近平滑假丝酵母ATCC 7330羰基还原酶CpCR的表达及酶学性质研究

通过构建重组菌Escherichia coli BL21(DE3)/pACYC Duet-1-cpcr,表达带有His标签的近平滑假丝酵母Candida parapsilosis ATCC 7330羰基还原酶CpCR基因,并采用Ni-Agarose亲和层析对重组酶CpCR进行分离纯化和酶学性质研究。重组酶CpCR的基因序列全长1 107 bp,含有368个氨基酸,分子质量在41 kDa左右,比酶活力为20 U/mg;该酶在4～33℃的温度范围稳定性较好,相对酶活力在80%以上,T₅₀值为37℃;该酶的pH适宜范围在6.2～7.5,在中性条件下稳定性最好; Cu²⁺对该酶有强烈的抑制作用,在1 mmol/L条件下,相对酶活力下降30%,在5 mmol/L条件下,相对酶活下降50%;该酶对底物苯甲醛、正丁醛亲和能力强于4-氯-3-酮基-丁酸乙酯(4-chlor-3-keto-butyrate-ethyl ester,COBE),对苯甲醛或正丁醛的催化效率是对COBE的20倍左右;该酶是一种NADPH依赖型的羰基还原酶。本研究为羰基还原酶CpCR分...     

Properties of enzymes which reduce dihydroxyacetone and related compounds in hansenula polymorpha CBS 4732

Hansenula polymorpha CBS 4732 grown on a variety of substrates contained very high activities of enzymes catalyzing the NADH-linked reduction of dihydroxyacetone, acetoin, diacetyl, acetol, methylglyoxal and acetone. The enzymes catalyzing these reductions have been purified and their kinetic properties are described. Three different enzymes were found responsible for the above-mentioned activities, namely: (1) dihydroxyacetone reductase; (2) acetone reductase; and (3) alcohol dehydrogenase. So far, the physiological function of dihydroxyacetone reductase and acetone reductase is obscure. The kinetic properties of dihydroxyacetone reductase and the regulation of the synthesis of this enzyme suggest that it does not function as a glycerol dehydrogenase.     

Radical‐scavenging properties of extracts from the white button mushroom,Agaricus bisporus

BACKGROUND: Agaricus bisporus is the premier cultivated edible mushroom but is usually considered to be of lesser value nutritionally and medicinally compared with other cultivated mushrooms. The objective of this study was to investigate the radical‐scavenging properties of methanolic extracts and the free radical‐processing enzyme activities of water extracts from A. bisporus fruit bodies. Analyses were performed on total fruit bodies from three strains and on separated stipe, cap and gills from one strain, all cultivated experimentally under the same conditions. RESULTS: EC₅₀ values of scavenging ability on 1,1‐diphenyl‐2‐picrylhydrazyl radicals (DPPH^?), scavenging ability on 2,2‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) radical cations (ABTS^?+) and reducing power of methanolic extracts from total fruit bodies ranged between 1.7 and 5.0 and were 1.1–1.9 times higher in a white hybrid than in two wild strains, cream and brown. The gills exhibited higher antioxidant activities relative to the stipe and cap. In water extracts, glutathione peroxidase activities were up to eight times higher than glutathione reductase activities and no difference was observed between strains. Catalase activity was highest in the brown wild strain. The only significant differences in free radical‐processing enzymes between the three parts of the mushroom were higher catalase activity in the gills and lower glutathione reductase activity in the stipe. CONCLUSION: The radical‐scavenging properties of the button mushroom are comparable to those of other edible mushrooms and dependent on the strain and on the section of the fruit body. Copyright © 2008 Society of Chemical Industry     

Purification and some properties of sulfur reductase from the iron-oxidizing bacterium Thiobacillus ferrooxidans NASF-1

