Survey of enzyme activity responsible for phenolic off-flavour production by <Emphasis Type="Italic">Dekkera</Emphasis> and <Emphasis Type="Italic">Brettanomyces</Emphasis> yeast

Volatile phenols are produced by Dekkera yeasts and are of organoleptic importance in alcoholic beverages. The key compound in this respect is 4-ethylphenol, responsible for the medicinal and phenolic aromas in spoiled wines. The microbial synthesis of volatile phenols is thought to occur in two steps, beginning with naturally occurring hydroxycinnamic acids (HCAs). The enzyme phenolic acid decarboxylase (PAD) converts HCAs to vinyl derivatives, which are the substrates of a second enzyme, postulated to be a vinylphenol reductase (VPR), whose activity results in the formation of ethylphenols. Here, both steps of the pathway are investigated, using cell extracts from a number of Dekkera and Brettanomyces species. Dekkera species catabolise ferulic, caffeic and p-coumaric acids and possess inducible enzymes with similar pH and temperature optima. Brettanomyces does not decarboxylate HCAs but does metabolise vinylphenols. Dekkera species form ethylphenols but the VPR enzyme appears to be highly unstable in cell extracts. A partial protein sequence for PAD was determined from Dekkera anomala and may indicate the presence of a novel enzyme in this genus.     

Human carbonyl reductase 4 is a mitochondrial NADPH-dependent quinone reductase

A protein encoded in the gene Cbr4 on human chromosome 4q32.3 belongs to the short-chain dehydrogenase/reductase family. Contrary to the functional annotation as carbonyl reductase 4 (CBR4), we show that the recombinant tetrameric protein, composed of 25-kDa subunits, exhibits NADPH-dependent reductase activity for o- and p-quinones, but not for other aldehydes and ketones. The enzyme was insensitive to dicumarol and quercetin, potent inhibitors of cytosolic quinone reductases. The 25-kDa CBR4 was detected in human liver, kidney and cell lines on Western blotting using anti-CBR4 antibodies. The overexpression of CBR4 in bovine endothelial cells reveals that the enzyme has a non-cleavable mitochondrial targeting signal. We further demonstrate that the in vitro quinone reduction by CBR4 generates superoxide through the redox cycling, and suggest that the enzyme may be involved in the induction of apoptosis by cytotoxic 9,10-phenanthrenequinone.     

不同提取液提取水稻幼苗质外体蛋白效果的比较

提取植物组织质外体蛋白质的主要困难是提取效率低且易被细胞质蛋白污染。为解决上述问题,以12天93-11水稻幼苗为试验材料,使用3种含不同浓度钾和钙离子的缓冲液作为提取液进行提取效果比较。3种提取液的相同成分都是0.1mol/L Tris-HCl pH 7.6,1mmol/L PMSF,区别点在于:Buffer A含0.2mol/L KCl;Buffer B含0.2mol/L CaCl₂;Buffer C含0.1mol/L KCl和0.1mol/L CaCl₂。结果表明,Buffer A的蛋白产率达到了(0.49±0.07)mg/g FW(叶片)和(0.83±0.06)mg/g FW(根部),比Buffer B和Buffer C分别提高了122.7%和53.1%(叶片)以及102.4%和59.6% (根部)。六磷酸葡萄糖脱氢酶活性检测的结果表明在这些蛋白质提取物中细胞质蛋白的污染率很低,可以控制在1%以下。这些实验结果说明通过优化提取液,建立了有效提取水稻幼苗质外体蛋白质的方法,可应用于植物质外体蛋白质组学研究。     

响应面法优化木薯淀粉发酵生产单细胞油脂工艺

采用响应面分析法对发酵性丝孢酵母菌株以木薯淀粉水解液为原料合成微生物油脂的培养条件进行优化。首先利用Plackett-Burman试验设计确定影响油脂产量的主要因素,在此基础上再利用Box-Behnken试验设计及响应面分析法对其进行条件优化。结果表明,发酵温度、C/N、pH对油脂产量具有显著影响,产油脂的最佳发酵条件为:发酵温度28.78°C、C/N 126.18、pH 6.69,油脂产量达到14.88g/L,比优化前提高了28.6%。同时,气相色谱分析表明,微生物油脂脂肪酸组成成分主要包括棕榈酸、硬脂酸、油酸、亚油酸酯等,是优良的生物柴油制备原料。     

Anaerobic aniline degradation via reductive deamination of 4-aminobenzoyl-CoA in Desulfobacterium anilini

