高温海藻糖合酶产生菌的诱变选育及产酶条件研究

通过紫外线复式诱变,筛选到一株高产高温海藻糖合酶的突变菌株 SNO2004-01-3,与出发菌株 Bacillus sp.SNO2004-01相比,其产酶能力提高了3.6倍.传代试验结果证明:突变菌株 SN02004-01-3具有良好的遗传稳定性.突变株发酵条件优化后表明:最适培养时间为16h,最适pH为7.0,最适生长温度为70℃.     

中性海藻糖酶基因缺失对面包酵母耐冷冻性的影响

研究了中性海藻糖酶基因(NTH1和NTH2)对面包酵母耐冷冻性的影响。通过分析中性海藻糖酶基因缺失突变株和亲本菌株(BY6-9α)的生长曲线、生物量、比生长速率、中性海藻糖酶酶活力、胞内海藻糖含量、胞内海藻糖降解速率、冷冻存活率、冷冻前产气量、比发酵力、冷冻后产气量和相对发酵力,结果表明,中性海藻糖酶基因对酵母生物量、比生长速率、胞内海藻糖含量和比发酵力均无显著影响,而相对于亲本菌株(BY6-9α),TL-101(nth1)和TL-201(nth1 nth2)的胞内海藻糖降解速率分别减慢了9.22%和15.60%,冷冻后CO2产生量分别提高了63.04%和65.22%,-20℃冷冻28 d后冷冻存活率分别提高了91.06%和103.01%,冷冻后相对发酵力分别提高了95.95%和116.04%,这充分说明敲除NTH1基因能明显改善酵母菌株的耐冷冻特性,而且酵母的耐冷冻特性与胞内海藻糖降解速率呈负相关。TL-102(nth2)的耐冷冻特性与亲本菌株无显著差异,说明单敲NTH2基因对酵母耐冷冻性无显著影响。     

NaF对酸性海藻糖酶酶活测定的影响及作用机制探讨

研究了氟化钠对酵母酸性海藻糖酶酶活力测定的影响。构建己糖转运蛋白基因缺失突变株TL-105(hxt14△)和TL-106(gal2△),分析氟化钠对菌株BY6-9α(亲本菌株)、TL-103(ath1△)、TL-104(agt1△)、TL-105(hxt14△)和TL-106(gal2△)酸性海藻糖酶酶活力、海藻糖酶分泌、海藻糖分泌、葡萄糖摄取和葡萄糖分泌的影响,结果表明,氟化钠对中性海藻糖酶、酸性海藻糖酶和海藻糖分泌均无影响,说明氟化钠对酸性海藻糖酶酶活力测定的影响与海藻糖酶和海藻糖分泌无关。在稳定期,柠檬酸法测得菌株TL-105(hxt14△)和TL-106(gal2△)的酸性海藻糖酶酶活力分别比亲本菌株提高了17.06%和300.23%,而柠檬酸氟化钠法测得菌株BY6-9α、TL-105(hxt14△)和TL-106(gal2△)的酸性海藻糖酶酶活力却相差不大,同时,在对数期,菌株BY6-9α和TL-103(ath1△)在酸性海藻糖酶酶活力测定前(氟化钠30℃诱导30 min)溶液中葡萄糖含量分别达到108.53±1.39和30.53±1.02 mg/L,这说明氟化钠能显著影响菌株对胞外葡萄糖的摄取并导致胞内葡萄糖的分泌,从而使测得的酸性海藻糖酶酶活力较高。     