Thiobacillus ferrooxidans strain NASF-1 grown aerobically in an Fe2+ (3%)-medium produces hydrogen sulfide (H2S) from elemental sulfur under anaerobic conditions with argon gas at pH 7.5. Sulfur reductase, which catalyzes the reduction of elemental sulfur (S0) with NAD(P)H as an electron donor to produce hydrogen sulfide (H2S) under anaerobic conditions, was purified 69-fold after 35-65% ammonium sulfate precipitation and Q-Sepharose FF, Phenyl-Toyopearl 650 ML, and Blue Sepharose FF column chromatography, with a specific activity of 57.6 U (mg protein)(-1). The purified enzyme was quite labile under aerobic conditions, but comparatively stable in the presence of sodium hydrosulfite and under anaerobic conditions, especially under hydrogen gas conditions. The purified enzyme showed both sulfur reductase and hydrogenase activities. Both activities had an optimum pH of 9.0. Sulfur reductase has an apparent molecular weight of 120,000 Da, and is composed of three different subunits (M(r) 54,000 Da (alpha), 36,000 Da (beta), and 35,000 Da (gamma)), as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This is the first report on the purification of sulfur reductase from a mesophilic and obligate chemolithotrophic iron-oxidizing bacterium.     

Purification of X-prolyl dipeptidyl aminopeptidase from Lactobacillus casei subspecies.

Prolyl dipeptidylaminopeptidases from two subspecies of Lactobacillus casei were purified and biochemically characterized. L. casei ssp. casei UL21 (a debittering strain) and L. casei ssp. rhamnosus UL26 (a non-debittering strain) were the source bacteria for this study. Purification of the enzymes from both the sources was effected by a gel filtration step through Sephacryl S-300 followed by ion-exchange chromatography through DEAE Sephacel. This rendered an electrophoretically homogeneous enzyme preparation. The purified enzymes from both the sources showed similar temperature optimum (45 degrees C) and pH optimum (7.0). Their activity profiles on various substrates and the nature of inhibition by different inhibitors were also found to be similar, indicating that this enzyme is perhaps not significantly involved in the debittering process during the maturation of cheese.     

Purification, characterization, and gene cloning of thermophilic cytochrome cd1 nitrite reductase from Hydrogenobacter thermophilus TK-6

A thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6 can grow autotrophically under anaerobic conditions by denitrification. One of the denitrification enzymes, cytochrome cd(1) nitrite reductase, was isolated and its gene was cloned from strain TK-6. The subunit molecular mass of the purified enzyme was 61.5 kDa and the isoelectric point was determined to be 9.3. The optimum temperature and pH for the enzymatic reaction were 70-75 degrees C and 6.5-7.0, respectively. The structural gene for the enzyme, nirS, is probably transcribed as a hexacistronic operon with the following genes encoding a putative diheme cytochrome c and the proteins required for biosynthesis of heme d(1). The NirS sequence was phylogenetically distinct from those of proteobacteria. The consensus -35 and -10 sequences were found in the putative nirS promoter region, but the consensus sequences for the DNR/NnrR-type or the NorR/FhpR-type nitric oxide sensing regulators were not found in this region.     

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.     

异养硝化-好氧反硝化菌株的分离、鉴定及其氨氮降解过程初探

该研究采用选择性培养基从汾河底泥中分离筛选异养硝化-好氧反硝化菌,通过形态观察、生理生化试验及分子生物学技术对其进行菌种鉴定,并对其氨氮（NH4+-N）降解过程进行动态监测,测定NH4+-N降解过程中关键酶的基因及酶活。结果表明,分离筛选得到一株异养硝化-好氧反硝化菌,编号为ZH-2,其被鉴定为醋酸钙不动杆菌（Acinetobacter calcoaceticus）。该菌在NH4+-N降解过程中存在NO3--N和NO2--N两种中心代谢产物,其基因组中含有3种关键酶（氨单加氧酶（Amo）、硝酸盐还原酶（Nap）和亚硝酸盐还原酶（Nir））基因,在NH4+-N降解过程,这3种关键酶的比酶活分别为45.69 mU/mg、3.54 mU/mg和6.91 mU/mg。综上,初步确定A. calcoaceticus ZH-2的NH4+-N降解过程是NH4+-N→NH2OH→NO2--N→NO3--N→NO2--N→NO→N2O→N2的经典降解途径,为该菌的进一步推广应用提供了理论依据。     

