用原生质体融合技术选育2—酮基—L—古龙酸高产菌株的研究

对革兰氏阳性的地衣芽孢杆菌（Ｂａｃｉｌｌｕｓｌｉｃｈｅｎｉｆｏｒｍｉｓ）Ｈ１９和革兰氏阴性的２－酮基－Ｌ－古龙酸产生菌Ｓ１９的原生质体的制备条件进行了研究,并采用聚乙二醇作诱导剂进行了两菌株的原生质体融合,用链霉素作为抗性标记对融合子进行了选择。从１７株产生２－酮基－Ｌ－古龙酸的融合子中选出了一株连续传代八次产酸高且产量稳定的融合子１５号。融合子１５号具有两个亲本菌株所具有的一些特性。     

L－山梨糖发酵生产2－酮基－L－古龙酸的最佳组合新菌系的生物特性的研究

从２２个不同组合的发酵Ｌ山梨糖生成２－酮基－古龙酸的组合菌系中选出了最佳组合新菌系Ｈ１９Ｓ１９。对该菌系的形态学及生理生化特性的研究表明,其中Ｈ１９为地衣芽枪杆菌（ＢａｃｉｌｌｓＬｉｃｈｅｎｉｆｏｒｍｉｓ）,是Ｓ１９的伴生菌;Ｓ１９是产生２－酮基－古龙酸的菌株,具有许多特点,其分类位置暂无法确定。     

2—酮—L—古龙酸还原酶分离纯化及其理化,酶学性质的研究

从发酵Ｌ－山梨糖的Ｇｌｕｃｏｎｏｂａｃｔｅｒ ｏｘｙｄａｎｓ和Ｂａｃｉｌｌｕｓ ｍｅｇａｔｅｒｉｕｍ２９８０混和菌株的无细胞抽提液中分离到了２－酮－Ｌ－古龙酸还原酶（ＫＧＲ）,测得其分子量为９０ｋＤａ。动力学性质研究表明它为一个典型的Ｍｉｃｈａｅｌｉｓ－Ｍｅｎｔｅｎ氏酶,对２－酮－Ｌ－古龙酸作用的Ｋｍ值为３．４２×１０＾－３ｍｏｌ,最适作用ｐＨ为６．５,最适作用温度为３０℃。２－酮－Ｌ－古龙酸还原     

新组合菌系SCB329—SCB933利用L—山梨糖发酵产生维生素C前体——2—酮基…

采用紫外照射,化学诱变和原生质融合等方法选育到一株性状更优良的突变株ＳＣＢ３２９,并与新筛选的一株芽孢杆菌ＳＣＢ９３３搭配组成新的组合菌系,产酸小菌ＳＣＢ３２９与其亲本菌株氧化葡萄糖酸杆菌性状相似,伴生大菌ＳＣＢ９３３属苏芸金芽孢杆菌（Ｂ．ｔｈｕｒｎｇｉｅｎｓｉｓ）,新组合菌系列Ｌ－山梨糖的发酵液提取后经纸层析,元素分析和红外吸收光谱等项鉴定,其发酵产物克系２－酮基－Ｌ－古龙酸,对新组合菌系的生物     

维生素C发酵中2—酮—L—古龙酸还原酶的研究

由于２酮Ｌ古龙酸还原酶（简称ＫＧＲ）的存在,理论上造成了Ｖｃ合成前体２酮Ｌ古龙酸（简称２ＫＬＧ）部分被还原成Ｌ艾杜糖酸［１,２］,影响了Ｖｃ二步发酵的产率。而从Ｌ山梨糖到２酮Ｌ古龙酸的转化中,除被ＫＧＲ催化的还原负反应外,均属...     

2-酮-L-古龙酸还原酶分离纯化及其理化、酶学性质的研究

从发酵Ｌ山梨糖的Ｇｌｕｃｏｎｏｂａｃｔｅｒｏｘｙｄａｎｓ和Ｂａｃｉｌｕｓｍｅｇａｔｅｒｉｕｍ２９８０混和菌株的无细胞抽提液中分离到了２酮Ｌ古龙酸还原酶（ＫＧＲ）,测得其分子量为９０ｋＤａ。动力学性质研究表明它为一个典型的ＭｉｃｈａｅｌｉｓＭｅｎｔｅｎ氏酶,对２-酮-Ｌ-古龙酸作用的Ｋ_ｍ值为３.４２×１０^－３ｍｏｌ,最适作用ｐＨ为６.５,最适作用温度为３０℃。２-酮-Ｌ-古龙酸还原酶的合成不受Ｌ-山梨糖和２-酮-Ｌ-古龙酸的诱导,故推测２-酮-Ｌ-古龙酸还原酶是Ｇｌｕｃｏｎｏｂａｃｔｅｒｏｘｙｄａｎｓ的一个组成酶。     