增强海藻糖胞内积累提高大肠杆菌耐受性与乙醇产率

为提高产乙醇大肠杆菌对发酵底物和乙醇的耐受性,并提高乙醇发酵性能,以大肠杆菌B0013-1031H为出发菌株,对其海藻糖代谢途径进行改造,获得了敲除海藻糖分解途径的突变株JC31和进一步加强海藻糖合成途径的突变株JC41。突变株JC31和JC41较出发菌株都具有更高的海藻糖合成与积累能力,其中JC41的胞内海藻糖含量可达出发菌株的12倍。与出发菌株相比,突变株JC31和JC41对葡萄糖和乙醇胁迫的耐受性显著提高。进一步引入乙醇合成途径,在葡萄糖质量浓度120 g/L的发酵条件下,菌株JC31-PA表现出最优的发酵性能,其最大乙醇产量为50. 6 g/L,较对照菌株提高了5. 42%;乙醇转化率为48. 72 g/100 g葡萄糖,较对照提高了12. 67%,达到理论转化率的95%。     

海藻糖合酶产生菌株的诱变选育

菌种是微生物工艺过程中的关键,在生产过程中起着举足轻重的作用。以海藻糖合酶产生菌株QD17为出发菌株,经过紫外诱变和化学诱变最终选育出一株高产海藻糖合酶突变株JL-2—24,其海藻糖生产能力由出发菌株的每g干菌体可转化麦芽糖生成海藻糖927．38mg增加到2458．5mg,提高了2倍。将紫外诱变、化学诱变与空白对照可以发现,亚硝基胍诱变的效果最好。诱变株JL-2—24生长良好,经10余代传代试验,传代稳定。     

海藻糖产生菌株的化学诱变选育

从土壤中分离纯化获得9株酵母菌株,并从中得到一株海藻糖产量较高酵母菌株,经初步菌种鉴定属瓶形酵母属。以其作为出发株,得出了以硫酸二乙酯(DES)诱变该株的致死率曲线,诱变剂用量为1%、诱变时间为50min时,该株的致死率达到75%。在该诱变条件下,采用三氯乙酸浸提酵母菌中的海藻糖,用硫酸蒽酮法在波长为620nm测定其海藻糖的含量。突变株经初筛和复筛,得到海藻糖高产菌株,其海藻糖含量较出发株高1.6倍,海藻样含量达到525μg/mL发酵液,干重比为19%,是目前报道海藻糖在酵母中干重比的1.2倍。     

海藻糖产生菌株发酵条件的研究

从土壤中分离得到的一株海藻糖产量较高酵母菌株,对其进行最佳摇瓶发酵条件的研究。最佳的发酵条件为：碳源为2%蔗糖,氮源为0.2%酵母膏和0.1%牛肉膏,发酵初始pH为4.5,接种量为8%,装液量为40mL。该摇瓶发酵酵母内海藻糖的产量为381.190μg/mL,比优化前的180.870μg/mL提高了2.1倍。     

酶法转化淀粉合成海藻糖的初步研究

海藻糖是一种新型多功能食品添加剂,目前其主要生产方法是以淀粉为底物进行酶法转化,本报道了从中国土样中筛选分离到的一株可产生相关酶的细菌的培养特性及初步鉴定结果。同时,对利用该菌株产生的酶转化淀粉合成海藻糖的反应条件进行了初步探讨。     

海藻糖含量与酿酒酵母酒精耐性的关系

对酿酒酵母(Saccharomyces cerevisiae BY-6)中性海藻糖酶缺失突变株(△nthl)的海藻糖含量和酒精耐性之间的关系进行了研究.结果表明,中性海藻糖酶缺失突变株细胞海藻糖含量较高,用18%vol的酒精处理4h后仍可保持高的细胞存活率,并且无呼吸缺陷型的出现,说明高海藻糖含量可以保护细胞膜,防止线粒体DNA丢失和胞内物质的外渗,与酵母的酒精耐性之间存在一定的关系.     

