常压室温等离子体诱变与微生物微液滴培养选育谷胱甘肽高产菌株

为提升野生毕赤酵母菌株BY-1产谷胱甘肽的能力,通过常压室温等离子体（atmospheric and room temperatureplasma,ARTP）诱变技术得到诱变菌株BY-1-26。进一步在摇瓶培养过程中添加1,2,4-三氮唑,提高诱变菌株产量。最后将1,2,4-三氮唑作为筛选因子,利用微生物微液滴培养（microbial microdroplet culture system,MMC）仪对诱变菌株进行适应性进化,获得了1株高产谷胱甘肽的突变株BY-2-24,并对其遗传稳定性进行研究。结果表明：出发菌株BY-1经过ARTP诱变处理、抗性梯度平板初筛、MMC适应性进化、摇瓶复筛等,可以选育高产谷胱甘肽突变株。突变株BY-2-24摇瓶产量达（312.13±2.62）mg/L,较出发菌株提高134.26%,且经过7次传代培养,仍然具有较好的遗传稳定性。同时,生物量提高118.33%,表明诱变菌株的生长能力得到提高。研究表明,ARTP与MMC联合应用作为一种简便高效的微生物诱变方式,可用于定向诱变筛选高性能微生物菌株,为高通量选育目标菌株提供了参考。     

Isolation and genetic determination of a Saccharomyces cerevisiae mutant that produces an endo-polygalacturonase

A Saccharomyces cerevisiae mutant that produces an endo-polygalacturonase (PGase) was isolated. The PGase gene was revealed to be located on chromosome X in both the mutant and its parental strain. The 5'-upstream region of the PGase gene in the mutant was entirely identical with that of its parent. Crossing of the mutant with a PGase-negative strain showed that the phenotype of PGase production was recessive. These results suggest that the mutant is rendered defective in a transacting factor that normally represses the expression of the PGase gene.     

Phenylpyrrole-resistance and aflatoxin production in Aspergillus parasiticus Speare

Mutants of Aspergillus parasiticus highly resistant to phenylpyrroles were isolated at a high mutation frequency, after UV-mutagenesis and selection on media containing fludioxonil. Studies on the effect of mutation(s) on the aflatoxin production resulted in the identification of two fludioxonil-resistant phenotypes: aflatoxigenic (FLD(afl)(+)) and non-aflatoxigenic (FLD(afl)(-)) mutant strains. Most of the FLD(afl)(+) mutant strains produced the aflatoxin B(1) at similar or even higher (up to 2.5-fold) concentrations than the wild-type parent strain on yeast extract sucrose medium. Interestingly, in most of these mutant strains the aflatoxigenic ability significantly increased (up to 4-fold) when the mutants were grown on fungicide-amended medium. However, a significant reduction in the aflatoxin production was observed in wheat grains by all FLD(afl)(+) mutant strains. Tests on the response of mutant strains to high osmotic pressure showed that most fludioxonil-resistant mutants were more sensitive to high osmolarity than the wild-type parent strain. Study of other fitness determining parameters showed that the mutation(s) for resistance to phenylpyrroles may or may not affect the mycelial growth rate, sporulation and conidial germination. However, in a number of aflatoxigenic-mutant strains these fitness parameters were unaffected or only slightly affected. Cross resistance studies with fungicides from different chemical groups showed that the mutation(s) for resistance to fludioxonil also highly reduced the sensitivity of mutant strains to the aromatic hydrocarbon and dicarboximide fungicides. No effect of phenylpyrroles resistance mutation(s) on fungitoxicity of triazoles, benzimidazoles, anilinopyrimidines, phenylpyridinamines, strobilurin-type fungicides and to the non site-specific inhibitors chlorothalonil and maneb was observed. The above mentioned data indicate, for the first time, the potential risk of increased aflatoxin contamination of agricultural products by the appearance and predominance of highly aflatoxigenic mutant strains of A. parasiticus resistant to aromatic hydrocarbon, dicarboximide and phenylpyrrole fungicides.     

烤烟叶数株高突变株的生长特征及DNA初步鉴定

把2001年烤烟大田自然条件下出现的多叶数、超株高的突变株,采用组织培养获得的再生苗,2002年在3个不同生态地点栽植进行生长观察,同时从细胞学、生理生化和遗传物质DNA的鉴定方面,以原品种株做对照.结果发现,在植株形态生长方面,突变株较对照株叶数仍表现出多叶数、超株高和晚花发育的特征性状;在叶细胞结构方面,突变株叶片气孔保卫细胞叶绿体个数极显著多于对照株;在生理生化特性指标方面,突变株叶绿素a、b和总量及可溶性蛋白质含量均较对照株高,且突变株和对照株的COD、POD同工酶及可溶性蛋白质SDS-PAGE电泳有酶谱带差异;提取叶细胞DNA的RAPD分析和开花期叶片mRNA差异显示的电泳图谱中,突变株与对照株对所选用的随机引物扩增产物条带数存在着数量差别.初步证明该变异株应是晚花或多叶数基因的突变体,为克隆烤烟叶数或晚花基因提供了依据.     