The initial reactions involved in anaerobic aniline degradation by the sulfate-reducing Desulfobacterium anilini were studied. Experiments for substrate induction indicated the presence of a common pathway for aniline and 4-aminobenzoate, different from that for degradation of 2-aminobenzoate, 2-hydroxybenzoate, 4-hydroxybenzoate, or phenol. Degradation of aniline by dense cell suspensions depended on CO₂ whereas 4-aminobenzoate degradation did not. If acetyl-CoA oxidation was inhibited by cyanide, benzoate accumulated during degradation of aniline or 4-aminobenzoate, indicating an initial carboxylation of aniline to 4-aminobenzoate, and further degradation via benzoate of both substrates. Extracts of alinine or 4-aminobenzoategrown cells activated 4-aminobenzoate to 4-aminobenzoyl-CoA in the presence of CoA, ATP and Mg²⁺. 4-Aminobenzoyl-CoA-synthetase showed a K _m for 4-aminobenzoate lower than 10 M and an activity of 15.8 nmol · min^-1 · mg^-1. 4-Aminobenzoyl-CoA was reductively deaminated to benzoyl-CoA by cell extracts in the presence of low-potential electron donors such as titanium citrate or cobalt sepulchrate (2.1 nmol · min^-1 · mg^-1). Lower activities for the reductive deamination were measured with NADH or NADPH. Reductive deamination was also indicated by benzoate accumulation during 4-aminobenzoate degradation in cell suspensions under sulfate limitation. The results provide evidence that aniline is degraded via carboxylation to 4-aminobenzoate, which is activated to 4-aminobenzoyl-CoA and further metabolized by reductive deamination to benzoyl-CoA.     

Lipase-coupling esterification of starch with octenyl succinic anhydride

Enzymatic modification of starch was conducted by lipase-coupling esterification with octenyl succinic anhydride (OSA). Parameters affecting the esterification were systematically studied. Products were characterized by Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction, differential scanning calorimetry (DSC) and viscosity analysis (VA). Optimum condition for lipase-coupling OSA starch preparation was as follows: starch pretreatment at 65 °C for 15 min, starch concentration 35%, amount of lipase and OSA, 0.6% and 3%, reaction pH, temperature and time, 8.0, 40 °C and 30 min respectively, which resulted in 0.0195 of the degree of substitution and 84.05 ± 2.07% of the reaction efficiency. FT-IR spectroscopy confirmed the formation of OSA starch. SEM and X-ray diffraction showed apparent surface change, but no crystalline change. DSC and VA results indicated the synthesized OSA starch gelatinized rapidly with high viscosity. Attractively, reaction time drastically reduced to 30 min, showing vast potential for scale production of OSA starch.     

硝酸还原酶抑制剂钨酸钠对油菜硝态氮积累的影响

采用溶液培养方法,选取硝酸盐积累差异明显的两个油菜品种（低硝态氮积累品种‘红油3号’和高硝态氮积累品种‘中双6号’,研究苗期根系硝酸还原酶（NR）活性被抑制以后两个油菜品种叶片、叶柄和根系中NR活性和硝态氮含量的变化。结果表明：1.0mmol.L-1的NR活性抑制剂Na2WO4对两个油菜品种的根系NR活性抑制效果最佳;根系NR活性被抑制以后,两个油菜品种的根系NR活性、硝态氮吸收速率均显著下降,而硝态氮含量却显著上升;且Na2WO4对‘中双6号’硝态氮吸收的抑制程度强于其对‘红油3号’的抑制。叶片和叶柄的NR活性变化不显著,但叶柄硝态氮含量显著下降,叶片硝态氮含量稳定,且这一趋势在低积累品种‘红油3号’中表现得更为明显。     

Quantitative Structure-Activity Relationship Study on Some Dihydropteridine Reductase Inhibitors

Abstract

The dihydropteridine reductase (DHPR) inhibitory potencies of some 4-phenyltetrahydropyridines, 4-phenylpiperidines, and 4-phenylpyridines, are analyzed in relation to their physicochemical and molecular properties. They are found to have significant correlation with Hammett constant s and the van der Waals volume V_w. The correlation is linear with s and parabolic with V_w. Hence, it is argued that DHPR inhibition involves dispersion interaction and is enhanced by electron donation from the substituents but hindered by steric effects produced by large substituents. It is also found that these electronic and steric effects are significant only when they are produced by substituents being at specific position in the molecules.     

