Purification and properties of dihydroxyacetone reductase and 2,3-butanediol dehydrogenase from Candida utilis CBS 621

Candida utilis CBS 621 contained four different enzymes capable of reducing carbonyl compounds such as dihydroxyacetone, acetoin, diacetyl, acetol, methylglyoxal and acetone, namely alcohol dehydrogenase, acetone reductase, dihydroxyacetone reductase and 2,3-butanediol dehydrogenase. The dihydroxyacetone reductase of C. utilis did not oxidize glycerol, thus providing evidence that this enzyme cannot function as a glycerol-2-dehydrogenase during growth of the yeast on glycerol. This enzyme may, however, play a role in the assimilation of 2,3-butanediol by C. utilis. The organism also contained a separate 2,3-butanediol dehydrogenase which was unable to reduce dihydroxyacetone. Both dihydroxyacetone reductase and 2,3-butanediol dehydrogenase were present at very high activities during growth of C. utilis on a variety of substrates, including 2,3-butanediol.     

产酸克雷伯氏菌乳酸脱氢酶基因敲除提高2,3-丁二醇产量

基于传统发酵工艺,通过在发酵阶段进行菌种强化结合在蒸馏烤酒阶段进行工艺优化（头尾酒浸提菌剂后返回地锅蒸馏）提升酱香型白酒基酒中四甲基吡嗪（TTMP）的含量。结果表明,采用菌种强化、工艺优化及二者相结合的方法分别能将酱香型白酒基酒中四甲基吡嗪的含量提高160.71%、85.75%和202.75%。感官评价结果表明,菌种强化结合工艺优化的技术对基酒的感官风味影响不大。该研究提供了一种适合企业落地应用的进一步提高酱香型白酒基酒中四甲基吡嗪含量的方法和思路。     

Metabolism of 2,3-butanediol in yeasts

The biochemistry and physiology of 2,3-butanediol metabolism has been studied in a number of selected yeast species. Candida utilis CBS 621 exhibited diauxic growth on 2,3-butanediol. The first phase was characterized bu the utilization of the two optically active stereoisomers and associated accumulatoin. In the second phase of growth the meso-form of 2,3-butanediol was utilized together with acetoin. An attempt is made to explain these phenomena on the basis of the substrate specificity of the two enzymes which oxidize 2,3-butanediol in C. utilis. Although whole cells oxidized acetoin and diacetyl at high rates, attempts to identify the enzymes responsible for these oxidations were unsuccessful. In C. utilis and other yeasts metabolism of 2,3-butanediol probably involves a cleavage of the substrate into C₂-units which are assimilated by the glyoxylate cycle. In the few yeasts which have been found to grow on 2,3-butanediol differences may be encountered with respect to the substrate specificity for the three stereoisomers of 2,3-butanediol. For example, Candida salmanticensis CBS 5121 showed no diauxis growth and utilized only two of three stereomers.     

利用玉米秸秆水解液发酵生产2,3-丁二醇

利用1株克雷伯氏菌(Klebsiella oxytocaZU-03)发酵玉米秸秆水解液生产2,3-丁二醇,使水解液中的己糖和戊糖都得到了有效利用。研究结果表明,当玉米秸秆水解液中Na2SO4浓度低于20 g/L时,对2,3-丁二醇的发酵无明显影响。乙酸和乙醇是主要的发酵副产物,当浓度分别超过20 g/L和5 g/L时,对发酵的抑制作用较明显。玉米秸秆水解液发酵生产2,3-丁二醇的适宜初始pH值为6.0-6.5,初始总糖浓度为80-100 g/L。在总糖浓度80 g/L(含葡萄糖47.25 g/L,木糖32.75 g/L),初始pH值6.0,温度30℃的条件下发酵64 h,总糖利用率达到99.36%,2,3-丁二醇的质量浓度为37.20 g/L,产率为0.468 g/g(总糖),达到理论最大产率的94%。     

代谢工程改造酿酒酵母生产2,3-丁二醇的研究进展

酿酒酵母（Saccharomyces cerevisiae）作为“细胞动力车间”能够生产生物燃料和工业产品,2,3-丁二醇（2,3-BD）是其重要的一 种次级代谢产物,广泛应用于工业化生产。 但野生型S. cerevisiae合成2,3-BD的效率低,制约着工业化生产进程,因此,应用代谢工程 法优化代谢途径来解决这一问题极其重要。 该研究对近年来利用代谢工程手段来提高S. cerevisiae 2,3-BD产量的策略进行了总结, 主要包括：过表达2,3-BD合成途径中的关键酶编码基因;敲除编码乙醇、甘油、乙酸等分支途径的关键酶基因;利用可再生能源合成 2,3-BD;应用辅因子工程手段对S. cerevisiae合成2,3-BD的代谢网络进行重新设计及合理改造等。 对S. cerevisiae未来的研究方向进行 了展望,为实现生物燃料2,3-BD的工业化生产提供了保障。     

