海洋源低温微生物产苹果酸脱氢酶发酵条件优化

苹果酸脱氢酶（MDH）是参与生物三羧酸循环（TCA）的关键酶,广泛用于临床肝脏相关疾病的诊断,并在生物制药、化工检测等领域具有极大的市场需求。该研究以墓画大洋芽孢杆菌（Oceanobacillus picturae）XJH-11为苹果酸脱氢酶（MDH）产生菌,基于单 因素试验,通过Plackett-Burman响应面试验并结合Box-Behnken试验设计对发酵条件进行响应面优化。结果表明,菌株XJH-11产MDH 的发酵培养基为葡萄糖10g/L、酵母膏20g/L、硫酸镁0.1g/L、初始pH值8.0,最佳发酵条件为发酵温度27℃、摇床转速150r/min、接种量5%、装液量125mL/500 mL。在此优化条件下,MDH酶活为43.67U/mL,比优化前提高了197.87%。     

丁香酚对金黄色葡萄球菌抗菌作用的探究

为了寻找代替化学防腐剂的绿色安全天然添加剂,研究了丁香酚对金黄色葡萄球菌的抑菌作用。以丁香酚为原材料,采用96孔板微量稀释法和琼脂平板稀释法确定最低抑菌浓度（MIC）和最低杀菌浓度（MBC）,通过菌落数直观地确定丁香酚对金黄色葡萄球菌的MIC值和MBC值;并以分光光度计法探究不同质量浓度丁香酚对金黄色葡萄球菌中苹果酸脱氢酶（MDH）和琥珀酸脱氢酶（SDH）酶活性的影响。结果表明,丁香酚对金黄色葡萄球菌的最低抑制浓度（MIC）为600 μg/mL,最低杀菌浓度（MBC）为700 μg/mL,其可将金黄色葡萄球菌菌体蛋白质质量浓度、苹果酸脱氢酶（MDH）酶活、琥珀酸脱氢酶（SDH）酶活分别降至0.033 mg/mL、8.28 U/mg、2.40 U/mL,原因可能是抑制了菌体三羧酸循环和电子链传递中相关关键酶的活性。     

晾制密度对雪茄烟叶非挥发性有机酸代谢的影响

为明确晾制密度对雪茄烟叶非挥发性有机酸代谢的影响,以德雪1号为试验材料,研究了竿距15、25和35 cm的晾制密度对晾房温湿度、叶片含水率、化学成分、非挥发性有机酸含量以及苹果酸脱氢酶（MDH）、琥珀酸脱氢酶（SDH）和柠檬酸合酶（CS）活性的影响。结果表明,随着晾制的进行,各处理含水率和苹果酸含量均呈下降趋势,柠檬酸和丙二酸含量呈上升趋势,草酸含量整体变化不大;MDH活性先升高后降低,CS活性一直升高,而SDH活性逐渐降低。晾制过程中,在竿距25 cm的晾制密度下,晾房内温湿度适宜,能提高雪茄烟叶MDH、SDH和CS活性以及柠檬酸和草酸含量,降低苹果酸和丙二酸含量,并且晾制后各化学成分较协调。因此,雪茄烟叶在晾制过程中,将晾制密度控制在竿距25 cm能提高烟叶非挥发性有机酸代谢能力,促进烟叶内在物质转化,提高烟叶品质。     