Synthesis and HMG CoA reductase inhibition of 4-thiophenyl quinolines as potential hypocholesterolemic agents

A series of novel 4-thiophenyl quinoline-based mevalonolactone derivatives were synthesized from ethyl 6,7,8-trisubstituted-4-chloro-quinoline-3-carboxylates by several reactions and evaluated for their ability to inhibit the rat HMG CoA reductase in vitro. It was found that substitution with a variety of thiophenyl groups at position 4 in quinoline resulted in retention or enhancement of the inhibition and the preferable groups were 4-isopropyl-thiophenyl and 3-methoxy-thiophenyl. (4R,6S)-6-[(E)-2-(6,7,8-trifluoro-4-isopropylthiophenyl-quinoline-3-yl)-ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (A16) and (4R, 6S)-6-[(E)-2-(6-fluoro-4,7-di-(3-methoxy-thiophenyl)-quinoline-3-yl)-ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (A23) were approximately three times more potent than rosuvastatin or pitavastatin in inhibiting HMG CoA reductase and selected as the hypocholesterolemic candidates for further evaluation.     

抑制西瓜蔓枯病菌的生防真菌筛选、鉴定及发酵条件优化

筛选对西瓜蔓枯病菌具有抑制作用的生防真菌,优化其发酵条件以提升发酵液抑菌效果。以平板对峙试验和摇瓶培养抑菌试验筛选拮抗真菌;根据形态学特征和18S rDNA序列分析进行菌株鉴定;通过扫描电镜观察对西瓜蔓枯病菌的寄生作用,以单因素试验和响应曲面法确定其最适发酵条件,在此基础上,通过盆栽试验研究其防病效果。结果表明:在所筛选到的9株生防真菌中M1菌株的抑菌率最高,其形态学特征与烟管菌(Bjerkandera adusta)相符,18S rDNA序列分析显示其与烟管菌在系统发育树上聚在一支,因此将菌株M1鉴定为黑管菌属(Bjerkandera)、烟管菌。在扫描电镜下观察到烟管菌能够直接穿透西瓜蔓枯病菌菌丝,对其发酵条件优化后确定了最佳条件组合:C/N为7.1,pH为7.4,装瓶量为44%,时间为19d,转速为180r/min,温度为29℃,在此条件下抑菌率为52.64%,高于未经优化的抑菌率。温室盆栽中对西瓜蔓枯病菌的防病效果达到74.3%,高于多菌灵处理。烟管菌作为生防真菌对西瓜蔓枯病菌具有较好的抑制作用,条件优化后进一步提升了抑菌效果,在生物防治中具有巨大应用潜力。     

家蝇抗菌肽AMPs17蛋白原核表达条件的优化及其抗真菌活性检测

目的:优化AMPs17重组蛋白的原核表达条件,分析重组蛋白的抗真菌活性。方法:比较不同的诱导温度(25℃、28℃、30℃、32℃、34℃)、异丙基硫代-β-D半乳糖苷(IPTG)诱导浓度(0. 025mmol/L、0. 05mmol/L、0. 1mmol/L、0. 3mmol/L、0. 5mmol/L、0. 8mmol/L、1. 0mmol/L)和诱导时间(12h、15h、18h、21h、24h)对AMPs17重组蛋白表达量的影响,筛选AMPs17重组蛋白的最佳表达条件;采用镍离子金属螯合剂亲和层析柱对重组蛋白进行纯化,SDS-PAGE和ImageJ图像分析系统对表达结果进行分析,Western blot对AMPs17重组蛋白进行鉴定,高效液相色谱分析重组蛋白的纯度,微量液体稀释法及菌落计数法检测其抗真菌活性。结果:在诱导温度为32℃、IPTG浓度为0. 05mmol/L的条件下诱导培养15h,AMPs17重组蛋白的表达量最高且最为稳定;HPLC色谱仪分析显示AMPs17重组蛋白纯度可达到90%以上;优化后的AMPs17重组蛋白能有效抑制白色念珠菌的生长。结论:优化了家蝇抗菌肽AMPs17的诱导表达条件,获得了高表达、稳定且具有抗真菌活性的蛋白质,为后续抗菌机制及应用研究提供一定的实验基础。     

Molecular determinants for the stereospecific reduction of 3-ketosteroids and reactivity towards all-trans-retinal of a short-chain dehydrogenase/reductase (DHRS4)

