Accumulation of 2-Keto-L-Gulonate at 33℃ by a Thermotolerant Gluconobacter Oxydans Mutant Obtained by Ion Beam Implantation

To obtain thermotolerant mutants of G. oxydans, which can enhance the transformation rate of L-sorbose to 2-Keto-L-gulonate （2-KLG） at 33℃ in a two-step process of vitamin C manufacture, ion beam was used as a mutation source. Gluconobacter oxydans GO and Bacillus megaterium B0 were used in this study. The original strain Gluconobacter oxydans GO was mutated by the heavy ion implantation facility at the Institute of Plasma Physics, Chinese Academy of Sciences. Several mutants including Gluconobacter oxydans GI13 were isolated and cocultured with Bacillus megaterium B0 at 33℃ in shaking flasks. The average transformation rate of the new mixed strain GI13-B0 in per gram-molecule reached 94.4% after seven passages in shaking flasks, which was increased by 7% when compared with the original mixed strain G0-B0 （Gluconobacter oxydans GO and Bacillus megaterium B0）. Moreover, the transformation rate of I13B0 was stable at 94% at temperatures ranging from 25℃ to 33℃, which would be of much value in reducing energy consumption in the manufacture of L-ascorbic acid, especially in the season of summer. To clarify some mechanism of the mutation, the specific activities of L-sorbose dehydrogenase in both GO and GI13 were estimated.     

Purification and Characterization of Organic Solvent and Detergent Tolerant Lipase from Thermotolerant Bacillus sp. RN2

The aim of this study was to characterize the organic solvent and detergent tolerant properties of recombinant lipase isolated from thermotolerant Bacillus sp. RN2 (Lip-SBRN2). The isolation of the lipase-coding gene was achieved by the use of inverse and direct PCR. The complete DNA sequencing of the gene revealed that the lip-SBRN2 gene contains 576 nucleotides which corresponded to 192 deduced amino acids. The purified enzyme was homogeneous with the estimated molecular mass of 19 kDa as determined by SDS-PAGE and gel filtration. The Lip-SBRN2 was stable in a pH range of 9-11 and temperature range of 45-60 °C. The enzyme was a non metallo-monomeric protein and was active against pNP-caprylate (C8) and pNP-laurate (C12) and coconut oil. The Lip-SBRN2 exhibited a high level of activity in the presence of 108% benzene, 102.4% diethylether and 112% SDS. It is anticipated that the organic solvent and detergent tolerant enzyme secreted by Bacillus sp. RN2 will be applicable as catalysts for reaction in the presence of organic solvents and detergents.     

耐热嗜碱蛋白酶基因的克隆、表达及其酶学性质分析

碱性蛋白酶是一类重要的工业酶制剂。为进一步提高碱性蛋白酶在高温碱性条件下的活力,增强其工业应用价值,本文从地衣芽胞杆菌CCTCC M2018539中首先通过PCR扩增获得碱性蛋白酶编码基因aprE539并克隆入表达载体pND-113中,在枯草芽胞杆菌WB600中实现碱性蛋白酶AprE539的表达;重组酶AprE539经硫酸铵沉降(30%~70%)、透析、DEAE阴离子交换和超滤获得纯酶,并以SDS-PAGE电泳测定AprE539的分子量大小。结果表明,AprE539的分子量大小为30 kDa。进一步对其酶学性质研究表明:AprE539的最适作用pH为11.0,最适作用温度为65~70℃;在pH6.0~12.0范围内具有较好的稳定性;在60℃保温1 h后酶活剩余70%;Cu²⁺、Mn²⁺、Ca²⁺和Mg²⁺对酶活有明显促进作用。AprE539在氨基酸序列上仅有2个氨基酸残基与碱性蛋白酶2709存在差异,但其酶学特征差异明显,为后续进一步探究其酶学性质差异性奠定基础。     

Cloning, Expression, and Characterization of Thermotolerant Manganese Superoxide Dismutase from Bacillus sp. MHS47

