产氨短杆菌与枯草杆菌发酵产肌苷比较试验

采用诱变得来的枯草杆菌GMI-741和产氨短杆菌GMA-2892在1.2L自控发酵罐上进行肌苷发酵试验,产氨短杆菌GMA-2802在种子培养基中培养15h后,菌浓度达1.0×1011个/ml,而同样条件下,枯草杆菌GMI-741菌浓度只有9.5×109个/ml.在1.2L自控发酵罐上发酵时,GMA-2802发酵周期54h,产肌苷达20.40g/L,发酵液主要原材料成本为441.1元/吨;GMI-741发酵周期60h,产肌苷达19.52g/L,发酵液主要原材料成本为559.1元/吨.     

产氨短杆菌GMA-2802 1.2L罐肌苷发酵试验

采用诱变得来的产氨短杆菌GMA－2802,在1.2L自控发酵罐上进行了5批发酵肌苷试验,发酵周期54小时,平均产肌苷20.4g/L。结果表明该菌档是一株具有较多优良特性的肌苷产生菌。     

原生质体融合选育肌苷产生菌产氨短杆菌GMA—2802

以肌苷产生菌产氨短杆菌ＧＭＡ－２７２２（Ａｄｅ－、 Ｇｕａ－、ＶＢ１－、Ｒｉｆｒ）和 ＧＭＡ－２７７６（Ａｄｅ－、 Ｂｉｏｔｉｎ－、ＶＢ１－、Ｓｍｒ）为出发菌株,经原生质体融合,将目的标志加以组合,获得遗传性状稳定、摇瓶发酵６１ｈ,产肌苷２５．４ｇ／Ｌ的融合子 ＧＭＡ－２８０２（Ａｄｅ－、Ｇｕａ－、Ｂｉｏｔｉｎ－、ＶＢ１－、 Ｒｉｆｒ、Ｓｍｒ）。     

新型肌苷产生菌──产氨短杆菌GMBA—800选育和特性的初步研究

采用多种方法诱变获得一株新型肌苷产生菌──产氨短杆菌（Ｂｒｅｖｉｂａｃｔｅｒｉｕｍａｍｍｏｎｉａｇｅｎｅｓ）ＧＭＢＡ－８００（具有腺嘌呤、生物素双重营养缺陷型和对８－氮杂鸟嘌呤、磺胺胍、６－流基嘌呤有抗性）,对其生长和发酵条件进行初步研究。肌苷产量从５ｇ／Ｌ提高到１８．４１ｇ／Ｌ,发酵周期从８４ｈ缩短为６３ｈ。菌株遗传性状稳定。     

新型肌苷新生菌——产氨短杆菌GMBA—800选育和特性的初步研究

采用多种方法诱变获得一株新型肌苷产生菌－－产氨短杆菌ＧＭＢＡ－８００,对其生长和发酵条件进行初步研究。肌苷产量从５ｇ／Ｌ提高到１８．４１ｇ／Ｌ,发酵周期从８４ｈ缩短为６３ｈ。菌株遗传性状稳定。     

采用产氨短杆菌合成辅酶A的研究

用产氨短杆菌鲜菌体在以腺嘌岭代替ATP的反应系统中酶促合成辅酶A(CoA),每毫升反应液的CoA产量达Z21单位(u);在不加任何核苷酸类物质的条件下,也可达185u;合成在5小时左右达高峰。CoA合成主要在细胞内进行。提出了连续式碳一阴离子交换树脂柱提纯CoA的新方法,既提高了收率,又避免了原锌汞齐法的毒物危害,方法简易可行。用本法制得的结晶品含CoA 231u／mg,收率17．0％;阴柱洗脱液不经结晶直接制成CoA注射液,提取率20．9％,经广州药检所检验,各项指标均符合国家标准。     

絮凝方法收集产氨短杆菌MA-2、黄色短杆菌MA-3条件的优化

采用壳聚糖作絮凝剂收集富含延胡索酸酶活力的产氨短杆菌MA-2、黄色短杆菌MA-3。研究了絮凝剂加入量及加入时混合方式与时间等因素对延胡索酸酶活力及分离效果的影响,并对壳聚糖絮凝收集产氨短杆菌MA-2、黄色短杆菌MA-3的过程进行了初步分析。     