大豆分离蛋白酶解液抗氧化性的近红外光谱定量测定

利用近红外光谱技术对四种蛋白酶(菠萝、碱性、木瓜和中性蛋白酶)的大豆分离蛋白水解样品进行测定,探索同一模型应用于不同酶的水解液抗氧化性测定的可行性。基于留一交叉验证方法,分析了采样密度和酶物质差异对全波长模型精度的影响,统计检验表明,采样密度与模型精度之间正相关,酶物质差异对单酶样本模型精度无显著影响,但对综合酶样本模型性能有显著影响。为了建立能够同时准确测定不同酶水解样品抗氧化性能力的综合酶样本预测模型,竞争性自适应重加权抽样(CARS)法用来对模型进行了优化,建模后,校正集R_(cv)和RMSECV分别为0.960 1和0.002 8,验证集的R_p和RMSEP为0.923 7和0.005 3,相对分析误差(RPD)为2.45,预测精度较好,说明建立针对不同酶的水解样品的同一模型是可行的。     

通过体外代谢途径构建确定乳酸合成关键酶

基于代谢控制分析理论提出了一种通过体外代谢途径构建和分析确定关键酶的方法并应用其确定乳酸高产菌株中的关键酶。首先获得高产菌株的粗酶液并测定葡萄糖到乳酸合成途径中各种蛋白的绝对浓度,进而通过向粗酶液中分别添加同等比例的各纯酶对途径进行扰动,并由扰动前后的途径通量变化计算出各酶的通量控制系数以确定关键酶。结果表明,该菌株乳酸合成途径中丙酮酸激酶和甘油醛-3-磷酸脱氢酶对途径通量影响最大,由此预测在该菌株基础上进一步过表达这两个酶对提高乳酸生成速率可能最为有效。     

A spheroplast rate assay for determination of cell wall integrity in yeast

The rate of formation of spheroplasts of yeast can be used as an assay to study the structural integrity of cell walls. Lysis can be measured spectrophotometrically in hypotonic solution in the presence of Zymolyase, a mixture of cell wall-digesting enzymes. The optical density of the cell suspension decreases as the cells lyse. We optimized this assay with respect to enzyme concentration, temperature, pH, and growth conditions for several strains of Saccharomyces cerevisiae. The level of variability (standard deviation) was 1–5% between trials where the replications were performed on the same culture using enzyme prepared from the same lot, and 5–15% for different cultures of the same strain. This assay can quantitate differences in cell wall structure (1) between exponentially growing and stationary phase cells, (2) among different S. cerevisiae strains, (3) between S. cerevisiae and Candida albicans, (4) between parental and mutated lines, and (5) between drug- or chemically-treated cells and controls. © 1998 John Wiley & Sons, Ltd.     

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.     

浙东腌冬瓜优势乳酸菌的分离及产酶特性分析

通过微生物分离和鉴定从浙东传统腌冬瓜中得到4株优势菌,分别为植物乳杆菌(Lz151)、发酵乳杆菌(Lz152)、棒状乳杆菌(Lz153)和戊糖乳杆菌(Lz154),通过酶学分析方法对4株乳酸菌的产生乳酸脱氢酶、亚硝酸盐还原酶、酯酶和转氨酶的特性进行研究。实验表明,4株乳酸菌都有乳酸脱氢酶、亚硝酸盐还原酶、酯酶和转氨酶活性,植物乳杆菌和发酵乳杆菌有较高的乳酸脱氢酶活力,分别达到122.34 U/m L和113.56 U/m L;发酵乳杆菌和戊糖乳杆菌有较高的亚硝酸盐还原酶活性,分别为17.62 U/m L和13.42 U/m L,而棒状乳杆菌具有最低的亚硝酸盐还原酶活力,为4.74 U/m L;植物乳杆菌和棒状乳杆菌比其他两种乳酸菌有较高的酯酶活性,达到11.53 U/m L和13.78 U/m L。此外,植物乳杆菌和戊糖乳杆菌有较高的转氨酶活性,分别为25.37 U/m L和23.19 U/m L。4株乳酸菌产酶的最适温度和p H存在菌种间差异,而且同一株菌产生各种酶的最适条件也不同,分离乳酸菌分别在30℃、35℃、40℃产各种酶的能力最高,而发酵乳杆菌、植物乳杆菌的最适产酶p H分别为6.0、7.0。     