利用菊芋菊粉制备R,R-2,3-丁二醇发酵工艺条件的优化

为了提高Paenibacillus polymyxa ZJ-9发酵菊芋菊粉制备R,R-2,3-丁二醇的产量、减少生产成本,考察了发酵温度、pH、接种量和溶氧(搅拌转速)对菌体生长繁殖、菊粉的利用率和R,R-2,3-丁二醇合成的影响。实验结果表明,Paenibacillus polymyxa ZJ-9发酵的最适温度、pH和接种量分别为30℃、6.0和8%。通过对不同搅拌转速条件下的菌体比生长速率μ_x和产物比合成速率μ_p分析,制定了一种两步溶氧控制策略发酵生产R,R-2,3-丁二醇:在发酵前24h,控制搅拌转速调控为240r/min有利于菌体生长,而24h后,控制发酵液搅拌转速调控为160r/min,能有效促进R,R-2,3-丁二醇合成,在上述条件下,R,R-2,3-丁二醇的产量高达27.83g/L,比恒定搅拌转速条件的最大值提高了10.15%。     

Enhanced 2,3-butanediol production by addition of acetic acid in Paenibacillus polymyxa

By the addition of 150 mM acetate into a batch culture at an initial pH of 6.8, the production of 2,3-butanediol (BDL) by Paenibacillus polymyxa reached 248 mM, yielding 0.87 mol.mol(-1) glucose, where the ratio of acetate consumed to glucose consumed (A/C ratio) was calculated as 0.35 mol acetate mol(-1) glucose. Therefore, a fed-batch culture was carried out by feeding glucose and acetate at a ratio of 0.35 mol acetate mol(-1) glucose. In the fed-batch culture performed at pH 6.8, BDL production reached 637 mM, yielding 0.81 mol.mol(-1) glucose, although the A/C ratio was only 0.18 mol acetate mol(-1) glucose. By decreasing pH to 6.3 in the fed-batch culture, BDL production reached 566 mM, yielding 0.88 mol.mol(-1) glucose and the A/C ratio was 0.32 mol acetate mol(-1) glucose. The optical purity of BDL, which was expressed as enantiomeric excess, was retained at more than 98% of the (R, R)-stereoisomer at the end of culture, which was comparable to that without acetate addition.     

Characterization of dimethyl sulphoxide reductase from brewing yeast

Dimethyl sulphoxide (DMSO) reductase is present in all brewing yeast strains tested, but is more readily detectable using activity stains on polyacrylamide gels than through direct spectrophotometric assay of crude extracts. The enzyme is detectable at higher activities in yeast cultivated at lower temperatures and this is because of increased expression of the primary gene that codes for the enzyme in yeast, MXR1. DMSO somewhat increases enzyme expression, notably in lager yeast by up to 70%, whereas methionine suppresses it. Both ale and lager strains produce enzymes of ~100,000 in molecular weight, but ion exchange chromatography divides the activity for both ale and lager yeasts into two fractions with distinct properties (heat tolerance, pH optimum, kinetic parameters). Copyright © 2017 The Institute of Brewing & Distilling     

植物乳杆菌亚硝酸盐还原酶基因在大肠杆菌中表达

构建乳杆菌亚硝酸盐还原酶基因的重组质粒并表达。以植物乳杆菌基因组DNA为模板,PCR扩增亚硝酸盐还原酶基因,连接到表达载体pET-32a(+)上,构建的重组载体转入大肠杆菌BL21(DE3)中,经IPTG诱导表达后,菌体总蛋白经SDS-PAGE鉴定显示重组质粒诱导表达产物有特异性蛋白条带。该表达产物经细菌亚硝酸盐还原酶试剂盒测定显示有明显的酶活性。实验结果为深入研究植物乳杆菌亚硝酸盐还原酶提供有益的参考。     

蜡样芽孢杆菌Bacillus cereus LJ01中亚硝酸盐还原酶的基因克隆、表达和纯化

利用克隆表达技术,对一种蜡样芽孢杆菌Bacillus cereus LJ01的亚硝酸盐还原酶（nitrite reductase,NiR）基因进行克隆、原核表达,利用Ni柱亲和层析和DEAE Sepharose Fast Flow交换层析对表达的重组NiR进行纯化,分析重组酶的性质。研究结果显示,该NiR含有一个1?623?bp的开放阅读框,编码540?个氨基酸序列,重组NiR的分子质量约为67?ku;该酶中同时存在铁离子和铜离子,且含量分别为51.0?mg/kg和184.5?mg/kg;圆二色谱结果显示α-螺旋结构在重组NiR中所占比例最大。     

