生黑葡萄糖酸杆菌转化高浓度山梨醇的条件优化

目前,国内维生素C主要采用二步发酵法生产,其中第一步为生黑葡萄酸杆菌(Gluconobacter melano-genus)将D-山梨醇转化为L-山梨糖。考察了该菌株在提高培养基中山梨醇浓度时的发酵特性和发酵条件。实验室摇瓶实验结果显示,通风量、发酵前期及后期pH值控制、接种种液类型都影响高浓度山梨醇摇瓶发酵的转化率。以35%山梨醇浓度发酵液做种子液明显优于生产上采用的三级种子液(12%~17%山梨醇浓度),培养基前期pH值5.0~6.0,后期pH值4.2~3.9,装液量180 mL,发酵周期在30 h之内,山梨醇转化率在98%以上。培养基山梨醇浓度由23%提高到35%,发酵周期延长8 h。上述实验结果对指导生产工艺优化具有重要意义。     

生黑醋酸杆菌在高浓度山梨醇下的半连续发酵特性

目的：调节生黑醋酸杆菌生物和代谢特性,以提高发酵效率。方法：通过改变种液特性,采用半连续培养的方式,对生黑醋酸杆菌在高醇浓度下的生长特性进行了研究。结果：通过优化可以提高VC一步发酵底物山梨醇浓度达38%,32h左右发酵率达95%,山梨糖产量达360mg/ml,半连续培养连续5批之间产糖稳定,没有明显差别。结论：通过优化,有效地提高了山梨糖的产率。     

生黑醋酸杆菌山梨醇高浓度发酵的培养特性

调节生黑醋酸杆菌的代谢特性,实现其在高浓度山梨醇下的发酵.改变发酵所用种液的培养特性以及在发酵中期补加山梨醇.在摇瓶培养条件下,通过上述方法可以将Vc一步发酵中底物山梨醇的浓度从23%提高到38%,培养30 h时发酵率达95%,山梨糖产量达360 mg/mL.在优化后的实验条件下山梨糖的发酵产率得到明显提高.     

1株产多糖芽胞杆菌的分子鉴定和诱变选育

以中国农业科学院北京畜牧兽医研究所鸡舍附近土壤为材料,采用稀释平板法对其中的菌株进行分离与纯化培养,并对其进行形态学鉴定和粗多糖提取量的检测,同时对分离得到的菌株进行紫外线和亚硝基胍的诱变。从土壤中分离得到1株产多糖的芽胞杆菌(编号P-30),结合形态学鉴定、16S rRNA序列分析和系统发育树分析结果,确定该菌株为短短芽胞杆菌(Bacillus brevis)。其16S rRNA GenBank登录号为HM185814。经过诱变选育后,获得3株(N-05、N-11、U-01)多糖产量为P-30的1.44、1.44和1.29倍的诱变菌株,具有良好的开发前景。     

L-精氨酸高产菌株的选育

以谷氨酸高产菌种LH谷氨酸棒杆菌为出发菌株,用化学试剂亚硝基胍(NTG)诱变,经结构类似物磺胺胍和摇瓶产酸筛选,获得一株产L-精氨酸的菌株LH425,在摇瓶发酵中,培养96h,产酸率38g·L-1。     

高产磷酯酶C菌株的诱变选育

在前面进行的高产磷酯酶C(PLC)菌株筛选、分类鉴定及其抗血小板功能研究的基础上,对选育出的高产PLC菌株BacilluscereusShenZhen7541进行了紫外线和Co60γ射线诱变处理,以期提高其产PLC的水平,从而有利于PLC的纯化制备。B.cereus7541经过三次紫外线照射及两次Co60γ射线辐射诱变处理,通过分离与卵黄琼脂杯碟法及NPPC法酶活检验筛选,最终获得了三株高产PLC突变菌株9287、9289和5612,其产PLC酶活水平分别达到14.878±1.428u/mL、16.450±0.793u/mL、16.400±0.967u/mL,较原始出发菌株B.cereus7541产酶水平(5.803±0.793u/mL)分别提高了2.879、3.236和3.226倍。经t值检验,三株菌产PLC水平与7541差异均达极显著(P<0.001)。     

植物乳杆菌荚膜缺陷型菌株的诱变选育

以自主分离和鉴定的产荚膜多糖植物乳杆菌C88为出发菌株,采用亚硝基胍诱变、墨汁负染和显微镜观察筛选获得一株荚膜缺陷型突变株,命名为植物乳杆菌C88M3,经多次传代突变菌株具有良好的遗传稳定性。通过16SrDNA序列分析、菌株生长曲线和RAPD分析比较了野生型菌株和荚膜缺陷型菌株在遗传特性和产荚膜情况方面的差异。通过化学诱变方法获得了乳杆菌荚膜缺陷型菌株,对进一步研究荚膜多糖在乳杆菌益生性中的功能和作用机制具有重要意义。     