欧文氏菌和棒杆菌的属间融合研究*

研究了用原生质体融合技术获得欧文氏菌和棒杆菌的融合细胞。串联发酵Ｄ－葡萄糖产生２－酮基－Ｌ－古龙酸的第一步发酵菌株欧文氏菌ＳＣＢ２４７经０．８ｍｇ／ｍＬ溶菌酶酶解０．５ｈ后,原生质体的形成率和再生率分别为９９．８％和２７．８％。第二步发酵菌株棒杆菌ＳＣＢ３０５８经预处理后由１．３ｍｇ／ｍＬ溶菌酶酶解２ｈ,原生质体的形成率和再生率分别为９９．５％和５６．３％。用携带氨苄青霉素抗性标记的ＳＣＢ２４７和经热灭活的ＳＣＢ３０５８为亲本,在４０％ＰＥＧ６０００和０．２ｍｏｌ／Ｌ新生磷酸钙等适宜条件下融合,融合频率为３．６×１０~(－６)。在非选择和选择培养基上连续传代十几次后,对融合子的单菌形态、染色结果、菌落形态、色泽、总蛋白量、同工酶、发酵性能等方面与亲本进行了比较。结果表明,融合子确系棒杆菌和欧文氏菌的融合细胞。摇瓶发酵结果显示,所得的３８株稳定的融合子中约４０％能转化葡萄糖为维生素Ｃ前体２－酮基－Ｌ－古龙酸。     

L—山梨糖脱氢酶的纯化及性质的研究

从５Ｌ罐发酵Ｌ－山梨糖的Ｇｌｕｃｏｎｉｂａｃｔｅｒ ｏｘｙｄａｎｓ ＳＣＢ３２９和Ｂａｃｉｌｌｕｓ ｔｈｕｒｉｎｇｉｅｎｓｉｓ ＳＣＢ９３３混合菌株中差速离心收集ＳＣＢ３２９菌体,破碎,离心获得无细胞抽提液,硫酸铵分级沉淀蛋白后依次经ＤＥＡＥ Ｃｅｌｌｕｌｏｓｅ ５２和Ｑ Ｓｅｐｈａｒｏｓｅ ＦＦ柱层析分得到了Ｌ－册梨糖脱氢酶（ＳＤＨ）,它能将Ｌ－册梨糖脱氢氧化为Ｌ－册梨酮,ＳＤＳ－ＰＡＧＥ电泳测     

新组合菌系SCB329—SCB933利用L—山梨糖发酵生产维生素C前体—2—酮基…

新组合菌系氧化葡萄糖酸杆菌ＳＣＢ３２９－苏芸金芽杆菌ＳＣＢ９３３能在较长时间内保持高的转化活力且具有极强的抗杂菌污染的特性。在一次投糖分批发酵的基础上,探索在控制溶氧、ＰＨ,温度等条件下,分批加入Ｌ－山梨糖发酵生产２－酮基－Ｌ－古龙酸新工艺。采用新工艺,既充分利用了菌系的优良特性,又避免了高糖浓度可能对菌系造成的不良影响。Ｌ－山梨糖最终浓度达到１４％（Ｗ／Ｖ）,产酸１２０－１３５ｇ／ｌ,转化率９０     

构建从葡萄糖直接发酵产生维生素C前体2-酮基-L-古龙酸的基因工程菌研究进展

构建从葡萄糖直接发酵产生维生素Ｃ前体２┐酮基┐Ｌ┐古龙酸的基因工程菌研究进展维生素Ｃ作为人体不可缺少的维生素和抗氧化剂在医药和食品工业上有重要用途,２－酮基－Ｌ－古龙酸（２－ＫＬＧ）是合成维生素Ｃ的重要前体,工业上大多采用“莱氏法”或“两步发酵法”生...     

