枯草杆菌(Bacillus subtilis)蛋白酶突变株蛋白酶和α-淀粉酶合成的关系

用同步辐射、离子注入和紫外辐射对枯草芽孢杆菌(Bacillus subtilis)183l菌株辐射诱变后,筛选到四个稳定的蛋白酶突变株。研究了B．subtilis 183l亲株及其蛋白酶突变株的蛋白海和α-淀粉酶合成情况,分析了蛋白酶和α-淀粉酶的合成关系。结果表明,B． subtilis 183l亲株及其突变株在以豆饼粉、玉米粉、麸皮等组成的发酵培养基中,37℃,120r／min培养条件下,各菌株最高α-淀粉酶活力相差不大;中性蛋白酶与α-淀粉酶的合成可能具有相互促进的作用。     

枯草芽孢杆菌抗药性突变株的筛选

从土样中分离出枯草杆菌,采用梯度平板法,利用链霉素,氯霉素,红霉素,四环素四种抗生素,对自然界中和经过紫外线诱变的菌株进行了抗药性突变株的筛选,我们发现分离出自然状态下的枯草杆菌没有抗药性,而经过紫外线诱变后,能够筛选出对链霉素具有抗药性的菌株.     

枯草杆菌JSIM-1018糖代谢突变株积累D-核糖研究

从收集的Bacillus,Brevibacterum,Corynebacterium,Pseudomonas,Arthrobacter等属菌种中进行D-核糖产生菌的筛选,对其中BacillusSM-18菌株用紫外线和甲基磺酸乙酯诱变,选育到莽草酸营养缺陷型突变株JSIM-1018．发酵产物经物理、化学鉴定为D-核糖．某些有机氮源如酵母粉、玉米浆、牛肉膏、蛋白陈以及某些芳香族氨基酸对突变株积累D-核糖有促进作用。在以葡萄糖为碳源的培养液中,摇瓶发酵D-核糖最高可达929／L,3000L发酵罐中     

酵母海藻糖酶缺失突变株的构建及其耐性

[目的]构建酵母海藻糖酶缺失突变株,并进行耐性分析,进一步研究海藻糖与酵母耐性之间的关系,为商业生产打下一定的基础.[方法]利用同源重组的方法,敲除了编码酸性海藻糖酶的ATH1基因和中性海藻糖酶的NTH1基因,构建了酸性海藻糖酶缺失突变株(△ath1)、中性海藻糖酶缺失突变株(△nth1)和双缺失突变株(△ath1△nth1),并进行了耐性分析.[结果]结合PCR和Southernblot的结果,验证了突变株构建的正确.所有突变株的海藻糖积累量和细胞密度均高于亲本,冷冻、高温、高糖和酒精耐性提高了.[结论]说明海藻糖含量与酵母耐性有一定的相关性.突变株耐性的改善,表明它们在酿造和烘焙产业中具有潜在的商业价值.     

枯草杆菌JSIM—1018糖代谢突变株积累D—核糖研究

从收集的Bacillus,Brevibacterum,Corynebacterium,Pseudomonas,Arthrobacter等属菌种中进行D-核糖产生菌的筛选,对其中BacillusSM-18菌株用紫外线和甲基磺酸乙酯诱变,选育到莽草酸营养缺陷型突变株JSIM-1018．发酵产物经物理、化学鉴定为D-核糖．某些有机氮源如酵母粉、玉米浆、牛肉膏、蛋白陈以及某些芳香族氨基酸对突变株积累D-核糖有促进作用。在以葡萄糖为碳源的培养液中,摇瓶发酵D-核糖最高可达929／L,3000L发酵罐中试D-核糖最高可达81．759／L,平均在64g／L以上,葡萄糖基本耗尽。     

红曲霉单产黄色素突变株的选育

在对红曲色素生产菌选育的过程中 ,得到了 9株在麦汁平板上不产红色素的突变株。对稳定性良好的 8株突变株进行固体发酵实验 ,两个样品呈黄色 ,其它呈白色。发酵样品在可见光谱范围内扫描 ,两黄色样品仅在 3 70nm处有一吸收峰 ,其它白色样品无吸收峰。单产黄色素突变株再经液体发酵实验 ,同样单产黄色素 ,说明这两株菌单产黄色素的性能是稳定的。     

质子自杀法选育克雷伯氏菌产乳酸突变株

以产酸克雷伯氏菌(Klebsiella oxytoca) M5al为出发菌株, 经亚硝基胍诱变处理, 运用质子自杀法选育, 从含0.17 mol/L NaBr-NaBrO3的初筛平板上选出44个具有稳定遗传性的单菌落, 然后结合培养基优化后的摇瓶发酵复筛, 获得3个产乳酸突变株, 其乳酸脱氢酶活性分别为出发菌株的50.6%、58.8%、61.3%。对其中乳酸脱氢酶活性最低的菌株在5 L自动发酵罐上进行批式发酵, 结果显示: 突变株乳酸产量大幅降低, 而乙酸、1,3-丙二醇的产量则显著增加。     