枯草芽孢杆菌B-903菌株的诱变选育

枯草芽孢杆菌B-903菌株是由河南省农业科学院植物保护研究所从郑州果园中分离得到,其代谢产生的抗菌物质对多种植物病原真菌具有较强抑制作用。以此菌株为出发菌株,进行亚硝基胍（NTG）和微波诱变处理,确定了,二者诱变处理的最佳处理剂量：NTG最佳处理浓度为200μg／mL,微波诱变为HI微波挡（850W、脉冲频率2450MHz）处理100s,筛选出13个高效突变株。经传代实验,2株高效突变株N1和W2抑菌圈直径分别稳定在26mm和24mm以上,比出发菌株提高21．8％和14．8％．     

降胆固醇嗜酸乳杆菌的诱变选育

通过诱变获得具有降胆固醇功能的优良嗜酸乳杆菌新菌株。利用亚硝基胍(作用浓度为1 g/L)对嗜酸乳杆菌进行诱变。突变菌株测定其耐渗透压和抗胆盐能力后,在含有0.3 g/L胆固醇的培养基中培养48 h,测定降胆固醇率。挑选优良突变菌株制成酸奶并喂养高脂大鼠模型,28 d后测定血清及粪便胆固醇指标。嗜酸乳杆菌突变后获得的60个突变菌株中有8株具有良好的耐渗透压和抗胆盐能力,其中突变株Y48的清除胆固醇能力最高,清除率达到(61.44±1.8)%。Y48发酵酸奶喂养高脂大鼠模型28 d后与对照大鼠模型相比,血清中TC、TG明显减少(P<0.05),粪便TC明显增加。通过诱变获得了优良的嗜酸乳杆菌突变菌株,为今后获得优质降胆固醇乳酸制品提供良好的候选菌种。     

Vc二步发酵一株新产酸菌的分子生物学特性

对最近分离到的一株能合成维生素C前体 - 2 -酮基 -L -古龙酸 (2 -KGA)的新产酸菌V6生物学和分子生物学特性进行了初步研究。该菌株为革兰氏阴性菌 ,细胞为短杆状 ,菌体大小为 0 .8- 1.0× 0 .4 - 0 .6 μm ,菌落为淡黄色 ,好氧 ,最适生长温度为 2 8～ 30℃ ,最适pH为 7.0～ 7.8,GCmol%含量为 5 3.1% ,不含质粒 ,能氧化葡萄糖、山梨醇和山梨糖合成 2 -KGA。 16SrDNA同源性分析发现 ,该产酸菌与以前报道的能合成 2 -KGA的三个属Ketogulonigenium属、Gluconobacter属和Acetobacter属的同源性分别是 98.9～ 99.3%、82～ 83%和 81～ 82 %。基于以上特性分析 ,该产酸菌在分类发育学上宜归为Ketogulonigenium属。     

维生素C二步发酵中离子注入诱变巨大芽孢杆菌的生物学效应

巨大芽孢杆菌BM279是经低能N 离子注入诱变原始菌株BM80而得到的维生素C高转化率伴生菌株。通过对离子注入前后出发菌和突变株的生理、生化等生物学特点比较,探讨了离子注入巨大芽孢杆菌对2-酮基-L-古龙酸(2KGA)高转化率的促进机理。离子注入对巨大芽孢杆菌自身的生长无明显影响,BM279呈现出与BM80基本一致的生长曲线;但BM279对混菌发酵体系中产酸菌GO29的细胞增殖有显著促进作用。BM279在混菌发酵过程中分泌较多的碱性物,有利于维持GO29生长、代谢的pH环境。BM279培养42 h,其胞外活性物质对GO29的糖酸转化活力较BM80有显著提高,且分泌时间较BM80推迟6 h。利用层析技术分别从BM80、BM279胞外液中纯化了L-山梨糖脱氢酶(L-sorbose dehydrogenase,SDH)激活蛋白(SSPBM80和SSPBM279),后者比活较前者高出50%,对GO29中的SDH酶活有更强促进作用。     

葡萄糖补加对VC二步发酵产古龙酸的影响

研究了葡萄糖的补加对维生素C二步发酵产酸的影响.摇瓶发酵实验结果表明,15%的接种量,接种至底物山梨糖浓度为8%的发酵培养基,发酵24h时,补加0.08%的葡萄糖,可提高发酵转化率5.2%.     

经太空诱变的莲藕的组织培养

1植物名称莲藕(Nelumbo nucifera)广昌太空莲3号,由江西省广昌白莲科学研究所提供. 2材料类别处于休眠期前期(10～12月)根状茎的项芽.     

Vitamin B12 Production by a Methanol-Utilizing Bacterium

Vitamin B(12) production by a newly isolated strain of a methanol-utilizing bacterium was studied. The maximal yield of the vitamin, 2.6 mg/liter of medium was attained by optimization.     