新组合菌系SCB329-SCB933利用L-山梨糖发酵产生维生素C前体——2-酮基-L-古龙酸的研究 Ⅰ.新组合菌系SCB329-SCB933的生物学特性

采用紫外照射、化学诱变和原生质融合等方法选育到一株性状更优良的突变株SCB329,并与新筛选的一株芽孢杆菌SCB933搭配组成新的组合菌系。产酸小菌SCB329与其亲本菌株氧化葡萄糖酸杆菌性状相似。伴生大菌SCB933属苏芸金芽孢杆菌（B.thuringiensis）。新组合菌系利用L-山梨糖的发酵液提取后经纸层析,元素分析和红外吸收光谱等项鉴定,其发酵产物确系2-酮基-L-古龙酸,对新组合菌系的生物学特性也进行了研究。     

Acidaminococcus gen. n., Acidaminococcus fermentans sp. n., Anaerobic Gram-negative Diplococci Using Amino Acids as the Sole Energy Source for Growth

Acidaminococcus gen. n. and the type species Acidaminococcus fermentans sp. n. were described. Amino acids, of which glutamic acid is the most important, could serve as the sole energy source for growth. Acetic and butyric acids and CO(2) were produced; propionic acid and hydrogen were not produced. Amino acid media supporting growth and the amino acid and vitamin requirements were described. Glucose was frequently not fermented or was weakly catabolized. Derivative products from glucose autoclaved in media, but not glucose itself, stimulated or were required for growth in amino acid media. A wide range of polyols and carbohydrates were not attacked. Lactate, fumarate, malate, succinate, citrate, and pyruvate were not used as energy sources for growth. Pyruvate completely suppressed growth. Cytochrome oxidase and benzidine reactions were negative; catalase, indole, acetyl methyl carbinol, and H(2)S were not produced; nitrate and sulfonthalein indicators were not reduced; ammonia was produced; gelatin liquefaction was negative or slow and partial; vancomycin (7.5 mug/ml) was resisted. Acidaminococcus was different from Veillonella in morphology, serology, nutrition, utilization of substrates, and accumulation of products in media supporting growth; Acidaminococcus resembled Peptococcus in utilization of glutamic acid and accumulation of similar products, but the two genera differed in morphology, gram reaction, serology, guanine plus cytosine content of deoxyribonucleic acid, and nutrition.     

Vc生产菌“神舟七号”搭载育种

选取3株性状不同的Vc二步发酵生产菌搭载于"神舟七号"飞船进行空间诱变,返回地面后,经富集培养和分离,获得近12000株诱变菌株。采用试管微量培养并监测pH值法作3次初筛,获300余株优良株,再经摇瓶发酵定酸、糖法作3次复筛,选出12株优良菌株。新菌株摇瓶发酵转化率提高了5%～7%。研究了新菌株生产的最适发酵条件,调整了部分工艺过程,应用于大生产,转化率提高4%～5%。     

Interspecific hybridization between Vigna radiata (L.) Wilczek and V. glabrescens

Summary Interspecific hybrids of the mungbean, Vigna radiata (L.) Wilczek (2n=22) and V. glabrescens (2n=44) were generated with the aid of embryo culture. V. glabrescens x V. radiata hybrids were recovered via germination of the immature embryos. Reciprocal hybrids were obtained via shoot formation from embryonic callus. The authenticity of the hybrids was determined by morphological characteristics, chromosome number, and isozyme patterns. The hybrids were highly sterile upon selfing, but backcrossing to the diploid parent yielded viable seeds. Some of the plants resembled the diploid parent morphologically while others resembled neither parent. The backcross plants were sufficiently fertile to give a large number of mature, selfed seeds. Plants obtained differed morphologically and in their isozyme patterns from either parent, indicating introgression. These progeny populations will be used as bridging materials to transfer pest resistance from the wild tetraploid to the cultivated mungbean.     

D-葡萄糖串联发酵产生维生素C前体—2-酮基-L-古龙酸——Ⅱ.菌株SCB3058产生2-酮基-L-古龙酸发酵条件的研究

维生素C(Vitamin C,简称Vc),又称L-抗坏血酸(L-Ascorbic acid)是人体必需的维生素,生理作用广泛,在医药和食品工业上均有重要地位。目前国内厂家多以我国发明的“二步发酵法”进行生产,即以D-山梨醇为原料生产2-酮基-L-古龙酸(以下简称2-KLG),然后制备维生素C。而近年来引起国内外普遍关注的是从D-葡萄糖串联发酵生产2-KLG的新工艺,以及采用基因工程技术,构建直接由D-葡萄糖转化生成2-KLG的基因工程菌的研究(图1)。1987年以来我国学者尹光琳等人采用了欧文氏菌(Erwinia sp.)和棒状杆菌(Corynebacterium sp.)进行串联发酵产生维生素C前体——2-酮基-L-古龙酸,并开展了一系列的研究。     