四川泡菜中植物乳杆菌亚硝酸盐还原酶基因克隆与表达

目的:对植物乳杆菌中亚硝酸盐还原酶基因进行克隆并表达,并测定酶的活力。方法:根据Gen Bank中亚硝酸盐还原酶（NIR）基因序列,设计引物。提取植物乳杆菌的DNA,采用PCR技术扩增nir基因,TA克隆构建获得重组质粒p MD19-nir,分析其核苷酸序列同源性。通过双酶切连接到表达载体p ET-32a（+）中,获得重组表达载体p ET-32a-nir,构建成功后转化到E.coli BL（DE3）中进行表达研究。经IPTG诱导表达,得重组蛋白,超声波破碎,提取粗酶液,测定酶活力。结果:克隆的目的基因序列长度为1638 bp。获得重组蛋白分子量为80 ku,酶活力为4.2 U/mg。结论:成功克隆了植物乳杆菌中的亚硝酸盐还原酶基因,成功的导入到E.coli BL（DE3）中,表达产物有酶活。      

木糖发酵高产2,3-丁二醇菌株的选育

以肺炎克雷伯氏菌(Klebsiella pneumoniae)CICC10011为出发菌株,进行紫外诱变选育。通过2,3-丁二醇抗性和产酸能力对诱变菌株进行初筛,发酵后23,-丁二醇产量检测进行复筛,获得6株高产2,3-丁二醇的肺炎克雷伯氏菌,其中产量最高的一株诱变菌U3-17,与出发菌株相比,产量提高了21.5%。在摇瓶批式补料发酵中,诱变株U3-17的23,-丁二醇产量达49.3 g/L。     

产2,3-丁二醇的酿酒酵母菌的筛选及其鉴定

酿酒酵母在自然界中广泛分布,尤其在发酵食品等领域应用广泛。2,3-丁二醇作为其利用葡萄糖发酵的产物之一,也有着极大地发展潜力。该研究利用肌酸显色法,对39株W系列菌株以及在六种不同地点分离出的菌株进行筛选,测定其乙偶姻产量,并结合形态学与分子生物学鉴定优选菌株。结果表明,菌株W141被鉴定为酿酒酵母（Saccharomyces cerevisiae）,发酵72 h时,2,3-丁二醇脱氢酶酶活力最高为0.025 7 U/mg,2,3-丁二醇产量最大为1.6 g/L,可作为2,3-丁二醇的生产菌株。     

An enzyme possessing both glutathione-dependent formaldehyde dehydrogenase and S-nitrosoglutathione reductase from Antrodia camphorata

Glutathione-dependent formaldehyde dehydrogenase (GFD or GSH-FDH) plays important roles in formaldehyde detoxification and antioxidation. A gene encoding GFD from Antrodia camphorata was identified based on sequence homology. The deduced amino acid sequence of 378 amino acid residues is conserved among the reported GFDs. To characterise the Ac-GFD, the coding region was subcloned into a vector pET-20b(+) and transformed into Escherichia coli. The recombinant GFD was expressed and purified by Ni²⁺-nitrilotriacetic acid Sepharose. This purified enzyme showed a single band on a 10% SDS–PAGE. The enzyme retained 50% GFD activity after heating at 50 °C for 5 min. The enzyme is bifunctional. In addition to the GFD activity, it also functions as an effective S-nitrosoglutathione reductase (GSNOR) presumably to safeguard against nitrosative stress. The K_m values for S-hydroxymethylglutathione and S-nitrosoglutathione were 1.20 and 0.28 mM, respectively.     

利用絮凝及双水相萃取分离纯化发酵液中的R,R-2,3-丁二醇

针对Paenibacillus polymyxa ZJ-9一步法发酵菊芋菊粉粗提液制备R,R-2,3-丁二醇的发酵液特点,利用壳聚糖对该发酵液进行絮凝研究。结果表明,壳聚糖分子量、壳聚糖用量、助凝剂海藻酸钠用量、pH和搅拌时间分别为:43.5 kDa、0.75 g/L、0.125 g/L、5.0和15 min时,絮凝效果最佳,在此工艺条件下,发酵液中菌体和蛋白质的絮凝率分别高达89.46%和78.93%,而R,R-2,3-丁二醇保留率约为98.54%。利用双水相萃取技术对絮凝后的发酵液中R,R-2,3-丁二醇进行了分离,结果表明,异丙醇/硫酸铵双水相体系萃取效果最好,当异丙醇和硫酸铵的用量分别约为33%和30%(w/w)时,R,R-2,3-丁二醇在上相的分配系数和萃取率最高,分别约为7.96和89.40%,且异丙醇/硫酸铵双水相体系能够有效萃取分离絮凝后的发酵液中不同含量的R,R-2,3-丁二醇。絮凝和双水相萃取技术分离发酵液中R,R-2,3-丁二醇具有针对性强、效率高、成本低等优点,适用于工业化生产。     

Molecular cloning and sequence analysis of the Zygosaccharomyces rouxiiLEU2 gene encoding a beta-isopropylmalate dehydrogenase.