提高乙酸耐受性酿酒酵母JJJ1突变株构建及转录组学分析

目的 构建JJJ1突变株以提高酿酒酵母在发酵过程中的乙酸耐受性,提高发酵效率。方法 本研究采用CRISPR-Cas9基因编辑技术构建酿酒酵母JJJ1Δ突变株,检测突变对酿酒酵母菌株的乙酸耐受性影响,基于转录组学分析突变株耐受乙酸的分子机制。结果 在含有5 g/L乙酸的液体培养基中,酿酒酵母JJJ1Δ存活率是野生型菌株的4.44倍,发酵72 h后酿酒酵母突变株JJJ1Δ的细胞生物量是野生型菌株的1.15倍,酿酒酵母JJJ1Δ的生长延滞期比野生型菌株缩短了30 h;转录组学研究表明,敲除JJJ1基因增强酿酒酵母的代谢、生物调控、膜流动性以及转运活性和电子转移活性,减少酿酒酵母细胞生理过程、细胞连接和拟核功能以及细胞结合功能。结论 敲除JJJ1基因的酿酒酵母突变株通过提高能量代谢和氨基酸合成,降低核糖体生物发生减少细胞生理活动,增强酿酒酵母菌株乙酸耐受性。     

A STUDY OF VIABILITY AND TREHALOSE METABOLISM IN SACCHAROMYCES CEREVISIAE VACUOLAR DEFICIENT MUTANT SKD2

The vacuolar deficient mutant SKD2 and a closely related vacuole complete strain SKD1ρ^? were studied to identify the cause of low trehalose accumulation in SKD2. The present work indicates that low intracellular trehalose levels in SKD2 result from reduced activity of enzymes of the trehalose-6-phosphate synthase/phosphatase complex rather than higher activity of trehalase in the cytosol caused by supposed ineffective compartmentalisation of vacuolar acid trehalase. In vitro studies using crude enzyme extracts from SKD2 and SKD1ρ^? also suggested that loss of viability of SKD2 might result from a fall in free phosphate levels as sugar phosphates accumulated .     

Site-directed mutagenesis improves the thermostability of a recombinant Picrophilus torridus trehalose synthase and efficiency for the production of trehalose from sweet potato starch

A new recombinant Picrophilus torridus TSase (PTTS) has the catalytic ability for the conversion of maltose to trehalose by intramolecular transglucosylation. For industrial applications, the high thermostability of the enzyme would be the most important property for reducing the microbial contamination and lower the production cost. Therefore, in this study, we substituted ten selected proline residues of PTTS which differ from two well-known thermostable TSases. Interestingly, we found that the N503 mutant type, namely N503P-PTTS, showed about 39% higher relative activity than that of the wild type at 65 °C for 120 min. The trehalose yield of mutant N503P-PTTS was 1.3-fold higher than that of the wild type with sweet potato starch as substrate at 50 °C for 4 h. This suggests that the proline site substitution technology used in this study is useful for altering enzyme properties and catalytic efficiency for possible industrial applications.     

Purification and characterization of an acid trehalase from Acidobacterium capsulatum

We purified an acid trehalase (EC 3.2.1.28, alpha,alpha'-trehalose glucohydrolase) from an acidophilic bacterium, Acidobacterium capsulatum. The enzyme was homogeneous based on polyacrylamide gel electrophoresis, and was composed of a single polypeptide chain with a molecular mass of 57 kDa. Maximum trehalase activity was observed at pH 2.5. The acid trehalase exhibited an apparent K(m) of 1.0 mM for trehalose at 30 degrees C and pH 3.0. The trehalase was located in the periplasmic space. The activity of the enzyme was activated by 1.0 mM MnCl2 or CoCl2, and inhibited by 1.0 mM PbCl2, HgCl2, NiCl2, p-chloromercuribenzoate, N-ethylmaleimide, monoiodoacetate, or EDTA. The enzyme showed high specificity for trehalose. It was found that an equimolar mixture of alpha-D-glucose and beta-D-glucose was formed on hydrolysis of trehalose by the trehalase.     