固定化产氨短杆菌MA-2、黄色短杆菌MA-3反应动力学的研究

多年来虽然有不少学者对固定化细胞生产Ｌ 苹果酸的方法进行过探讨[1～ 6] ,但对过程动力学的研究报道并不多见[2 ,7] ,在富马酸铵转化体系中的表观动力学及本征动力学模型还未见报道 ,本文对富马酸铵转化体系中固定化产氨短杆菌ＭＡ 2、黄色短杆菌ＭＡ 3细胞的动力学进行了探讨 ,测定了两种固定化细胞的表观动力学常数 ,并进一步求解了相应的本征动力学常数 ,这一结果便于从理论上指导富马酸铵转化过程的工业化生产。1　材料和方法1 1　试剂富马酸 ,工业级 ,苏州合成化工厂 ,碳酸钙 ,工业级 ,泗联化工厂。1 2　菌株本文所用的菌株是由我院…     

短乳杆菌DM9218肌苷水解酶基因的异源表达及活性检测

目的 探讨短乳杆菌DM9218肌苷水解酶基因A0008的异源表达及其对肌苷的分解活性检测。方法 克隆来源于短乳杆菌DM9218基因组的肌苷水解酶基因A0008,构建原核表达载体,转入大肠埃希菌BL21诱导重组蛋白表达并纯化,进行体外酶活检测。结果 成功构建了肌苷水解酶A0008-pET28a原核表达载体,表达并纯化出重组蛋白,酶活结果显示该重组蛋白具有水解肌苷的能力。结论 短乳杆菌DM9218基因A0008可能编码肌苷水解酶并参与DM9218对肌苷的分解。     

Isolation and characterization of two distinct membrane fractions from the Gram-positive nucleotide producer Brevibacterium ammoniagenes ATCC 6872

Abstract Bouyant density gradient centifugation of 'crude membranes' of the coryneform bacterium Brevibacterium ammoniagenes yielded two distinct membrane fractions which differed significantly in equilibrium densities (1.15 and 1.18 g/cm³), NADH dehydrogenase activity (0.29 and 0.09 μmol·min⁻¹·mg⁻¹), protein composition and association of ribosomes. As determined by one dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) proteins unique to either the free membrane (FM) fraction or the denser ribosome-containing complexed membrane (CM) fraction were identified.     

Genomic organization of purK and purE in Brevibacterium ammoniagenes ATCC 6872: purE locus provides a clue for genomic evolution

Abstract From the genomic library of Brevibacterium ammoniagenes ATCC6872, the purE locus encoding 5'-phosphoribosyl-5aminoimidazole (AIR) carboxylase (EC 4.1.1.21) was cloned and its nucleotide sequence was determined. From the sequence analysis, two distinct open reading frames (ORFs) in the sequence of the purE locus were identified as purK and purE genes ( purK-purE ). An in vivo translation experiment reconfirmed the purK and purE genes to be independent. The genomic organization in the purE locus of B. ammoniagenes is opposite to that of the bacteria Escherichia coli and Bacillus subtilis . However, it coincides with the fused genes ( purKE ) of higher organisms Saccharomyces cerevisiae, Schizosaccharomyces pombe and Vigna aconitifolia . This suggests that the purE locus might be an intermediate form for genomic evolution of bacteria to higher organisms.     

The derepression of enzymes of de novo pyrimidine biosynthesis pathway in Brevibacterium ammoniagenes producing uridine-5-monophosphate and uracil

A mutant of Brevibacterium ammoniagenes producing large quantities of UMP and uracil is described. The mutations render bacteria braditrophic for arginine, sensitive to adenine, resistant to rifampicin and pyrimidine analogues 5-fluorouracil, 5-fluorouridine, azauracil and thiouracil. The activities of enzymes involved in the UMP biosynthesis, i.e. orotate phosphoribosyltransferase, orotate-5-monophosphate decarboxylase, dihydroorotate oxidase, are 4-, 3.5- and 4.5-fold higher in the mutant than in the parent strain when grown in minimal medium. The synthesis of these enzymes in mutant cells is not repressed in the presence of exogenous Ura. True revertants, which completely restore the wild-type phenotype, occur among the Arg+ clones. The nature of the mutation is discussed.     

