弹性蛋白酶产生菌的化学诱变选育

从湖北工业大学巡司河附近的土壤和污水中采集到的3个样品，经富集培养，分离到14株能产生弹性蛋白酶的菌株，反复初筛、复筛后，分离到一株产胞外弹性蛋白酶较高且稳定的细菌，经初步鉴定属枯草芽孢杆菌（Bacillus subtilia）。该菌的生长温度为37℃，属革兰氏阳性菌，要求培养基的pH为7．0，对该菌进行化学诱变，诱变剂选用硫酸二乙酯（DES）。诱变处理后绘制致死曲线。诱变浓度为1％，诱变时间为50min时，该菌的致死率迭到77．5％。在此诱变剂量下，选透明圈直径与菌落直径之比最大的菌落进行酶活测定，在波长为495nm同一稀释倍数下，诱变前该菌酶活为74．25U／mL。诱变后的酶活为98．55U／mL，较出发菌株提高了33％。     

产克拉维酸的棒状链霉菌突变株的选育

以棒状链霉菌(Streptomyces clavuligerus),B71为出发菌株,通过紫外线诱变育种,采用琼脂块法初筛和摇瓶发酵复筛,选育出2株甘油耐受性正向突变株S.clavuligerus B71—14和S.clavuligerus B71-49,在摇瓶条件下,其克拉维酸产量与出发菌株S.davuligerus B71相比,分别提高了90.6%和88.8%,并具有良好的遗传稳定性。     

Site-directed mutation of β-galactosidase from Streptococcus thermophilus for galactooligosaccharide-enriched yogurt making

β-Galactosidase is one of the most important enzymes used in dairy processing. It converts lactose into glucose and galactose, and also catalyzes galactose to form galactooligosaccharides (GOS), so-called prebiotics. However, most of the β-galactosidases from the starter cultures have low transgalactosylation activities, the process that results in galactose accumulation in yogurt. Here, a site-directed mutation strategy was attempted, to genetically modify β-galactosidase from Streptococcus thermophilus. Out of 28 Strep. thermophilus strains, a β-galactosidase gene named bgaQ, encoded for high β-galactosidase hydrolysis activity (BgaQ), was cloned from the strain Strep. thermophilus SDMCC050237. It was 3,081 bp in size, with 1,027 deduced amino acid residuals, which belonged to the GH2 family. After replacing the Tyr⁸⁰¹ and Pro⁸⁰² around the active sites of BgaQ with His⁸⁰¹ and Gly⁸⁰², the GOS synthesis of the generated mutant protein BgaQ-8012 increased from 20.5% to 26.7% at 5% lactose, and no hydrolysis activity altered obviously. Subsequently, the purified BgaQ or BgaQ-8012 was added to sterilized milk inoculated with 2 starters from Strep. thermophilus SDMCC050237 and Lactobacillus delbrueckii ssp. bulgaricus ATCC11842. The GOS yields with added BgaQ or BgaQ-8012 increased to 5.8 and 8.3 g/L, respectively, compared with a yield of 3.7 g/L without enzymes added. Meanwhile, the addition of the BgaQ or BgaQ-8012 reduced the lactose content by 49.3% and 54.4% in the fermented yogurt and shortened the curd time. Therefore, this study provided a site-directed mutation strategy for improvement of the transgalactosylation activity of β-galactosidase from Strep. thermophilus for GOS-enriched yogurt making.     

MPMS诱变结合抗生素抗性选育多杀菌素高产菌株

采用多功能等离子体诱变系统(MPMS)对菌株ASAGF 13-8-1进行诱变,并通过链霉素、庆大霉素、利福平、氯霉素四种抗生素抗性筛选多杀菌素高产菌株。利用96孔板发酵培养结合生物检测的快速方法进行高产菌株高通量初筛,摇瓶发酵进行复筛。通过5轮复筛验证,获得1株产量较出发菌株提高28.68%且遗传稳定的突变菌14-2。比较MPMS诱变和MPMS诱变结合0.9μg/mL链霉素、14μg/mL庆大霉素、400μg/mL利福平、2μg/mL氯霉素的四重抗性筛选对出发菌株ASAGF 13-8-1的作用效果,结果显示MPMS诱变结合抗生素抗性筛选更有利于多杀菌素高产菌株的选育。     

消除葡萄糖效应提高酵母降糖速度的研究

葡萄糖阻遏效应是造成啤酒发酵缓慢的原因之一,利用葡萄糖结构类似物可以筛选出抗葡萄糖阻遏效应的菌株,从而提高酵母的发酵速度,常见的葡萄糖结构类似物有2-脱氧-D-葡萄糖和N-乙酰基-D-葡糖胺。本试验以降糖速度为发酵速度指标,利用固体培养以及液体发酵的方法来选择合适的葡萄糖结构类似物,并用紫外诱变法筛选出降糖速度快的突变菌株28号,通过遗传稳定性试验研究了所选菌株的稳定性。     