Molecular characterization of the probiotic strain Bacillus cereus var. toyoi NCIMB 40112 and differentiation from food poisoning strains

Bacillus cereus var. toyoi strain NCIMB 40112 (Toyocerin), a probiotic authorized in the European Union as feed additive for swine, bovines, poultry, and rabbits, was characterized by DNA fingerprinting applying pulsed-field gel electrophoresis and multilocus sequence typing and was compared with reference strains (of clinical and environmental origins). The probiotic strain was clearly characterized by pulsed-field gel electrophoresis using the restriction enzymes Apa I and Sma I resulting in unique DNA patterns. The comparison to the clinical reference strain B. cereus DSM 4312 was done with the same restriction enzymes, and again a clear differentiation of the two strains was possible by the resulting DNA patterns. The use of the restriction enzymes Apa I and Sma I is recommended for further studies. Furthermore, multilocus sequence typing analysis revealed a sequence type (ST 111) that was different from all known STs of B. cereus strains from food poisoning incidents. Thus, a strain characterization and differentiation from food poisoning strains for the probiotic strain was possible.     

酶法改变甘草苷糖醛酸基提高其甜度的研究(Ⅱ)——能水解甘草苷糖醛酸基酶的提纯与酶性质研究

以酶法改变甘草苷糖醛酸基提高其甜度为目的,对新分离筛选的甘Ｍ－２和甘Ｍ－６霉菌产的β－葡萄糖醛酸苷酶进行了分离提纯和酶学方面的研究。结果表明,两株菌产的β－葡萄糖醛酸苷酶被６０％饱和硫酸铵沉淀较好,经ＤＥＡＥ－ＣｅｌｕｏｓｅＤＥ５２离子交换层析柱梯度洗脱,得到纯酶,提纯倍数分别为１０．６７和６．１５倍,收率分别为３３．０％和２４．２％,其中甘Ｍ－２菌产β－葡萄糖醛酸苷酶只能将甘草苷水解成甘草次酸（ＧＡ）;甘Ｍ－６菌产β－葡萄糖醛酸苷酶水解甘草苷主要变成甜度很高的单葡萄糖醛酸基甘草苷（ＧＡＭＧ）;两种酶在ＳＤＳ－聚丙烯酰胺凝胶电泳都得到电泳单点,其酶蛋白相对分子质量分别为６００００和４２０００;酶反应最适ｐＨ分别为５．０和６．０,最适温度均为４０℃,均在ｐＨ４．０～８．０和２０～７０℃范围内相对稳定。     

Kinetic Characterization of Chimeric Cyclomaltodextrin Glucanotransferase from Genes of Two Alkalophilic Bacillus

The chimeric CGTase, which was constructed genetically with two β-CD forming type enzyme genes of different species of alkalophilic Bacillus, was characterized by comparing with the actions of parent enzymes. The final composition of CDs in the reaction digest from starch by the chimeric enzyme, especially in the yield of α-CD, was different from those by parent enzymes, and total yields of CDs by these enzymes were almost the same. The chimeric enzyme catalyzed the formation of β-CD as a major action from starch, the rapid synthesis of α-CD from other CDs and maltooligosaccharides and the slow degradation and interconversion of α-CD to others. From these results, the role of second and third segments in the enzyme molecules was discussed.     