重组羧肽酶原B突变体C383A的纯化与性质研究

目的纯化羧肽酶原B突变体C383A并研究其酶学性质。方法用DEAE-FF离子交换树脂纯化C383A,测定其动力学参数,温度对酶活性的影响、温度稳定性、最适pH以及pH稳定性等酶学性质,并与野生型比较。结果纯化后获得高纯度突变体C383A,与野生型相比,催化效率及稳定性均有提高,但与底物的亲和力降低,随着温度的上升催化速率的增长率低于野生型。结论将重组羧肽酶原B第383位Cys突变为Ala可对酶的性质产生影响,与野生型比较,C383A提高了酶的催化效率及温度和pH稳定性。     

Characterization of a flavinogenic mutant of methanol yeast Candida boidinii and its extracellular secretion of riboflavin

A flavinogenic mutant was derived from Candida boidinii by mutagenesis. The mutant was smaller than the wild type, did not grow on a minimal medium, and required l-tryptophan, l-leucine, inositol, and nicotinate for growth. The mutant was defective in the oxidative pentose phosphate pathway, lacking glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The specific activities of the transaldolase and transketolase of the mutant were higher than those of the wild type. These high activities might direct the flux of the carbon source to the nonoxidative pathway with formation of a large amount of pentose phosphates, increasing riboflavin synthesis. Under microaerobic conditions at 25 degrees C, 90 mg/l riboflavin was obtained.     

The study of polymorphism rs9939609 FTO gene in patients with overweight and obesity

In 94 persons living in the Moscow region, with a body mass index >25 kg/m2 were identified on the polymorphism rs9939609 FTO gene. The results showed that 83% of them were carriers of the mutant allele, and 43%--contained the mutant allele in the homozygous state. Carriers of the mutant allele rs9939609 FTO gene were distinguished by higher absolute and relative values of fat mass and triglycerides in serum.     

高产琥珀酸放线杆菌的诱变选育

对琥珀酸放线杆菌（Actinobacillus succinogenes mdrh-5）进行紫外-亚硝基胍复合诱变,采用变色圈、氟乙酸耐受和CuSO₄快速分析结合的筛选方法,获得1株高产琥珀酸突变菌株。与野生菌相比,突变株琥珀酸产量提高了43%,副产物乙酸降低了66%,突变株遗传性质稳定。     

Domesticating brewing yeast for decreasing acetaldehyde production and improving beer flavor stability

High level of acetaldehyde produced by yeast in beer industry, especially in China, is still an important issue. To obtain a brewing strain with low acetaldehyde, a novel approach, based on domestication by disulfiram–ethanol plates, was developed. A mutant D-A-14 with lower acetaldehyde was obtained with UV mutagenesis. Through longtime domestication, the mutant D-A-14 produced 60.57 % less (2.20 mg/L) than the Wt strain MA12. Besides, the ratio of higher alcohols to esters in beer fermented by the mutant D-A-14 (3.7) was lower than that of MA12 (5.8), which exhibited harmonious flavor. For further study, DNA microarray technique was exploited to analyze the changes in genes involved in metabolism between the Wt and mutant. The data of DNA microarray were consistent with the decrease in acetaldehyde, organic acids, and the ratio of higher alcohols to esters of mutant D-A-14. Therefore, the newly screened strain has the potential to be applied in the brewing industry.     

Seed protein characterisation and nitrogen fixation rates in the chickpea mutant hyprosola and its parent

The amino acid composition of the seed proteins of Hyprosola, a high protein, high yielding mutant of chickpea (Cicer arietinum L), differed little from that of its parental cultivar, Faridpur-1. Oser's essential amino acid index was 76-5 in the mutant and 76-7 in the parent. Hyprosola seed has higher crude protein, extractable protein, globulin and albumin contents than Faridpur-1. The mutant had an albumin/globulin ratio of 0-23 compared with the parental value of 0-21. The mutant oligomeric proteins differed quantitatively and qualitatively from the parent on cellulose acetate electrophoretograms. The polypeptide composition of globulins and albumins fractionated by SDS-PAGE differed markedly in the intensity of many bands but not in the mobility of any polypeptides. Nitrogen fixation (acetylene reduction) rates were similar in the two cultivars, and in both hydrogen evolution utilised 45 % of the electron flow through nitrogenase.     