A DNA fragment carrying the LEU2 gene of osmotolerant yeast Zygosaccharomyces rouxii was isolated. The sequenced DNA fragment (2630 bp) contained two ORFs; one of them (1086 bp long, predicting a protein of 362 amino acids) shared a high degree of similarity with LEU2 genes of other yeast species. The cloned DNA fragment fully complemented the leu2 mutations of Saccharomyces cerevisiae and Z. rouxii.     

高效液相色谱法检测枯草芽孢杆菌发酵液中乙偶姻和2,3-丁二醇含量

枯草芽孢杆菌可发酵代谢产生乙偶姻和2,3-丁二醇(两者可逆转化),本研究建立了用高效液相色谱法同时检测发酵液中乙偶姻和2,3-丁二醇的方法。对比了不同色谱柱和流动相配比对乙偶姻和2,3-丁二醇含量的测定结果,进一步检测枯草芽孢杆菌发酵液中乙偶姻和2,3-丁二醇含量。结果表明:采用Bio-Rad Aminex HPX-87H Column分析;流动相:0.005 mol/L的硫酸溶液;流速:0.5 mL/min;柱温:28℃;示差检测器,在30 min内完成样品检测,乙偶姻和2,3-丁二醇标准曲线相关系数分别为0.9997和0.9998,精密度标准偏差分别为1.76%和1.74%,稳定性标准偏差分别为1.2%和1.6%,回收率为98.93%~102.10%,相对标准偏差≤ 2%。此方法简单准确,检测目标物浓度为50 g/L,检测结果重现性好,发酵液中乙偶姻和2,3-丁二醇含量呈负相关,乙偶姻的含量随着2,3-丁二醇的增加而降低,该方法适合枯草芽孢杆菌发酵液中乙偶姻和2,3-丁二醇的发酵检测。     

Characterization of Ypr1p from Saccharomyces cerevisiae as a 2-methylbutyraldehyde reductase

The metabolism of aldehydes and ketones in yeast is important for biosynthetic, catabolic and detoxication processes. Aldo-keto reductases are a family of enzymes that are able to reduce aldehydes and ketones. The roles of individual aldo-keto reductases in yeast has been difficult to determine because of overlapping substrate specificities of these enzymes. In this study, we have cloned, expressed and characterized the aldo-keto reductase Ypr1p from the yeast Saccharomyces cerevisiae and we describe its substrate specificity. The enzyme displays high specific activity towards 2-methylbutyraldehyde, as well as other aldehydes such as hexanal. It exhibits extremely low activity as a glycerol dehydrogenase. The enzyme functions over a wide pH range and uses NADPH as co-factor. In comparison to other mammalian and yeast aldo-keto reductases, Ypr1p has relatively high affinity for D,L-glyceraldehyde (1.08 mM) and hexanal (0.39 mM), but relatively low affinity for 4-nitrobenzaldehyde (1.07 mM). It displays higher specific activity for 2-methylbutyraldehyde than does yeast alcohol dehydrogenase and has a K(m) for 2-methyl butyraldehyde of 1.09 mM. The enzyme is expressed during growth on glucose, but its levels are rapidly induced by osmotic and oxidative stress. Yeast in which the YPR1 gene has been deleted possess 50% lower 2-methylbutyraldehyde reductase activity than the wild-type strain. This suggests that the enzyme may contribute to 2-methyl butyraldehyde reduction in vivo. It may therefore play a role in isoleucine catabolism and fusel alcohol formation and may influence flavour formation by strains of brewing yeast.     

Characterization of the second form of NADH-cytochrome b5 reductase gene from arachidonic acid-producing fungus Mortierella alpina 1S-4

The second type of cytochrome b5 reductase (Cb5R-II) gene was characterized in the arachidonic acid-producing fungus Mortierella alpina 1S-4. Its cDNA (897 bp) and predicted amino acid (298 aa) sequences show more than 70% similarity to the previously isolated first type of Cb5R. Highly conserved exon-intron organization suggests that the two genes evolved from the duplication of a common ancestral gene. Cb5R-II has a flavin-binding domain at its highly hydrophobic N-terminal and an NADH-binding domain at the C-terminal. In comparison with Cb5R genes from other sources, high homology (46-54%) was found for yeast and plant genes. Phylogenetic analysis revealed that microbial and plant Cb5R genes represent a gene family evolved from one prototype and are different from mammalian Cb5R genes.