二氧化硫对葡萄采后有机酸含量及苹果酸代谢途径的调控作用

为探究二氧化硫(sulfur dioxide,SO_2)对木纳格葡萄采后果实中有机酸含量及苹果酸代谢调控的影响。基于高效液相色谱(high performance liquid chromatography,HPLC)测定果实贮藏过程中酒石酸、L-苹果酸、L-抗坏血酸和柠檬酸含量的变化,采用转录组测序(RNAsequencing,RNA-Seq)分析SO_2调控果实苹果酸代谢的主要途径。并通过测定苹果酸脱氢酶基因(malate dehydrogenase,MDH)、细胞质苹果酸脱氢酶基因(cytoplasmic malate dehydrogenase,cyt MDH)、线粒体苹果酸脱氢酶基因(mitochondrial malate dehydrogenase,mt MDH)、乳酸脱氢酶基因(L-lactate dehydrogenase,LDH)、NADP-苹果酸酶基因(NADP-dependent malic enzyme,NADP-ME)、丙酮酸脱羧酶1基因(pyruvate decarboxylase1,PDC1)、丙酮酸脱羧酶2基因(pyruvate decarboxylase2,PDC2)和乙醇脱氢酶基因(alcoholdehydrogenase,ADH)的表达差异性,进一步验证转录组测序的结果。结果表明:贮藏第60d,SO_2处理组果实中酒石酸、L-抗坏血酸和柠檬酸分别比CK组高0.13 mg/g、0.34 mg/100 g、0.02 mg/g;L-苹果酸比CK组低0.64 mg/g。SO_2可以维持木纳格葡萄果实中的酒石酸、L-抗坏血酸和柠檬酸含量,通过上调mt MDH、LDH、NADP-ME、PDC1和下调MDH、cyt MDH、PDC2、ADH的相对表达量,促进果实中L-苹果酸含量的分解,保持果实的风味特征。本研究为研究SO_2对鲜食葡萄采后果实有机酸代谢的调控作用提供理论依据。     

丁香酚对金黄色葡菌球菌抗菌作用的探究

为了寻找代替化学防腐剂的绿色安全天然添加剂,研究了丁香酚对金黄色葡萄球菌的抑菌作用。以丁香酚为原材料,采用96孔板微量稀释法和琼脂平板稀释法确定最低抑菌浓度(MIC)和最低杀菌浓度(MBC),通过菌落数直观地确定丁香酚对金黄色葡萄球菌的MIC值和MBC值;并以分光光度计法探究不同质量浓度丁香酚对金黄色葡萄球菌中苹果酸脱氢酶(MDH)和琥珀酸脱氢酶(SDH)酶活性的影响。结果表明,丁香酚对金黄色葡萄球菌的最低抑制浓度(MIC)为600μg/mL,最低杀菌浓度(MBC)为700μg/mL,其可将金黄色葡萄球菌菌体蛋白质质量浓度、苹果酸脱氢酶(MDH)酶活、琥珀酸脱氢酶(SDH)酶活分别降至0.033 mg/mL、8.28 U/mg、2.40 U/mL,原因可能是抑制了菌体三羧酸循环和电子链传递中相关关键酶的活性。     

温度对Zymomonas mobilis糖代谢关键酶活力和代谢流量的影响

以运动发酵单胞菌(Zynwmonas mobilis)ATCC31821为模式菌株,研究不同温度条件对其葡萄糖代谢关键酶活力的影响.采用全自动发酵罐,在整个发酵过程中通过充入氮气调节发酵液的溶氧量(DO)=0%,添加0.5mol/LNaOH溶液控制pH=5.5,发酵温度分别控制为25、30、35、40℃,发酵24h,测定其糖代谢网络中ED、HMP、TCA等途径的关键酶活力和代谢物成分.结果表明,在发酵温度为30～35℃时,乙醇脱氢酶(ADH)、葡萄糖-6-磷酸脱氢酶(G-6-PDH)、丙酮酸脱羧酶(PDC)、葡萄糖激酶(GK)、丙酮酸激酶(PK)和甘油醛-3-磷酸脱氢酶(GD-3-PDH)的活力较高,菌体的ED途径代谢活跃,碳素流量增加,乙醇生产量和糖转化率较高,而TCA途径的苹果酸脱氢酶(MDH)和异柠檬酸脱氢酶(ICDH)等活力较低,进入TCA途径的碳素流量明显减少;发酵温度为25、40℃时,TCA途径的酶活力升高,ED途径的酶活力减弱,生成乙醇的代谢流量减少,因此温度是z.mobilis发酵过程中调控菌体细胞生长和糖代谢的一个重要因素. 、葡萄糖-6-磷酸脱氢酶(G-6-PDH)、丙酮酸脱羧酶(PDC) 葡萄糖激酶(GK)、丙酮酸激酶(PK)和甘油醛-3-磷酸脱氢酶(GD-3-PDH)的活力较高,菌体的ED途径代谢活跃,碳素流量增加,乙醇生产量和糖转化率较高,而TCA途径的苹果酸脱氢酶(MDH)和异柠檬酸脱氢酶(ICDH)等活力较低,进入TCA途径的碳素流量明显减少;发酵温度为25、40℃时,TCA途径的酶活力升高,ED途径的酶活力减弱,生成乙醇的代谢流量减少,因此温度是z.mobilis发酵过程中调控菌体细胞生长和糖代谢的一个重要因素.葡萄糖-6-磷酸脱氢酶(G-6-PDH)、丙酮酸脱羧酶(PDC) 葡萄糖激酶(GK)、     