产氢菌的复合诱变选育及突变株HCM-23的产氢特性

以厌氧产氢细菌Clostridium sp. H-61为原始菌株, 先后经亚硝基胍(NTG)、紫外(UV)诱变, 选育得到1株高产突变株HCM-23。在葡萄糖浓度为10 g/L的条件下, 其产氢量为3024 mL/L, 比原始菌株提高了69.89%; 其最大产氢速率为33.19 mmol H2/g DW·h, 比原始菌株(19.74 mmol H2/g DW·h)提高了68.14%。经过多次传代试验, 稳定性良好。其发酵末端产物以乙醇和乙酸为主, 属于典型乙醇型发酵代谢类型。其最适产氢初始pH为6.5, 最适生长温度为36℃, 以蔗糖为最佳碳源。与原始菌株相比, 突变株HCM-23的产氢特性发生了改变, 如生长延滞期延长, 可利用无机氮源等。     

发酵法生产鸟苷的生产性试验

以鸟苷生产菌Ｂａｃｉｌｌｕｓｓｕｂｉｔｉｌｉｓ２０６６为生产菌株,经摇瓶、２０Ｌ自控发酵罐、５００Ｌ发酵罐和２５０００Ｌ生产罐逐级放大试验,分别达到１５．６ｇ／Ｌ,１７．７ｇ／Ｌ,１３ｇ／Ｌ和１１ｇ／Ｌ的产量。     

Fermentative Production of Guanosine by 8-Azaguanine Resistant of Bacillus subtilis

Two forms of the restriction enzyme HindIII were alternated with each other under some physiological or biochemical conditions. Addition of a low amount of phase T7 to the culture of HindIII-producing Haemophilus influenzae Rd, resulted in appearance of some amounts of the P2 fraction of HindIII, which was eluted with a high concentration of KCI from a phosphocellulose column. Higher amounts of T7 caused a decrease of the P2 fraction; finally the alternative PI fraction of HindIII, which was eluted with a lower concentration of KCI, remained exclusively.

Addition of disaccharides such as maltose and trehalose to the bacterial extract, yielded more P2, although the disaccharides inhibited to this enzyme. Urea showed an interesting distribution of these two forms of HindIII. Phosphocellulose chromatography in the presence of 2 m urea generated a broad peak of HindIII Activity. Addition of 4 m urea, on the contrary, showed only one active peak of this enzyme. The HindIII could be purified by the following DEAE-cellulose chromatography.

These results indicate the presence of only one kind of HindIII molecule, which was alternated between free and bound forms, and a certain kind of factor that would equilibrate these two forms.     

肌苷产生菌缺失核苷水解酶活性突变株选育及其发酵生产试验

以枯草芽孢杆菌ＪＳＩＭ－１０１９为出发菌株,经物理、化学诱变剂连续处理,获得一株缺失核苷水解酶活性的突变株ＪＳＩＭ－Ｂ－１９８。该突变株不能降解肌苷,应用于发酵生产中,肌苷产量显著增加。在２００００升发酵罐中,连续１０罐批,平均产肌苷８．３８ｇ／Ｌ,对糖转化率２１．９８％,发酵周期５０ｈ￣６０ｈ,该菌株遗传性能稳定,已在工业生产中应用。     

肌苷和鸟苷生产菌中嘌呤核苷合成途径三段基因序列的分析

为了研究肌苷和鸟苷生产菌中与产苷有关的嘌呤核苷合成途径的遗传背景,选择了pur操纵子的启动子序列、编码SAMP合成酶的purA基因和编码GMP合成酶的guaA基因,设计合适的引物,分别从野生菌、一株肌苷低产菌和肌苷鸟苷高产菌中扩增出相应片段,经克隆和测序后,对它们进行比较和分析。分析结果表明两株生产菌的purA基因发生了1个碱基缺失,导致阅读框发生移码突变;而鸟苷高产菌在pur操纵子的启动子部分和操纵子抑制蛋白结合区域发生了近10％的突变,可能影响整个操纵子的表达调控。     

Production of Guanosine by Psicofuranine and Decoyinine Resistant Mutants of Bacillus subtilis

Growth of Bacillus subtilis AG169 that produced large amounts of xanthosine and guanosine was inhibited by psicofuranine. When AG169 was mutated to resistance against psicofuranine, a mutant, GP–1, which yielded more guanosine was obtained. Psicofuranine did not inhibit growth of GP–1 any more. The guanosine 5′-monophosphate (GMP) synthetase activities were then assayed. In GP–1, the specific activity decreased about half, the complete loss of repression by GMP was found, and the inhibition by GMP was slightly loosed, when compared with those of AG169.