DHRS4, a member of the short-chain dehydrogenase/reductase superfamily, reduces all-trans-retinal and xenobiotic carbonyl compounds. Human DHRS4 differs from other animal enzymes in kinetic constants for the substrates, particularly in its low reactivity to retinoids. We have found that pig, rabbit and dog DHRS4s reduce benzil and 3-ketosteroids into S-benzoin and 3α-hydroxysteroids, respectively, in contrast to the stereoselectivity of human DHRS4 which produces R-benzoin and 3β-hydroxysteroids. Among substrate-binding residues predicted from the crystal structure of pig DHRS4, F158 and L161 in the animal DHRS4 are serine and phenylalanine, respectively, in the human enzyme. Double mutation (F158S/L161F) of pig DHRS4 led to an effective switch of its substrate affinity and stereochemistry into those similar to human DHRS4. The roles of the two residues in determining the stereospecificity in 3-ketosteroid reduction were confirmed by reverse mutation (S158F/F161L) in the human enzyme. The stereochemical control was evaluated by comparison of the 3D models of pig wild-type and mutant DHRS4s with the modeled substrates. Additional mutation of T177N into the human S158F/F161L mutant resulted in almost complete kinetic conversion into a pig DHRS4-type form, suggesting a role of N177 in forming the substrate-binding cavity through an intersubunit interaction in pig and other animal DHRS4s, and explaining why the human enzyme shows low reactivity towards retinoids.     

Assay of UDP, GDP, CDP, and ADP reductase activities by column chromatography on polyethyleneimine cellulose

Deoxyribonucleosides were separated from ribonucleosides by chromatography on polyethyleneimine cellulose columns (Pasteur pipettes. The deoxyribonucleosides were quantitatively eluted with 25 mM boric acid in less than 10 ml while the ribonucleosides were retained. The ribonucleosides were eluted with 1 M NaCl. This method was utilized to assay for GDP, UDP, ADP, and CDP reductase activities after hydrolysis of the substrate and product nucleotides to the corresponding nucleosides. All four reductase activities were assayed using identical conditions of column size, eluting solution (25 mM boric acid), and elution volume. The use of polyethyleneimine cellulose columns with boric acid can be adapted to other enzyme assays such as purine nucleoside phosphorylase and for the isolation of deoxyribonucleotides from cellular extracts.     

4,4,4-三氟乙酰乙酸乙酯羰基还原酶产生菌的筛选及产酶条件研究

对实验室保藏菌种进行筛选,得到一株葡萄汁酵母Saccharomyces uvarum SW-58,对其产酶条件进行优化,其发酵培养基组成:醋酸钠60 g/L,玉米浆30 g/L,KH2PO46 g/L,MgSO4.7H2O 1 g/L;培养条件为:发酵温度30℃,初始pH 6.0,发酵周期36 h。4,4,4-三氟乙酰乙酸乙酯羰基还原酶酶活最高可达388.1 U/L,产物的浓度由优化前的3.5 g/L提高到4.6 g/L,所得产物的光学纯度由优化前的60.8%e.e.提高到85.0%e.e。     

Properties and tissue distribution of a novel aldo-keto reductase encoding in a rat gene (Akr1b10)

A recent rat genomic sequencing predicts a gene Akr1b10 that encodes a protein with 83% sequence similarity to human aldo-keto reductase (AKR) 1B10. In this study, we isolated the cDNA for the rat AKR1B10 (R1B10) from rat brain, and examined the enzymatic properties of the recombinant protein. R1B10 utilized NADPH as the preferable coenzyme, and reduced various aldehydes (including cytotoxic 4-hydroxy-2-hexenal and 4-hydroxy- and 4-oxo-2-nonenals) and α-dicarbonyl compounds (such as methylglyoxal and 3-deoxyglucosone), showing low K_m values of 0.8-6.1 μM and 3.7-67 μM, respectively. The enzyme also reduced glyceraldehyde and tetroses (K_m = 96-390 μM), although hexoses and pentoses were inactive and poor substrates, respectively. Among the substrates, 4-oxo-2-nonenal was most efficiently reduced into 4-oxo-2-nonenol, and its cytotoxicity against bovine endothelial cells was decreased by the overexpression of R1B10. R1B10 showed low sensitivity to aldose reductase inhibitors, and was activated to approximately two folds by valproic acid, and alicyclic and aromatic carboxylic acids. The mRNA for R1B10 was expressed highly in rat brain and heart, and at low levels in other rat tissues and skin fibroblasts. The results suggest that R1B10 functions as a defense system against oxidative stress and glycation in rat tissues.