Determination of 1,5-anhydro-D-glucitol on the basis of its inhibitory effect on trehalase activity

1,5-Anhydro-D-glucitol (1,5-AG) was found to inhibit trehalase and trehalose phosphorylase activities competitively, because of its structural similarity with D-glucose. Trehalase from Nocardia sp., one of the most 1,5-AG-sensitive enzymes, was used in the determination of 1,5-AG concentration, which is a useful marker for the diagnosis of diabetes. A good linear relationship was observed between 1,5-AG concentration in the range of 0.02 to 1.0 mM and the extent of trehalase inhibition by 1,5-AG. The 1,5-AG concentration range could be determined by estimating enzymatically the amount of the reaction product, D-glucose, produced by the trehalase.     

Production of Trehalose from Starch by Thermostable Enzymes from Sulfolobus acidocaldarius

The optimum conditions for the production of trehalose from starch were investigated using two thermostable enzymes, maltooligosyl trehalose synthase (MTSase) and maltooligosyl trehalose trehalohydrolase (MTHase), from Sulfolobus acidocaldarius ATCC 33909. The optimum pH was 5.5 and the optimum temperature was 55—57°C using isoamylase from Pseudomonas amyloderamosa as a debranching enzyme. The addition of CGTase to the reaction mixture during the saccharification process caused an increase in trehalose and a decrease in maltose and maltotriose. Isoamylase was better than pullulanase as a debranching enzyme. The yield of trehalose was independent of the type of starch used. Under optimum conditions, the yield of trehalose from corn starch at 30% concentration was more than 82%.     

常温常压等离子诱变结合玉米秸秆水解液驯化酿酒酵母生产生物乙醇

因玉米秸秆水解液抑制物中的糠醛和乙酸通常会抑制酿酒酵母的活力,造成乙醇产量下降。该实验为了获得抗水解抑制物并且提高乙醇产量的优良菌株,以酿酒酵母xp为出发菌株,通过常温常压等离子诱变(atmospheric and room-temperature plasma mutagenesis,ARTP)技术,得到突变体库,并以玉米秸秆水解液为筛选压力,经过25代驯化培养,筛选出优良菌株xp2。该菌体的生物量DCW最高为3. 71 g/L,较原菌的3. 10 g/L上升了19. 68%。生长对数期为6～22 h,稳定期为22～42 h,比原菌的对数期明显提前。稳定期持续时间延长4 h,生长性能优势明显。发酵上清液中的糠醛1. 43 g/L,乙酸1. 21 g/L,较出发菌株发酵上清液的糠醛3. 78 g/L,乙酸1. 65 g/L,分别降低了62. 17%和26. 67%,乙醇产量和平均得率分别为38. 7 g/L和0. 806 g/(L·h),较出发菌株的30. 3 g/L和0. 631 g/(L·h),提高了17. 12%和27. 73%。该实验表明改造菌性状优良,转化糠醛和乙酸的能力明显...     

Wx基因对小麦淀粉合成关键酶活性的影响

为研究Wx基因的缺失对小麦淀粉合成关键酶活性的影响,本试验以8个Wx小麦近等基因系为材料,在灌浆期的10、20、30和40 d取籽粒对ADP-葡萄糖焦磷酸化酶(AGPase)、可溶性淀粉合成酶(SSS)、颗粒结合型淀粉合成酶(GBSS)和淀粉分支酶(SBE)活性进行了测定。结果表明:野生型的AGPase、SSS、GBSS和SBE活性在花后所有时期均最高(GBSS花后40 d除外,处于中间水平),而WxABD型4种淀粉合成相关酶活性几乎在整个灌浆期均低于其余基因型,尤其是WxABD型的GBSS活性尽管也呈现先升高后降低、20 d左右达到高峰的趋势,但其升高或降低的幅度很小,其活性相对较为稳定。研究证明小麦Wx基因缺失对淀粉合成关键酶活性影响较大,依次为Wx-D1Wx-A1WxB1;淀粉合成关键酶活性在花后逐渐增加,以花后20 d最大,此后逐渐降低,到40 d达最低值;WxABD型缺失所有Wx基因,淀粉合成酶活性在花后各时期最低,其中GBSS活性远低于其余基因型且在花后各时期变化较小。     