Isolation and characterization of Schizosaccharomyces pombe mutants lacking aminopeptidase activity

A mutant strain of the fission yeast Schizosaccharomyces pombe defective in aminopeptidase I was isolated by screening for lack of activity against the chromogenic substrate lysine-beta-naphthylamide in isolated colonies. Tetrad dissection of sporulated diploids heterozygous for the wild-type and mutant allele resulted in a 2:2 segregation of mutant and wild-type phenotype indicating a single chromosomal gene mutation. Gene dosage experiments indicated that the mutation might reside in the structural gene of aminopeptidase I. No vital consequences of aminopeptidase I deficiency on cell life and sporulation could be detected. However, the enzyme seems to be involved in protein degradation under conditions of nutrient deprivation.     

Effect of Bacillus subtilis spo0A mutation on cell wall lytic enzymes and extracellular proteases, and prevention of cell lysis

The Bacillus subtilis spo0A mutant is an adequate host for extracellular protein production (e.g., alpha-amylase). However the mutant was prone to cell lysis. SDS-PAGE and zymography of cell wall lytic proteins indicated that the spo0A mutant contained high amounts of two major autolysins (LytC [CwlB] and LytD [CwlG]) and two minor cell wall lytic enzymes (LytE [CwlF] and LytF [CwlE]). On the other hand, the expression of eight extracellular protease genes was very poor or absent in the spo0A mutant. An eight-extracellular-protease-deficient mutant (Dpr8 strain) was constructed and the strain also exhibited cell lysis. The autolysins from the spo0A mutant were degraded by the supernatant of the wild type but not degraded by that of the Dpr8 mutant. These results suggest that the extensive cell lysis of the spo0A mutant was partially caused by the stability of autolysins via the decrease of the extracellular proteases. The introduction of a major autolysin and/or SigD mutations into the spo0A mutant was effective for preventing cell lysis.     

基于代谢途径提高红曲黄色素生成及控制橘霉素产生的研究

基于红曲霉菌合成代谢红曲色素和橘霉素的代谢机理,研究了几种化学物质对红曲霉突变菌株合成代谢红曲黄色素和橘霉素的影响。研究结果表明,一定量的生物素、乙酸钙和乙酸锌有利于红曲霉突变菌株合成代谢黄色素。不论辛酸添加量多少和添加时间或早或晚,对于红曲霉突变菌株的生长及其合成代谢黄色素都有明显不利的影响,但是一定量辛酸的添加有利于红色素的合成代谢,在一定程度上也能明显消除橘霉素的合成代谢。     

凝乳酶高产菌株的超声波-紫外复合诱变育种及其发酵条件的优化

以编号为Jl-1的黑曲霉为出发菌株,通过超声波和紫外诱变处理,在酪蛋白培养基上以凝乳圈直径为指标进行初筛,在基础固态发酵培养基上进行复筛,选育得到一株具有良好遗传稳定性的突变菌株.其经固态发酵凝乳酶产量为凝乳活力600U/mL,较出发菌株Jl-1提高57%.菌株经8次传代培养,凝乳酶产量仅降低7.1%.在此基础上应用单因素试验和正交设计对菌株产凝乳酶的固态发酵条件进行了系统研究和优化,得到最佳的试验组合为发酵温度28℃.发酵时间为6d,加水量为20mL,加样量为4份.采用此条件,凝乳酶产量达828U/mL,比Jl-3优化前提岛38%.     

耐Mn~(2+)高产柠檬酸菌株的选育及其特性分析

诱变选育耐锰离子(Mn2+)的高产柠檬酸菌株,并研究了突变株的特性。采用紫外和亚硝基胍复合诱变,以含不同浓度Mn2+的培养基进行筛选,同时考察了不同浓度Mn2+对突变株柠檬酸发酵、关键酶酶活力及呼吸途径的影响。在以葡萄糖为唯一碳源进行摇瓶发酵时,突变株柠檬酸产量比野生菌株提高了92.4%,突变株UV-141对高浓度Mn2+有一定的耐受性,且Mn2+对其发酵生产柠檬酸有促进作用,与不添加Mn2+时相比,添加300 mg/L Mn2+时,柠檬酸产量及葡萄糖最大比消耗速率分别提高了107%和16.7%;乌头酸酶最高酶活下降了15.8%,NAD+-(NADP+-)异柠檬酸脱氢酶对Mn2+不敏感且在整个发酵过程中始终处于较低水平,有利于柠檬酸的积累;当侧系呼吸链(AOX)受到水杨基氧肟酸(SHAM)抑制时柠檬酸产量显著下降,而添加Mn2+则能够削弱其对柠檬酸产量的影响。突变株柠檬酸产量明显高于野生菌株,Mn2+能促进突变株葡萄糖代谢加快、乌头酸酶酶活下降,同时能削弱SHAM对AOX途径的影响,从而促进柠檬酸的大量积累。     