Furthermore, as growth of GP–1 was strongly inhibited by decoyinine, decoyinine resistant mutants were derived from GP–1. Of these mutants, two strains, MG–1 and MG–4, were resistant to decoyinine completely and showed the exclusive accumulation of guanosine in high yields, i.e. 16.0 and 15.5 g of guanosine per liter with weight yields of 20.0 and 19.4% of consumed sugar, respectively. GMP synthetase activity of MG–1 increased remarkably in comparison with that of GP–1 or AG169, and the inhibitions by GMP, psicofuranine and decoyinine were completely released in MG–1. Namely, the psicofuranine and decoyinine resistances seemed to cause mainly variations of GMP synthetase, and as results, the conversion of xanthosine 5′-monophosphate (XMP) to GMP proceeded more smoothly, and a larger amount of guanosine was accumulated.     

一株可积累鸟苷菌株的纯培养条件研究

菌株Bacillus.subtilis.S3 68是以鸟苷生产菌株B .subtilis.A0 66为出发菌经诱变所得。对该菌株进行培养条件研究的过程中 ,发现该菌株可以在摇瓶纯培养条件下积累鸟苷。试验结果表明 :发酵过程中 ,腺嘌呤的用量 0 .3 5mg/ml时 ,发酵液中鸟苷积累量最大 ,培养基中腺嘌呤的用量高于或低于 0 .3 5mg/ml均不利于鸟苷产物的积累 ;培养基中味精、硫酸铵、硫酸镁、磷酸二氢钾及Mn2 +用量显著影响发酵液中鸟苷积累水平 ;培养基中生物素、蛋氨酸、精氨酸、组氨酸、氯化钙及Fe2 +、Zn2 +用量与鸟苷积累的相关性不显著     

Purification and Characterization of NfrA1, a Bacillus subtilis Nitro/flavin Reductase Capable of Interacting with the Bacterial Luciferase

ipa-43d is a hypothetical gene identified by the Bacillus subtilis genome project (Mol. Microbiol. 10, 371-384 1993; Nature 390, 249-256 1997). The ipa-43d protein overexpressed in E. coli was purified to homogeneity and its properties were analyzed biochemically. The ipa-43d protein was found to be tightly associated with FMN and to be capable of reducing both nitrofurazone and FMN effectively. Although the ipa-43d protein catalysis obeys the ping-pong Bi-Bi mechanism, catalysis mode was changed to the sequential mechanism upon coupling with the bioluminescent reaction. Database search showed that B. subtilis possessed four genes (ipa-44d, ytmO, yddN, and yvbT), encoding proteins similar in amino acid sequence to LuxA and LuxB of Photobacterium fischeri, and, in particular, ipa-44d is immediately adjacent to the ipa-43d gene on the chromosome.     

不同启动子调控羰基还原酶基因在枯草芽胞杆菌中的表达水平

为了实现羰基还原酶基因mldh在枯草芽胞杆菌中的高效表达,以摩氏摩根菌MorganellamorganiiCMCC（B）49208染色体DNA为模板,PCR扩增得到目的基因mldh,分别与启动子PQ和启动子p43进行连接,构建不同启动子组合的表达载体PHY—p43-mldh、PHY—PQ—mldh、PHY—p43-p43-mldh和PHY—p43-PQ—mldh,化学法转化B．subtilisWb600后对重组茵细胞破碎液进行SDS-PAGE分析及全细胞生物转化反应实验发现,4种重组茵的转化能力差异显著,其中重组菌B．subtilisWb600（PHY—p43-p43-mldh）进行全细胞转化反应,转化液中d-伪麻黄碱的浓度最高,达到142．1mg／L,底物转化率为78．25％,成功实现了羰基还原酶基因mldh在枯草芽胞杆菌中的高效表达。     

柠檬酸钠对枯草杆菌生长代谢及肌苷积累的影响

研究柠檬酸钠对枯草杆菌生长代谢及产苷的影响 ,在基础料中添加浓度为 0 2g L的柠檬酸钠 ,肌苷产量提高 18% ,肌苷对葡萄糖得率增加 38%。通过分析糖代谢途径中关键酶的酶活 ,结果表明添加柠檬酸钠改变了一些关键酶的活力 ,可降低糖酵解途径中 6_磷酸果糖激酶和丙酮酸激酶的活力 ,从而减弱了糖酵解途径的通量。     

Nucleotide mutations in purA gene and pur operon promoter discovered in guanosine- and inosine-producing Bacillus subtilis strains

The promoter region of the pur operon, which contains 12 genes for inosine monophosphate biosynthesis from phosphoribosylpyrophosphate, and the purA gene, encoding the adenylosuccinate synthetase, were compared among wild-type and three purine-producing Bacillus subtilis strains. A single nucleotide deletion at position 55 (relative to translation start site) in purA gene was found in a high inosine-producing strain and in a high guanosine-producing strain, which correlates with the absence of adenylosuccinate synthetase activity in these strains. Within the pur operon promoter of high guanosine-producing strain, in addition to a single nucleotide deletion in PurBox1 and a single nucleotide substitution in PurBox2, there were 4 substitutions in the flanking region of the PurBoxes and 32 nucleotide mutations in the 5′ untranslated region. These mutations may explain the purine accumulation in purine-producing strains and be helpful to the rational design of high-yield recombinant strains.