呼吸代谢参与粉红单端孢侵染厚皮甜瓜果实不同阶段的防御反应

目的：探讨三羧酸（tricarboxylic acid cycle，TCA）循环和磷酸戊糖途径（pentose phosphate pathway，PPP）在病原真菌侵染果实中的作用。方法：用粉红单端孢（Trichothecium roseum）接种‘玉金香’厚皮甜瓜果实，观察（22±2）℃、相对湿度55%～60%条件下病斑直径的变化，测定接种果实病健交界处组织TCA循环和PPP关键酶活力以及中间产物含量的变化，分析不同侵染阶段TCA循环和PPP发挥的作用。结果：果实病斑直径在接种后24 h内无明显变化，48 h时明显增大，72 h时显著高于对照组（P＜0.05）。与对照组相比，接种粉红单端孢显著降低了早期果实的异柠檬酸脱氢酶活力（P＜0.05），而显著提高了苹果酸脱氢酶（malate dehydrogenase，MDH）活力（P＜0.05），24 h时MDH活力是对照组的2.14 倍；显著提高了早期果实烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide，NAD）和还原型烟酰胺腺嘌呤二核苷酸含量（P＜0.05），24 h时分别高出对照组50.37%和50.31%；激活了果实的NAD激酶，显著提高了后期葡萄糖-6-磷酸脱氢酶和6-磷酸葡萄糖酸脱氢酶的活力（P＜0.05），48 h时分别是对照组的1.83 倍和2.51 倍；显著促进了后期果实烟酰胺腺嘌呤二核苷酸磷酸和葡萄糖-6-磷酸的合成（P＜0.05）；显著降低了早期和中期果实核糖-5-磷酸异构酶活力和核酮糖-5-磷酸含量（P＜0.05）。结论：粉红单端孢侵染厚皮甜瓜早期诱导了果实组织TCA循环，后期促进了PPP，说明TCA循环参与了更多果实早期的抗病防御，而PPP在后期寄主防御反应中发挥更大的作用。     

利用玉米芯水解液产苹果酸的戴尔根霉突变菌株选育及代谢分析

诱变筛选发酵玉米芯水解液高产苹果酸的根霉属菌株,并对其进行代谢分析,研究其高产苹果酸的机理。对分离得到的菌株进行ITS序列鉴定,进一步利用软X射线辐射对菌种进行诱变筛选、对出发菌株和突变菌株的相关酶活力进行测定及代谢通量分析。利用软X射线辐射结合丙烯醇平板筛选乙醇脱氢酶缺陷型菌株,得到突变株-1,发现其乙醇脱氢酶基因的3 处密码子突变为“TAA”,阻断了突变菌株的乙醇代谢途径。进而利用软X射线辐射结合氟乙酸平板筛选乙醛酸循环缺陷型菌株,得到复合突变株-2,降低了副产物富马酸及琥珀酸的产量。复合突变株-2的葡萄糖-6-磷酸脱氢酶中几处NADP（H）结合位点发生突变,增加了糖酵解和磷酸戊糖两种途径的相互作用,促进了其戊糖代谢。经过两步分离筛选,得到的复合突变株-2能发酵玉米芯水解液高产苹果酸,且减少了副产物乙醇、富马酸、琥珀酸的生成。复合突变株-2的苹果酸产量占代谢物总产量的比例由出发菌株的71%增加到91%,苹果酸产量增大1 倍,研究成果对工业化利用突变菌株生产苹果酸具有重要意义。     

碳酸酐酶的应用研究现状及其酶活检测方法述评

碳酸酐酶（CA）是一类催化二氧化碳和水生成碳酸氢根和氢离子的可逆反应的锌酶.它广泛存在于动植物及微生物体中,且在生物加工过程中起重要作用.由于碳酸酐酶的特性,它正被广泛应用于诸如生物检测、天然活性物质的筛选、生物传感器、CO2捕集和生理诊断等领域.对碳酸酐酶的应用现状以及碳酸酐酶的酶活测定方法进行了综述.     

