGA was produced by d-gluconate oxidation catalyzed by NADP-dependent cytoplasmic 5KGA reductase from Gluconobacter species at fairly alkaline pH such as 10.     

Synthesis of α-ketoglutaric acid by Yarrowia lipolytica yeast grown on ethanol

The ability of yeast to synthesize α-ketoglutaric acid (KGA) from ethanol has been studied. Thiamine-auxotrophic yeasts of different genera and species may be able to produce KGA; the main condition of synthesis is growth limitation by thiamine. Using a model culture, mutant Yarrowia lipolytica N 1, the principal conditions affecting KGA oversynthesis were identified. These were: thiamine concentration in medium and in cells, nitrogen and oxygen concentration in medium, and pH level. A KGA concentration of 49 g/l and a yield from ethanol consumed of 42% were achieved. Based on the results of the analysis of the activities of the key enzymes participating in ethanol metabolism and KGA synthesis, a concept of the mechanism of KGA biosynthesis by Y. lipolytica yeast is suggested and discussed. Received: 1 March 1999 / Received revision: 28 June 1999 / Accepted: 5 June 1999     

Efficient callus formation and plant regeneration of goosegrass [<Emphasis Type="Italic">Eleusine indica </Emphasis>(L.) Gaertn.]

Efficient methods in totipotent callus formation, cell suspension culture establishment and whole-plant regeneration have been developed for the goosegrass [ Eleusine indica (L.) Gaertn.] and its dinitroaniline-resistant biotypes. The optimum medium for inducing morphogenic calli consisted of N6 basal salts and B5 vitamins supplemented with 1-2 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg l(-1) glycine, 100 mg l(-1) asparagine, 100 mg l(-1) casein hydrolysate, 30 g l(-1) sucrose and 0.6% agar, pH 5.7. The presence of organogenic and embryogenic structures in these calli was histologically documented. Cell suspension cultures derived from young calli were established in a liquid medium with the same composition. Morphogenic structures of direct shoots and somatic embryos were grown into rooted plantlets on medium containing MS basal salts, B5 vitamins, 1 mg l(-1) kinetin (Kn) and 0.1 mg l(-1) indole-3-acetic acid (IAA), 3% sucrose, 0.6% agar, pH 5.7. Calli derived from the R-biotype of E. indica possessed a high resistance to trifluralin (dinitroaniline herbicide) and cross-resistance to a structurally non-related herbicide, amiprophosmethyl (phosphorothioamidate herbicide), as did the original resistant plants. Embryogenic cell suspension culture was a better source of E. indica protoplasts than callus or mesophyll tissue. The enzyme solution containing 1.5% cellulase Onozuka R-10, 0.5% driselase, 1% pectolyase Y-23, 0.5% hemicellulase and N(6) mineral salts with an additional 0.2 M KCl and 0.1 M CaCl(2) (pH 5.4-5.5) was used for protoplast isolation. The purified protoplasts were cultivated in KM8p liquid medium supplemented with 2 mg l(-1) 2,4-D and 0.2 mg l(-1) Kn.     

The active transport of 2-keto-D-gluconate in vesicles prepared from Pseudomonas purida.

The transport of 2-keto-D-gluconate (alpha-D-arabino-2-hexulopyranosonic acid; 2KGA) in vesicles prepared from glucose-grown Pseudomonas putida occurs by a saturable process with a Km of 110.0 +/- 2.9 microM and a Vmax. of 0.55 +/- 0.04 nmol X min-1 X (mg of protein)-1. The provision of phenazine methosulphate/ascorbate or L-malate leads to an accumulation of intravescular 2KGA, a decrease in the Km value to 50 +/- 2.1 microM and 35 +/- 2.9 microM respectively and no change in the Vmax. In the presence of electron donors the transport of 2KGA is inhibited by the respiratory poisons antimycin A, rotenone and the uncoupler 2,4-dinitrophenol. 2KGA transport is also competitively inhibited by 4-deoxy-4-fluoro-2-keto- or 3-deoxy-3-fluoro-2-keto-D-gluconate with Ki values of 50 microM and 160 microM respectively. The carrier system for 2KGA is repressed in vesicles from cells grown on succinate. Such vesicles transport 2KGA by non-specific physical diffusion with a Km value of infinity in the absence or presence of electron donors. Vesicles from glucose or succinate grown cells, in the presence of phenazine methosulphate/ascorbate at pH 6.6, generate a proton-motive force (delta p) of approx. 140 mV. The delta p, composed of proton gradient (delta pH) and a membrane potential (delta psi), is collapsed in the presence of dinitrophenol. Based on the results obtained with valinomycin, nigericin and carbonyl cyanide m-chlorophenylhydrazone, the active transport of 2KGA at pH 6.6 is coupled predominately to the delta pH component of delta p.