Role in anaerobiosis of the isoenzymes for Saccharomyces cerevisiae fumarate reductase encoded by OSM1 and FRDS1

Saccharomyces cerevisiae possesses both a cytoplasmic and a mitochondrial fumarate reductase, encoded by FRDS1 and OSM1, respectively. While previous studies have shown that mutants lacking FRDS1 and OSM1 cannot grow under anaerobiosis (Arikawa et al., 1998), the physiological role of fumarate reductase (FR) remains poorly understood. Here, we report that an osm1 frds1 mutant is unable to grow anaerobically, even with glutamate as a sole nitrogen source, when succinate can be produced by the TCA oxidative branch. We also show that the growth of the mutant is not restored by adding acetoin, an alternative sink for NADH oxidation, but it is at least partly restored by the addition of oxygen or menadione, which can oxidize FADH(2) in addition to NADH. These data indicate that the growth inhibition of the mutant is due to an inability to reoxidize FAD, rather than an indirect effect on NADH or an inability to produce succinate per se. During anaerobic growth, FRDS1 expression was two to eight times higher than that of OSM1, and fumarate reductase activity was higher in the osm1 mutant than in the frds1 mutant. FRDS1 expression was induced by anaerobiosis, and this induction was abolished in a rox1 mutant. We conclude that the formation of succinate is strictly required for the reoxidation of FADH(2) during anaerobiosis, and that it is regulated through the control of FRDS1 expression when oxygen is limiting. Based on these data, we discuss the potential role of fumarate reductase in the regeneration of the FAD-prosthetic group of essential flavoproteins.     

Soybean Flour Lipoxygenase Isozymes Effects on Wheat Flour Dough Rheological and Breadmaking Properties

The effects of soy flour from LOX null mutant isolines and purified LOX isozymes on the rheological and breadmaking properties of a commercial hard wheat flour were investigated. Wheat flours were fortified with either 3% soy mutant flours alone, or in the presence of 0.2% and 1% linoleic acid. Purified LOX 2 isozyme had the greatest effect among LOX isozymes on dough extensibility and strength. Linoleic acid substrate addition reduced dough extensibility and strength. The L2L3 null isoline mutant for L1 resulted in the largest increase in bread volume. A reduction in bread firmness occurred after 5 day storage for all three single null mutant-containing samples in the presence of 1.0% linoleic acid substrate.     

ε-聚赖氨酸高产菌株的选育

依据传统诱变理论和白色链霉菌生物合成ε 聚赖氨酸的特点 ,确立了以枯草芽孢杆菌为敏感菌株 ,抗性突变株筛选和抑菌圈法相结合的初筛方法。在优化诱变条件的基础上 ,以白色链霉菌为出发菌株 ,经紫外线与硫酸二乙酯诱变 ,选育出一株遗传性状稳定 ,遗传标记为AECr+Glyr的ε 聚赖氨酸生产菌株 ,其产量可达 0 79g/L。     

紫外诱变法选育酒酒球菌乙醇胁迫耐受菌株及其发酵性能研究

该研究以酒酒球菌（Oenococcus oeni）SD-2a为研究对象,采用紫外诱变法选育乙醇胁迫耐受突变菌株,评价其在乙醇胁迫条件下的生长能力,并测定其产β-葡萄糖苷酶活性及其在模拟酒中苹果酸、乳酸含量及活菌数的变化。结果表明,分离筛选到3株乙醇胁迫耐受性好的突变菌株,分别编号为UVe1、UVe2、UVe3,在高体积分数乙醇（12%和14%）胁迫环境下,3株突变菌株的生长速度均显著高于出发菌株SD-2a（P＜0.05）;突变菌株UVe2的β-葡萄糖苷酶活性显著高于出发菌株SD-2a和对照菌株L-450（P＜0.05）;在模拟酒中,3株突变菌株的苹果酸降解与乳酸生成速度均显著高于出发菌株SD-2a（P＜0.05）,且突变菌株UVe1和UVe2的存活率显著高于出发菌株SD-2a（P＜0.05）;综上,突变菌株UVe2具有优良商业酒酒球菌的潜力。     

鸟苷生产菌的选育及其发酵条件优化

以枯草芽孢杆菌T1 0 0 1为出发菌株 ,经紫外线、硫酸二乙酯逐级诱变处理 ,选育出腺嘌呤缺陷型 (Ade- )菌株 ,然后经蛋氨酸亚砜 (MSO)、8 氮鸟嘌呤 ( 8 AG)结构类似物平板定向筛选 ,获得鸟苷高产菌株TA2 0 8。通过模式识别法对发酵培养基的组成进行优化 ,同时对发酵条件如温度、pH值等进行了探索。在最优条件下 ,在 5L自控发酵罐上发酵 60h ,可产鸟苷 2 3 .68g/L。