柠檬醛对酸腐菌线粒体形态和功能的影响

本研究探讨了柠檬醛对酸腐菌线粒体形态、三磷酸腺苷（adenosine triphosphate,ATP）合成和三羧酸（tricarboxylic acid cycle,TCA）循环的影响。扫描电子显微镜结果显示,柠檬醛处理后,酸腐菌线粒体出现扭曲、坍塌甚至破裂的现象。酸腐菌线粒体结构的破坏导致其胞内ATP流失,胞外ATP含量增加。经最小抑菌浓度和最小杀菌浓度的柠檬醛处理后,酸腐菌TCA循环中柠檬酸合酶、α-酮戊二酸脱氢酶、异柠檬酸脱氢酶、琥珀酸脱氢酶和苹果酸脱氢酶活力以及柠檬酸含量都呈下降趋势。结论：柠檬醛处理影响了酸腐菌线粒体的形态和功能,从而抑制其生长。     

Involvement of enzymes of amino acid metabolism and tricarboxylic acid cycle in bovine oocyte maturation in vitro

Few studies demonstrate at a biochemical level the metabolic profile of both cumulus cells and the oocyte during maturation. The aim of the present study was to investigate the differential participation of enzymatic activity in cumulus cells and in the oocyte during in vitro maturation (IVM) by studying the activity of enzymes involved in the control of amino acid metabolism, alanine aminotransferase (ALT) and aspartate aminotransferase (AST); and the tricarboxylic acid (TCA) cycle, isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH). No NAD-dependent isocitrate dehydrogenase (NAD-IDH) activity was recorded in cumulus-oocyte complexes (COCs). ALT, AST, NADP-dependent isocitrate dehydrogenase (NADP-IDH) and MDH enzymatic units remained constant in cumulus cells and oocytes during IVM. Specific activities increased in oocytes and decreased in cumulus cells as a result of IVM (P<0.05). Similar activity of both transaminases was detected in cumulus cells, unlike in the oocyte, in which activity of AST was 4.4 times greater than that of ALT (P<0.05). High NADP-IDH and MDH activity was detected in the oocyte. Addition of alanine, aspartate, isocitrate + NADP, oxaloacetate or malate + NAD to maturation media increased the percentage of denuded oocytes reaching maturation (P<0.05), in contrast to COCs in which differences were not observed by addition of these substrates and co-enzymes. The activity of studied enzymes and the use of oxidative substrates denotes a major participation of transaminations and the TCA cycle in the process of gamete maturation. The oocyte thus seems versatile in the use of several oxidative substrates depending on the redox state.     

pH值对运动发酵单胞菌葡萄糖代谢酶活性影响的研究

以运动发酵单胞菌(Zymomonas mobilis)ATCC31821为模式菌株,研究pH值条件对其葡萄糖代谢关键酶活力的影响.结果表明:发酵液pH5.5时,胞内乙醇脱氢酶、丙酮酸脱羧酶、葡萄糖激酶、葡萄糖-6-磷酸脱氢酶的活力较高,而异柠檬酸脱氢酶活性较低,能促进乙醇的生成;pH 5.0时,苹果酸脱氧酶的酶活力较低,使糖酵解反应向另一个方向发生偏移,促进乙醇的形成.pH 4.0～6.5时,丙酮酸激酶和甘油醛-3-磷酸脱氢酶的酶活力水平相差不大,说明pH值对这2种酶的活性影响甚微.因此,pH5.0～5.5,代谢途径(如糖酵解途径、ED途径等)中的胞内代谢酶活性增强,有利于乙醇的产生.     

A new family of NAD(P)H-dependent oxidoreductases distinct from conventional Rossmann-fold proteins

A new family of NAD(P)H-dependent oxidoreductases is now recognized as a protein family distinct from conventional Rossmann-fold proteins. Numerous putative proteins belonging to the family have been annotated as malate dehydrogenase (MDH) or lactate dehydrogenase (LDH) according to the previous classification as type-2 malate/L-lactate dehydrogenases. However, recent biochemical and genetic studies have revealed that the protein family consists of a wide variety of enzymes with unique catalytic activities other than MDH or LDH activity. Based on their sequence homologies and plausible functions, the family proteins can be grouped into eight clades. This classification would be useful for reliable functional annotation of the new family of NAD(P)H-dependent oxidoreductases.     