Nitro‐blue tetrazolium staining of starch‐synthesizing enzymes in developing rice grains

An in situ nitro‐blue tetrazolium (NBT) staining method was used to locate the activities of enzymes involved in starch synthesis in developing grains of field grown rice (Oryza sativa L.) cultivar Tainung 67 and its early ripening mutant SA 419. The results indicated that all the tested enzymes, including sucrose synthase, invertase, hexokinase, ADP‐glucose pyrophosphorylase, UDP‐glucose pyrophosphorylase and starch synthase, were detectable in developing rice endosperms by using NBT staining. The activity of these enzymes was also measurable by using chemical assays. The expression of enzyme activity, as indicated by the formation of blue formazan precipitate, in growing grain of Tainung 67 were visualized in entire endosperm at 11 days after anthesis (DAA). However, NBT staining shifted from central to peripheral region of endosperm after 18 DAA, and the staining disappeared at 25 DAA. Similar staining patterns were also observed in the growing grains of mutant SA 419, but the shift of staining from central to peripheral endosperm occurred at 11 DAA. Electron microscopy examinations showed that the growing patterns of starch granules, sampled from central and peripheral regions of endosperm, varied between two cultivars, with SA 419 growing faster than Tainung 67. Both cultivars showed that the shifts of NBT staining pattern coincided with the changes in the growing pattern of starch granules located in central and peripheral regions of endosperms. The NBT staining results showed that the activities of sucrose to starch conversion enzymes in mutant SA 419 declined and disappeared earlier than its wild‐type Tainung 67 and therefore it ripened earlier than cultivar Tainung 67. Copyright © 2005 Society of Chemical Industry     

出芽短梗霉发酵生产聚苹果酸的代谢通量及关键酶活性分析

野生型出芽短梗霉菌株TKPM00006及其诱变菌株CGMCC30007在相同条件下,使用5 L罐进行聚苹果酸发酵,分析这2株菌在相同发酵状态下发酵中后期代谢网络的代谢流分布和关键酶活变化,对出芽短梗霉合成聚苹果酸的机理进行探究。结果表明,菌株TKPM00006和菌株CGMCC30007的菌体生长情况相似,但产酸量分别为20.54 g/L和30.2 g/L。.通过代谢通量分析及关键酶活性的测定可知,丙酮酸羧化途径及乙醛酸途径是PMLA合成的主要途径,TCA循环途径在发酵后期比较弱,该结论通过添加代谢抑制剂及中间代谢物实验加以证明。酶活分析同时还证明了高产菌株比出发菌株的PMLA合成能力强主要是因为丙酮酸羧化途径的加强。根据实验分析可在丙酮酸节点进行靶点改造或通过发酵调控改变丙酮酸节点处碳架的分配,通过加强丙酮酸羧化途径来减少因副产物的生产而造成的碳架流失,达到增加聚苹果酸生物合成的目的。     

产脂肪酶菌株的常压室温等离子体诱变及高通量筛选方法的建立

脂肪酶由于可催化的反应种类和底物类别多,且具有位置选择性和异构体选择性等特点而常用于结构酯的合成和油脂改性等领域。丝孢酵母(Trichosporon sp.)是一种产脂肪酶菌种,但生产能力偏低是限制该菌实现工业化生产的重要因素。本研究利用常压室温等离子体(ARTP)对丝孢酵母野生菌进行诱变处理,并建立了96孔板培养结合对硝基苯酚棕榈酸酯(P-NPP)法测定酶活力的高通量筛选方法,实现了60个突变菌株的初筛。以酶活力为筛选指标时,突变率和正突变率分别为51.7%和28.3%。8株初筛菌株的摇瓶发酵结果显示,A13和A5的产酶提高最显著,培养96 h后分别比野生菌增加2.64倍和1.54倍,且2个突变菌株的遗传稳定性良好。对比研究发现,突变菌株A13相较野生菌的最大优势在于提前24 h便能达到最高产酶量。