噬菌体对国内谷氨酸生产菌的鉴别

利用噬菌体的宿主专一性,对收集的国内用于生产的谷氨酸生产菌进行鉴别,发现一株7338菌株对T6—13噬菌体敏感,而对7338的噬菌体不敏感。根据上述事实,工厂中为防止噬菌体侵袭,轮换使用不同菌株时,提出要了解菌株对不同噬菌体的敏感性,以免感染造成巨大经济损失。     

交流示波极谱滴定法测定谷氨酸含量

在ＫＣｌ底液中,用ＫＯＨ标准溶液直接滴定谷氨酸至极谱图形上切口出现指示终点,此方法简单、快速,终点直观,结果准确。     

Phylogenesis of Brain Glutamic Acid Decarboxylase from Vertebrates: Immunochemical Studies

Brain high-speed supernatants from various lower and higher vertebrates were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, electroblot on nitrocellulose membranes, and immunolabelling using an anti-glutamic acid decarboxylase (anti-GAD) antiserum prepared from rat antigen. Rat brain extracts showed two distinct immunolabelled bands (MW 59,000 and 62,000 daltons). The molecular weight of the native enzyme was 120,000 daltons. The immunoblot pattern was not affected by a 3-h incubation of the homogenate. In the substantia nigra, the decrease in the immunolabelling of both bands corresponded very closely to the decrease of GAD activity following lesioning of the striato-nigral pathway. Moreover, experiments with preadsorbed antiserum showed that both subunits have common antigenic determinants. The immunolabelling was consistently more intense over the lightest band. The autoradiography of immunoprecipitated rat brain GAD, iodinated prior to electrophoresis, revealed two radiolabelled bands corresponding to the two immunolabelled ones. Their radioactivity was found in a one-to-five ratio which closely paralleled their respective immunolabelling intensity. Thus, the two subunits recognized by the antiserum are not present in stoichiometric proportions in the rat brain high-speed supernatant. These findings suggest the existence of two homodimeric GAD with common antigenic determinants which are present in different amounts. Immunoprecipitation curves of brain GAD from rat, mouse, rabbit, monkey, human, quail, frog, and trout were similar, with a less than 10-fold maximum shift in affinity for GAD. GAD immunoblots from the various higher vertebrates showed a pattern similar to that obtained in rat.(ABSTRACT TRUNCATED AT 250 WORDS)     

d－生物素代替玉米浆发酵生产谷氨酸的研究

用ｄ－生物素代替玉米浆的摇瓶发酵实验,筛选出高产酸菌株,测定了ｄ－生物素的亚运量可变范围。比较了单一玉米浆、单一ｄ－生物素与两者各半量发酵结果及不同接种量对产酸的影响。采用ｄ－生物素代替半量玉米浆的中糖发酵工艺应用于生产。结果证明此工艺可行,降低了成本,提高了产酸率,平均产酸率达７．８１％,平均转化率达５３．２％。     

Production of Proteases from Brevibacterium Linens

Protease formation in submerged cultivations of Brevibacterium linens was studied. The effect of several proteinaceous materials on the production of proteolytic enzymes was investigated in mineral media containing 0.2% malt extract for bacterial growth. The addition (0.5%) of yeast extract or enzymatically hydrolyzed casein considerably increased the amount of protease formed, whereas ammonium salts supplied additionally in most cases had a repressive effect on enzyme formation. Furthermore, the kinetic of protease formation was determined in a highly instrumented fermenter system. Respiration activity indicated several phases of bacterial growth. Most of the proteolytic activity was synthesized during active growth; there was only a small increase in the stationary phase. A total proteolytic activity of 36 U/ml was formed in 24 hr. Concentration of α-amino nitrogen decreased steadily and ammonium ions accumulated during bacterial growth. Electrophoretic analysis revealed the occurrence of one leucine aminopeptidase (26 kDa monomer) and several proteases. There is a broad spectrum of proteolytic active proteins in the range of 11-66 kD which may be caused by some auto-degrading effects.