Zn2+对制麦中苹果酸脱氢酶和呼吸消耗的影响

研究了在制麦过程中添加Zn2+对苹果酸脱氢酶(MDH)活性和大麦呼吸消耗的影响.结果表明在制麦过程中添加浓度为0.50mmol/L、0.75mmol/L、1.00mmol/L的Zn2+均可有效的抑制MDH活力,进行动力学分析得出Zn2+大麦MDH的抑制类型为非竞争性抑制;添加浓度为0.75mmol/L、1.00mmol/L的Zn2可降低制麦过程中的呼吸消耗,其中添加1.00mmol/L Zn2+的试验组的呼吸消耗率最低,相对于对照组,两种大麦的呼吸消耗分别降低了5.22％和10.7％.同时研究了Zn2对于麦芽中的α、β淀粉酶和果胶酶活力影响,表明对α、β淀粉酶有促进作用,对果胶酶影响不明显.     

Role of the isocitrate dehydrogenases and other Krebs cycle enzymes in lactating bovine mammary gland

The role of the isocitrate dehydrogenases and other Krebs cycle enzymes in bovine mammary metabolism was studied by investigation of their distribution between cytosol and mitochondria. Citrate synthase was used as a marker for mitochondrial disruption, and distributions were normalized to this enzyme. Aconitase, fumarase, and NAD+:malate dehydrogenase were distributed between the mitochondria and the cytosol; evidence for the possible involvement of an aspartate:malate shuttle was also found. The NADP+:isocitrate dehydrogenase is predominantly cytosolic with a small but significant amount of mitochondrial component. Using the dye dichlorophenol-indophenol, a low level of NAD+:isocitrate dehydrogenase activity was observed in bovine mammary tissues. This assay also allows for detection of the enzyme in fresh mitochondria from a variety of other bovine tissues (heart, liver, kidney, and brain). Activities of the isocitrate dehydrogenases were also examined as a function of gestation and lactation. The NAD+:isocitrate dehydrogenase is apparently depressed during gestation with the NADP+ form of the enzyme (cytosolic) elevated postpartum. These results indicate that a substantial portion of Krebs cycle activity may become extramitochondrial in bovine mammary gland at the onset of lactation.     

Purification and characterization of malate dehydrogenase from Corynebacterium glutamicum

The malate dehydrogenase (MDH) (EC 1.1.1.37) from Corynebacterium glutamicum (Brevibacterium flavum) ATCC14067 was purified to homogeneity. Its amino-terminal sequence (residues 1 to 8) matched the sequence (residues 2 to 9) of the MDH from C. glutamicum (GenBank accession no. CAC83073). The molecular mass of the native enzyme was 130 kDa. The protein was a homotetramer, with a 33-kDa subunit molecular mass. The enzyme was almost equally active both for NADU and NADPH as coenzyme on the bases of the k(cat) values at pH 6.5 which is the optimum pH for the both coenzymes. Plotting of the logarithms of the 1/Km, k(cat), and k(cat)/K(m) values with respect to oxalacetate against pH lead to speculation that imidazolium is possibly a functional group in the active center of the enzyme. Citrate activated the enzyme in the oxidation of malate to oxalacetate and inhibited it in the reverse reaction.     

PURIFICATION AND CHARACTERIZATION OF A MALATE DEHYDROGENASE FROM PHASEOLUS MUNGO

A malate dehydrogenase (MDH) was identified and isolated from the seeds of the mung bean (Phaseolus mungo). The procedure entailed extraction, ammonium sulfate precipitation, ion exchange chromatography on CM‐Sephadex and high performance liquid chromatography on POROS HS‐20. The purified protein exhibited a molecular mass of 38 kDa in SDS‐polyacrylamide gel electrophoresis under both nonreduced and reduced conditions. The pI was 9.7 by isoelectric focusing. The specific activity of the MDH was estimated to be 199 U/mg. The enzyme expressed its optimum activity at pH 7.2, 35C, and showed stable activity below 40C. The Km for oxaloacetate was 112 µM. The partial N‐terminal amino acid sequence data analysis of the first 20 amino acids of the mung bean MDH revealed 95 and 80% homology with two reported MDH from soya bean (Glycine max) and potato (Solanum tuberosum), respectively.