Galactonolactone Dehydrogenase Requires a Redox-Sensitive Thiol for Optimal Production of Vitamin C

The mitochondrial flavoenzyme l-galactono-γ-lactone dehydrogenase (GALDH) catalyzes the ultimate step of vitamin C biosynthesis in plants. We found that recombinant GALDH from Arabidopsis (Arabidopsis thaliana) is inactivated by hydrogen peroxide due to selective oxidation of cysteine (Cys)-340, located in the cap domain. Electrospray ionization mass spectrometry revealed that the partial reversible oxidative modification of Cys-340 involves the sequential formation of sulfenic, sulfinic, and sulfonic acid states. S-Glutathionylation of the sulfenic acid switches off GALDH activity and protects the enzyme against oxidative damage in vitro. C340A and C340S GALDH variants are insensitive toward thiol oxidation, but exhibit a poor affinity for l-galactono-1,4-lactone. Cys-340 is buried beneath the protein surface and its estimated pK_a of 6.5 suggests the involvement of the thiolate anion in substrate recognition. The indispensability of a redox-sensitive thiol provides a rationale why GALDH was designed as a dehydrogenase and not, like related aldonolactone oxidoreductases, as an oxidase.l-Ascorbate (vitamin C) is the most consumed vitamin on earth. It is a multifunctional antioxidant that is particularly abundant in plants where it can reach millimolar concentrations, representing over 10% of the soluble carbohydrate content. l-Ascorbate is a cofactor for a number of enzymes and it is a major constituent of the intracellular redox buffer. Its main function in plants is to scavenge reducing equivalents originating from respiration and photosynthetic activity, protecting proteins, unsaturated fatty acids, and DNA from irreversible oxidative damage (Smirnoff and Wheeler, 2000).The terminal step of l-ascorbate biosynthesis in plants is catalyzed by the mitochondrial flavoenzyme l-galactono-γ-lactone dehydrogenase (GALDH; l-galactono-1,4-lactone:ferricytochrome c oxidoreductase; EC 1.3.2.3). GALDH mediates the two-electron oxidation of l-galactono-1,4-lactone into l-ascorbic acid with the concomitant reduction of cytochrome c (Scheme 1):Open in a separate windowScheme 1.Besides from producing l-ascorbate, the exploitation of the electron transport chain by GALDH is important for the proper functioning of plant mitochondria (Alhagdow et al., 2007). Furthermore, it has been reported that GALDH is required for the correct assembly of respiratory complex I (Pineau et al., 2008).GALDH and other aldonolactone oxidoreductases are two-domain proteins with a conserved vanillyl-alcohol oxidase (VAO)-type FAD domain (Fraaije et al., 1998; Leferink et al., 2008a). Most aldonolactone oxidoreductases are hydrogen peroxide-producing oxidases containing covalently bound FAD, while GALDH reacts poorly with molecular oxygen and contains noncovalently bound FAD (Leferink et al., 2008b). Aldonolactone oxidoreductases have been isolated from various sources, but they are not well characterized. No crystal structure is available, and little is known about the nature of the active site and the catalytic mechanism. Several aldonolactone oxidoreductases, including GALDH from plants (Mapson and Breslow, 1958; Ôba et al., 1995; Østergaard et al., 1997; Imai et al., 1998; Yabuta et al., 2000), l-gulono-1,4-lactone oxidase from animals (Nishikimi, 1979), d-arabinono-1,4-lactone oxidase from yeast (Huh et al., 1994), and trypanosomal aldonolactone oxidoreductases (Logan et al., 2007), are sensitive toward inactivation by thiol-modifying agents. In line with this, we previously found that recombinant GALDH from Arabidopsis (Arabidopsis thaliana) is slowly inactivated during storage and that the activity can be completely restored by treatment with the reducing agent dithiothreitol (DTT; Leferink et al., 2008b).So far nothing is known about the nature of the thiol inactivation, and to our knowledge the effect of oxidants on the activity of aldonolactone oxidoreductases has not been studied before. Here we investigated the susceptibility of GALDH to oxidative stress and identified a critical Cys in the substrate binding site that makes the enzyme vulnerable toward irreversible inactivation.     

Fermentation of glucose by Acetobacter melanogenus.

Growing cultures of Acetobacter melanogenus ATCC 9937 concerted D-glucose to 2,5-diketo-D-gluconic acid with D-gluconic acid and 5-keto-D-gluconic acid as intermediates. The 2,5-diketo-D-gluconic acid was isolated from the fermented medium by treatment with an anion exchange resin.     

空间诱变高粱突变体的研究

1996年经返回式卫星搭载处理的纯系高粱品种晋粮5号（CK）的种子经次年播种后,获得少量矮秆早熟突变体（SP3）。此后连续二年播种,该突变体矮秆早熟性状稳定。该突变体与未经搭载的对照相比,有如下不同之处：（1）成熟期缩短15 d左右,株高降低40 cm。（2）穗长增加3.9 cm ,每穗籽粒数比对照多400粒。（3）SP3平均千粒重为34 g,对照为27 g。（4）SP3叶片变窄,变短,增厚,叶面积减少了43%～22%。（5）SP3穗轴长度比对照增加30%,各节间长度比对照缩短了67%～15%。（6）SP3种子中亮氨酸含量比对照增加15%,可溶性糖含量比对照增加25%,单宁含量降低了30%。