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.     

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%。     

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.     

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。     

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。     

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。     

木糖发酵产2，3-丁二醇培养基的优化

以2,3-丁二醇得率作为考察指标,运用单因素试验和4因素3水平响应面实验设计方法,研究木糖、氮源、无机盐和金属离子对2,3-丁二醇得率的影响。结果表明,培养基的最佳浓度组合为:木糖为83.4 g/L、酵母粉为20.3 g/L、KH2PO4为10 g/L、K2HPO4为7.2 g/L(、NH4)2SO4 2 g/L、柠檬酸钠4 g/L、MgSO4.7H2O 0.05 g/L、MnSO4.7H2O为0.005 g/L、ZnSO4.7H2O 0.01 g/L、FeSO4.7H2O0.005 g/L、CaCl2 0.057 g/L。利用优化后的培养基进行发酵,2,3-丁二醇得率达0.348 g/g,与初始条件相比,提高了16%;实验结果与模型预测值拟合一致。     

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.     

亚硝酸还原酶的分离纯化及其性质研究

经硫酸铵分级沉淀、DEAE-Cellulose柱层析、SephadexG-75凝胶过滤层析,由巨大芽孢杆菌(B.megaterium MPF-906)分离纯化得到亚硝酸还原酶(nitrite reductase,NiR),分子量为35ku.NiR反应的最适温度和pH值分别为40℃和6.5.热稳定性较好,80℃保温4h后仍有50％的酶活力.在pH5.5～9.0均较稳定,残余酶活都在60％以上.Cu2+、Ba2+能提高酶活力;Mn2+、Pb2+对酶活力有明显抑制作用;Na+、Fe3+、M2+、Al3+对其有轻微抑制作用;Ca2+、Zn2+对酶活力影响不大.亚硝酸钠为底物,该酶的Km=8.6mmol/L,Vmax=4.1U/mg,该酶的适电子供体为抗坏血酸20mol/L、0.1mol/L的连二亚硫酸钠和0.075mol/L的草酸钠.     

Characterization of D-galacturonate reductase purified from the psychrophilic yeast species Cryptococcus diffluens

An efficient method for the direct extraction of yeast genomic DNA from agave must was developed. The optimized protocol, which was based on silica-adsorption of DNA on microcolumns, included an enzymatic cell wall degradation step followed by prolonged lysis with hot detergent. The resulting extracts were suitable templates for subsequent qPCR assays that quantified mixed yeast populations in artisan Mexican mezcal fermentations.     

Sequence and analysis of an aldose (xylose) reductase gene from the xylose-fermenting yeast Pachysolen tannophilus

A xylose reductase gene was isolated from the xylose-fermenting yeast Pachysolen tannophilus as a cDNA clone by selecting clones that hybridized specifically to xylose-inducible messenger RNA. Use of the cDNA clone as a probe in Northern hybridizations identified a xylose-inducible mRNA species large enough to encode a 36kDa xylose reductase protein known to be produced by this yeast. A corresponding genomic clone was isolated as a 3kb EcoRI fragment that specifically hybridized to the cDNA clone. The sequence of the cDNA and the largest open reading frame of the genomic clone are identical. The predicted translation product exhibits: (1) significant sequence identity with a previously published N-terminal amino acid sequence from purified P. tannophilus xylose (aldose) reductase protein exhibiting NADH/NADPH-dependent activities (aldose reductase, EC 1.1.1.21); (2) identity with a protein composed of 317 amino acid residues with a calculated molecular mass of 36·2kDa, equivalent to that reported for purified P. tannophilus xylose reductase; and (3) considerable sequence similarity to, and features of, a superfamily of oxidoreductases. This sequence is deposited as GenBank Accession Number U40706.     

枯草芽孢杆菌B.subtilisML中性蛋白酶基因的克隆及鉴定

以蛋白酶高产菌株Ｂ．ｓｕｂｔｉｌｉｓ ＭＬ为供体菌，提取其染色体ＤＮＡ，经限制性内切酶ＢａｍＨＩ完全酶切后，插入枯草杆菌载体ｐＮＱ１２２的相同酶切位点，连接后转化中性碱性蛋白酶基因双缺陷型菌株Ｂ．ｓｕｂｔｉｌｉｓＤＢ１０４。从含有氯霉素的酪蛋白平板上获得了２０个具有蛋白酶活性的克隆。