发酵液中酮基还原酶活性测定方法的构建

本研究以4-氯乙酰乙酸乙酯（COBE）为底物,还原型辅酶Ⅱ （NADPH）为辅酶,应用紫外-可见光分光光度计法测定发酵液中酮基还原酶活性,通过连续测定NADPH消耗量来计算酶活力,得到了最佳反应体系：NADPH浓度为0.2 mmol/L,COBE浓度为1.0 mmol/L,磷酸缓冲溶液浓度为100 mmol/L、pH值为6,反应温度为40℃.在此条件下平行检测5次酶活,相对标准偏差（RSD）为0.48％.此方法操作简便,耗时短,精确度高,重复性好,可作为发酵液中酮基还原酶活性测定方法加以推广.     

INFLUENCE OF DIFFERENT FACTORS ON ACTIVITY AND STABILITY OF ß-GALACTOSIDASE FROM LACTOBACILLUS ACIDOPHILUS1

Eight Lactobacillus acidophilus strains were tested for β-galactosidase activity. The results show that β-galactosidase is an inducible enzyme in this organism. Wide variations in enzyme properties of the strains were observed. The temperature and pH optima of the enzymes were 40–45C and 6.5–7.0, respectively. At 0.1 mM concentration, the enzyme activity in strain 4495 was slightly stimulated by 2-mercaptoethanol, partially inhibited by EDTA but remained more or less unaffected with thiomersal and ascorbic acid. In contrast, all the modulators completely inhibited the enzyme activity in strain 301. The enzyme in strain 301 was stable for 80 min at 50C, whereas the activity in strain 4495 was completely lost in 40 min. However, the enzyme was stable over a pH range of 5.8–8.0 and up to 60 days at 4 ± 1C in both strains. The K_m values for β-galactosidase from both the strains were same, whereas V_max for the enzyme from strain 301 was about 2-fold higher than that of strain 4495.     

Influence of enzymes on the evolution of fructosans in sourdough wheat processes

 The effects of white wheat flour, sourdough starters (multiform and pure strain) and enzymes (amylase/pentosanase and/or lipase) on amounts and composition of fructosans during a breadmaking process were investigated. Samples yielded a crude extract of fructosans close to 3%, of which about 30% was pure fructosans. In dough and bread samples the presence of yeast invertase split a considerable amount of fructosans, and further treatment with this enzyme during the measurement procedure did not yield more fructosans. Treatment with an inulinase showed the presence of inulin-type linkages in flour, the amount depending on the flour source. As expected, fermentation reduced the amount of fructosans. Different trends were observed with each starter, and some interactions appeared between type of flour and enzyme addition. Characterisation of fructosans by size exclusion chromatography revealed five fractions with apparent molecular weight (MW) between 25000 and 192 Da; fructosans isolated from dough and bread samples had the same MW distribution but a different relative proportion of each fraction, and differed from those obtained from flour. Received: 27 April 1999     

Survey of hydrogen sulphide production by wine yeasts

Twenty-one strains of commercial wine yeasts and 17 non-Saccharomyces species of different provenance were surveyed for their ability to produce hydrogen sulphide in synthetic grape juice medium indicator agar with different nitrogen sources, as well as in natural grape juice. Bacto Biggy agar, a commercially available bismuth-containing agar, was used to compare our results with others previously reported in the literature. Under identical physiological conditions, the strains used in this study displayed similar growth patterns but varied in colony color intensity in all media, suggesting significant differences in sulphite reductase activity. Sulphite reductase activity was absent for only one strain of Saccharomyces cerevisiae. All other strains produced an off-odor to different extents, depending significantly (P <0.05) on medium composition. Within the same species of some non-Saccharomyces yeasts, strain variation existed as it did for Saccharomyces. In natural musts, strains fell into three major groups: (i) nonproducers, (ii) must-composition-dependent producers, and (iii) invariable producers. In synthetic media, the formation of sulphide by strains of S. cerevisiae results from the reduction of sulphate. Therefore, this rapid screening methodology promises to be a very useful tool for winemakers for determining the risk of hydrogen sulphide formation by a given yeast strain in a specific grape juice.