Halomonas glaciei sp. nov. isolated from fast ice of Adelie Land,Antarctica

Eleven psychrophilic bacteria were isolated from a solid layer of fast ice in the middle of Pointe-Geologie Archipelago, Adelie Land, Antarctica. The 11 isolates based on the phenotypic characteristics, chemotaxonomic and phylogenetic analysis have been identified as members of the genus Halomonas. All the isolates at the 16S rDNA sequence level were identical, possessed the 15 conserved nucleotides of the family Halomonadaceae and four nucleotides of the genus Halomonas. Therefore, the 16S rDNA sequence of DD 39 was used for calculating the evolutionary distances and for phylogenetic analysis. It was observed that DD 39 formed a robust cluster with H. variabilis, from which it differed by 0.7%. Further DNA-DNA hybridization studies indicated low DNA-DNA homology (15%) between H. variabilis and DD 39. Between the 11 Antarctic isolates the homology was >85%. In addition it was observed that DD 39 was different from H. variabilis in that it was psychrophilic, could tolerate only up to 15% sodium chloride, could not hydrolyse esculin, could not reduce nitrate, was urease negative, could not utilize glycerol as a carbon source, and was resistant to ampicillin and erythromycin and sensitive to nalidixic acid. In addition, it also exhibited distinct differences with respect to high content of C(16:1) and low levels of cyclo-C(17:0) and cyclo-C(19:0). DD 39 also differed from all the other reported species of Halomonas with respect to many phenotypic characteristics. It is proposed therefore that DD 39 should be placed in the genus Halomonas as a new species that is Halomonas glaciei. The type strain of H. glaciei is DD 39(T) (MTCC 4321; JCM 11692).     

NADH peroxidase activity of rubrerythrin

P. S. Alban et al. (J. Appl. Microbiol. (1998) 85, 875-882) reported that a mutant H2O2-resistant strain of Spirullum (S.) volutans showed constitutive overexpression of a protein whose amino acid sequence and molecular weight closely resembled that of a subunit of rubrerythrin, a non-heme iron protein with no known function. They also reported that the mutant strain, but not the wild-type, showed NADH peroxidase activity. Here we demonstrate that rubrerythrin and nigerythrin from Desulfovibrio vulgaris and rubrerythrin from Clostridium perfringens show NADH peroxidase activities in an in vitro system containing NADH, hydrogen peroxide, and a bacterial NADH oxidoreductase. The peroxidase specific activities of the rubrerythrins with the "classical" heme peroxidase substrate, o-dianisidine, are many orders of magnitude lower than that of horseradish peroxidase. These results are consistent with the phenotype of the H2O2-resistant strain of S. volutans. The reaction of reduced (i.e., all-ferrous) rubrerythrin with excess O2 takes several minutes, whereas the anaerobic reaction of reduced rubrerythrin with hydrogen peroxide is on the millisecond time scale and results in full oxidation of all iron centers to their ferric states. Rubrerythrins could, thus, function as the terminal components of NADH peroxidases in air-sensitive bacteria and archaea.     

Radiation-Resistant Mutants of Salmonella typhimurium LT2: Development and Characterization

A series of repeated exposures to gamma irradiation with intervening outgrowth of survivors was used to develop radioresistant cultures of Salmonella typhimuium LT2. Stepwise increases in resistance to both ionizing and ultraviolet irradiation were obtained independently of the presence or absence of integrated P22 prophage. Single clonal isolates, representing parent and radioresistant populations, retained the general characteristics of the LT2 parent, including serological properties, phage typing, antibiotic sensitivities, mouse virulence, and most biochemical test reactions. Resistant cells were generally larger and contained 1.8 to 2.1 times more ribonucleic acid and protein than parent cells, but deoxyribonucleic acid (DNA) contents were similar. Heterogeneity in the populations with respect to release of H(2)S, utilization of carbon sources, and growth on minimal medium is considered to be ancillary, rather than causally related, to increased radioresistance. The resistant isolates displayed an increased ability to reactivate gamma-irradiated P22 phage. DNA polymerase I and polynucleotide-joining enzyme activities were elevated in extracts of radioresistant cells relative to parent cells. It is suggested that the observed increases in radioresistance result from a selection of mutations leading to an increased capacity to repair DNA.