高产无色素普鲁兰糖突变菌株P1012的选育及发酵性能研究

本研究旨在选育出高产普鲁兰多糖且黑色素分泌缺失的菌株,为普鲁兰的发酵生产提供宝贵的菌种资源。研究采用三种诱变剂(紫外线(UV)、亚硝基胍(NTG)、硫酸二乙酯(DES))对出发菌株茁芽短梗霉P23进行多轮诱变处理。通过从PDA平板上挑选出色素低且黏度大的菌落作为候选菌株,并经红外光谱(FT-IR)分析其胞外多糖的构型。结果筛选出13株候选菌株,其中菌株P1012于PDA平板上培养7 d形成的菌落为白色,96 h发酵液呈乳白色,且吸光值(OD654 nm表示色素的相对含量)达到0.048,其胞外多糖经红外光谱检测可初步分析为普鲁兰多糖,与出发菌株P23相比,菌株P1012主要表现在细胞合成色素上的缺失。发酵培养后,测得普鲁兰产量为28.01 g/L,糖转化率达到56.02%,糖转化率比出发菌株高出28.8%。表明菌株P1012可以作为生产普鲁兰多糖的候选菌株。     

酒曲根霉的诱变育种

本试验对根霉RA-1菌株进行NaNO2诱变和紫外线诱变,得到一株低聚糖酶活力较高的菌株,酶活力达到4360(U/g干曲),比出发菌株提高403.7%,液化酶比出发菌株提高到486.2%,达到1953(U/g干曲),糖化酶提高到出发菌株的567.8%,达到2.55×105(U/g干曲),将该菌株进行斜面传代,产酶稳定。     

Antioxidative Activity and Safety of 50% Ethanolic Red Bean Extract (Phaseolus radiatus L. var. Aurea)

ABSTRACT: This study evaluated the antioxidative activities of 50% ethanolic extract from red bean (Phaseolus radiatus L. var. Aurea). The antioxidative activities, including α,α‐diphenyl‐β‐picryl‐hydrazyl (DPPH) radicals scavenging effects, Fe²⁺‐chelating ability, and reducing power, were studied in vitro. The antioxidative activity was found to increase with the concentration of the extract to a certain extent and then level off as the concentration further increased. Compared with commercial antioxidants, the red bean extract showed less scavenging effect on the DPPH radical and less reducing power than α‐Tocopherol and BHT, but better Fe²⁺‐chelating ability. No mutagenic effect toward any tester strains was found in the 50% ethanolic extract of red bean.     

醇脱氢酶KpADH的127位点对催化活性和对映选择性的影响

醇脱氢酶可用于合成手性化合物,在医药、材料等领域应用广泛。来源于多孢克鲁维酵母（Kluyveromyces polysporus）的醇脱氢酶KpADH同时具有还原(4-氯苯基)-(吡啶-2-基)-甲酮（CPMK）和氧化异丙醇、1,4-丁二醇的活性。通过分子对接和结构分析,发现Y127是紧靠底物的关键位点。本文通过对127位点定点饱和突变来提高催化活性和对映选择性,突变体Y127V和Y127I还原CPMK的比活力达到95.0U/mg和84.0U/mg,分别是野生型的6.5倍和5.8倍,突变体Y127L催化CPMK生成(R)-(4-氯苯基)-(吡啶-2-基)-甲醇[(R)-CPMA]的e.e.值由82％提高到99.2％。同时,127位点的突变提高了对醇类底物的氧化活性,突变体Y127I对异丙醇的比活力是野生型的1.46倍,而Y127C更青睐于对1,4-丁二醇的催化氧化,其比活力是野生型的3.00倍。分子间作用力分析表明,Y127L与底物CPMK间增加的氢键和π-π作用力稳定了底物构象,进一步提高了还原CPMK的对映选择性。本文为醇脱氢酶KpADH的分子改造和机制解析提供了指导,提高了该酶的工业应用潜力。     

紫外诱变筛选磺胺敏感性菌株及在牛奶磺胺抗生素检测中的应用

本研究利用紫外诱变方法筛选到两株具有磺胺敏感性的菌株H1、H2。DNA鉴定为芽孢杆菌属嗜热脂肪地芽孢杆菌种。将H1、H2作为指示菌,进行牛奶磺胺残留总量检测,发现这两株菌对不同种类的磺胺药物具有不同的灵敏度。当H1、H2处理后芽孢悬液菌落总数比为3:4时,检测试剂盒有较好的灵敏度,分别为磺胺:30μg/L、磺胺嘧啶:30μg/L、磺胺甲嘧啶:30μg/L、磺胺二甲嘧啶:30μg/L、氨苯磺胺:60μg/L、磺胺噻唑:45μg/L、磺胺氯哒嗪:80μg/L、磺胺甲噁唑:75μg/L、磺胺胍:75μg/L。在此比例下对两株菌同时传代,探究菌株传代对检测试剂盒灵敏度的影响,结果表明以第四代菌为指示菌时,该检测试剂盒丧失对氨苯磺胺和磺胺胍的敏感性;第六代丧失对磺胺氯哒嗪、磺胺甲噁唑的敏感性;对于磺胺、磺胺嘧啶、磺胺甲嘧啶、磺胺噻唑的敏感性可稳定到第七代。通过与国内外商业试剂盒对比,本试剂盒灵敏度高于国内试剂盒,检测时间优于国外试剂盒。