A superoxide dismutase gene from thermotolerant Bacillus sp. MHS47 (MnSOD47) was cloned, sequenced, and expressed. The gene has an open reading frame of 612 bp, corresponding to 203 deduced amino acids, with high homology to the amino acid sequences of B. thuringiensis (accession no. EEN01322), B. anthracis (accession no. NP_846724), B. cereus (accession no. ZP_04187911), B. weihenstephanensis (accession no. YP_001646918), and B. pseudomycoides. The conserved manganese-binding sites (H28, H83, D165, and H169) show that MnSOD47 has the specific characteristics of the manganese superoxide dismutase (MnSOD) enzymes. MnSOD47 expressed an enzyme with a molecular weight of approximately 22.65 kDa and a specific activity of 3537.75 U/mg. The enzyme is active in the pH range 7-8.5, with an optimum pH of 7.5, and at temperatures in the range 30-45 °C, with an optimum temperature of 37 °C. Tests of inhibitors and metal ions indicated that the enzyme activity is inhibited by sodium azide, but not by hydrogen peroxide or potassium cyanide. These data should benefit future studies of MnSODs in other microorganisms and the biotechnological production of MnSOD47, and could also be used to develop a biosensor for the detection of antioxidants and free radical activity. In the future, this basic knowledge could be applicable to the detection of cancer risks in humans and therapeutic treatments.     

Screening of thermotolerant yeast by low-energy ion implantation for cellulosic ethanol fermentation

One thermotolerant mutant, ST-1559, was selected after mutagenizing with ion beam implantation. The results showed that ST-1559 could grow well under 45°C,and its ethanol yield reached 109.46 g/L when the glucose content was 250 g/L in the fermentation medium. When corn straw which was pretreated by NaOH was used as substrate in fermentation experiment under 45°C, it produced 13.48 g/L ethanol, and 82.17% of the cellulose was achieved. These results suggest that ST-1559 had advantage in simultaneous saccharification and fermentation.     

架空线路输电能力计算

基于对架空输电线路的导线选型方法与导线载流量计算方法的分析,得出了决定导线输电能力的主要因素,给出了 220、500 kV架空输电线路常用导线热稳极限载流量的推荐值,并提出了提高江苏电网架空线路输电能力的措施与建议。     

三株野生型耐高温酵母的耐性对比研究

对三株野生型产酒精耐高温马克斯克鲁维酵母(Kluyveromyces marxianus)在不同温度、pH、酒精浓度、糖浓度下的生理耐性进行了研究,并与安琪耐高温酵母进行了比较,得出的结论为:三株野生型耐高温酵母K. marxianus GX-17,K. marxianus HN-79,K.marxianus HN-138高温条件下的生长和产酒精能力明显高于安碘酵母;K marxianus GX-17对pH变化的适应力较安琪酵母强;野生型耐高温酵母的耐糖能力略低于安琪酵母;安琪酵母的耐酒精能力明显高于野生型耐高温酵母.     

特定底物技术快速检测水中大肠菌群相关指标

大肠菌群(Coliforms)、耐热大肠菌群(thermotolerant coliforms)和埃希氏大肠杆菌(E.Coli)都是判断水体是否存在粪便污染的重要微生物指标。文中介绍了一种特定底物技术可在 24h之内同时检测大肠菌群和埃希氏大肠杆菌数量,如果提高培养温度至44.5±0.2℃,则可检测耐热大肠菌群数量。     

产酯酵母和耐高温酵母原生质体形成与再生条件研究

选取脱壁预处理时间、蜗牛酶浓度、酶解时间三因素设计三因素三水平正交试验来研究产酯酵母和耐高温酵母原生质体形成与再生条件。结果表明,产酯酵母原生质体形成与再生最佳条件为：预处理20min、1%蜗牛酶酶解30min;耐高温酵母为：预处理10min,1.5%蜗牛酶酶解60min。     

耐高温酵母菌的筛选及其乙醇发酵特性

采用平板分离法,从29 份新鲜采集的土样中分离获得64 株耐高温酵母菌株,并对其在高温条件下的乙醇发酵性能进行了分析比较。26S rDNA D1/D2区域序列测定和生理特征分析结果表明,这些酵母菌株在亲缘关系上可归类于6 个属7 个种,分别为热带假丝酵母（Candida tropicalis）（占总分离株的39.1%）、马克斯克鲁维酵母（Kluyveromyces marxianus）（占23.4%）、东方伊萨酵母（Issatchenkia orientalis）（占29.7%）、季也蒙毕赤酵母（M. guilliermondii）（占1.6%）、Kazachstania bovina（占1.6%）、Candida palmioleophila（占1.6%）和酿酒酵母（Saccharomyces cerevisiae）（占3.1%）。其中,马克斯克鲁维酵母的耐高温能力和乙醇发酵能力最强;40 ℃条件下发酵72 h后,发酵液中乙醇体积分数最高可达6.56%,显著高于相同条件下其他耐高温酵母的乙醇产量。上述结果表明,马克斯克鲁维酵母在高温乙醇发酵过程中具有明显优势,可作为利用生物质发酵生产乙醇的优良候选菌株。