假肠膜明串珠菌甘露醇脱氢酶基因的克隆及表达

利用聚合酶链式反应从假肠膜明串珠菌中扩增出甘露醇脱氢酶（mannitol dehydrogenase,MDA）基因的结构基因,克隆入表达载体pETDuet-1,构建了甘露醇脱氢酶表达质粒pETDuet-1-mdh,将其转化进入大肠杆菌BL21(DE3),经异丙基-β-D-硫代半乳糖苷诱导表达。假肠膜明串珠菌甘露醇脱氢酶结构基因长度为1 017 bp,重组甘露醇脱氢酶基因在大肠杆菌内成功表达,其蛋白质分子质量为36.0 kD;重组甘露醇脱氢酶活力为0.15 U/mg pro,高于假肠膜明串珠菌中甘露醇脱氢酶活力0.03 U/mg pro。     

Pilot-scale production of exopolysaccharide from Leuconostoc pseudomesenteroides XG5 and its application in set yogurt

Exopolysaccharide from Leuconostoc pseudomesenteroides XG5 (XG5 EPS) is a linear dextran that is built by glucose units via α-1,6 glycosidic bond. The primary objective of this study was to investigate the yield of XG5 EPS and its application in set yogurt. In laboratory scale, the culture conditions of XG5 EPS production were optimized using the L9 (3³) orthogonal test. Here, the optimized yield of XG5 EPS was 26.02 g/L under the conditions of 100 g/L sucrose, initial pH 7.0, 25°C incubation, and 100 rpm for 36 h in a shaking flask. Based on the optimized parameters of laboratory scale, a pilot fed-batch fermentation was performed in a 50-L bioreactor with an adjusted agitation speed of 20 rpm. The XG5 EPS yield reached 40.07 g/L in fed-batch fermentation, which was 54% higher than that achieved in laboratory scale. In addition, XG5 EPS was added into set yogurt to investigate its effect on the stability of set yogurt. Our data demonstrated that the XG5 EPS improved the water-holding capacity, texture profile, and viscosity of set yogurt during cold storage compared with the controls. In particular, addition of 0.5% XG5 EPS increased the structure of 3-dimensional network of set yogurt, which eventually improved the physical stability of the set yogurt. Overall, this study provided new insights for exploring the pilot scale production and application of dextran.     

Potential and limitations of MALDI-TOF MS for discrimination within the species Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides

The discriminatory power of MALDI-TOF MS (matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry) was evaluated for differentiation of bacterial strains within Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides species. Protein fingerprints were generated with MALDI-TOF MS for 24 Leuconostoc strains and analyzed with ClinProTools at species level and below. A treatment of bacterial cells prior to MALDI-TOF MS analysis was optimized applying different lysozyme concentrations. A pre-treatment with a lysozyme concentration of 12.5 μg ml^?1 for 30 min exposure time enhanced the reproducibility of the spectra but did not influence the cluster analysis in ClinProTools. The cluster analysis resulted in the identification of seven different peak patterns shared among twelve strains of L. pseudomesenteroides and eight peak patterns shared among twelve strains of L. mesenteroides. The protein fingerprints of 24 Leuconostoc strains were sufficiently diverse for a reliable discrimination of half of the analyzed starter cultures at strain level. The other half of the strains could only be identified at cluster level. The discrimination at subspecies level was not possible on the basis of MALDI-TOF MS profiling. The MALDI-TOF MS methodology delivered interesting information about the diversity of bacterial isolates belonging to the two species L. mesenteroides and L. pseudomesenteroides but had its limitations for subspecies discrimination of unknown isolates as well as strain identification.     

西藏灵菇中产胞外多糖假肠膜明串珠菌发酵性能及流变学特性研究

以分离自西藏灵菇发酵液的3株产胞外多糖的假肠膜明串珠菌为研究对象,对其产胞外多糖能力、发酵性能及流变学特性进行了研究。结果表明:本研究分离得到的3株假肠膜明串珠菌均具有较高的产孢外多糖的能力,菌株R5的胞外多糖产量最高,达到454.67 mg/L;三株菌生长过程符合细菌生长典型规律,产酸时期主要在菌株的对数生长期,适于发酵乳制品生产,其中菌株R5发酵酸乳的组织状态、风味的感官评分为86分,明显优于其他两株菌。流变学特性表明,三株菌制备的发酵乳的表观黏度都随剪切时间的延长而降低,呈现剪切稀释的流动特征,黏度大小依次为R5>R2>R1;均能够形成触变环,为正触变性流体,R1发酵乳与R5发酵乳触变环面积相近,分别为2301.72、2924.09 1/s Pa,较R2发酵乳(4697.82 1/s Pa)小;三株菌制备的发酵乳的G’值(弹性模量)都高于G″值(粘性模量),均是弹性模量占优势,表现出类固体特性,菌株R5发酵出的酸乳具有较高的弹性和黏性。通过比较三株菌的发酵性能与流变学特性,表明菌株R5相较于其他两株菌具有较强的产胞外多糖的能力,较高的表观黏度,较好的粘弹性,结构恢复能力较强,发酵的酸乳具有更好的组织结构,具有一定的应用潜力。      

Purification and characterization of a bacteriocin produced by Leuconostoc mesenteroides E131

Leuconostoc mesenteroides E131, isolated from Greek traditional fermented sausage, prepared without the addition of starters, produces a bacteriocin which is active against the pathogen Listeria monocytogenes. The bacteriocin was purified by 50% ammonium sulphate precipitation, cation exchange, and reverse-phase chromatography. Bacteriocin is active at pH values between 4.0 and 9.0 and retains activity after incubation for 1 h at 100 °C. Proteolytic enzymes inactivated the bacteriocin after 1 h of incubation, while renin resulted in full inactivation only after 24 h. Lipase resulted in full inactivation after 4 h. Applying molecular methods, it was determined that the bacteriocin produced, named as mesenterocin E131, was identical to mesenterocin Y105 and was expressed during the exponential growth phase.     

Purification and characterization of NAD-specific 6-phosphogluconate dehydrogenase from Leuconostoc lactis SHO-54

The 6-phosphogluconate dehydrogenase (EC 1.1.1.44) from Leuconostoc lactis SHO-54 was purified with an overall yield of 38% and a specific activity of 140.0 units/mg protein. The enzyme had a tetrameric structure and a molecular mass of 32.8 kDa. The amino acid composition of the purified enzyme was determined, and the enzyme contained no sulfhydryl amino acids. The K(m) values for 6-phosphogluconate and NAD were 0.95 mM and 0.32 mM, respectively.     

Purification and characterization of a p-coumarate decarboxylase and a vinylphenol reductase from Brettanomyces bruxellensis

The presence of Brettanomyces bruxellensis has been correlated with an increase of phenolic aromas in wine. The production of these aromas results from the metabolization of cinnamic acids, present in the wine, to their ethyl derivatives. Hence, the participation of two enzymes has been proposed: a p-coumarate decarboxylase (CD) and a vinylphenol reductase (VR). Both enzymes were purified and characterized from B. bruxellensis. In denaturing conditions, the CD enzyme had a molecular mass of 21 kDa, while in native conditions its mass was 41 kDa. The optimal activity was obtained at a temperature of 40 degrees C and a pH of 6.0. For p-coumaric acid, the K(m) value and V(max) were 1.22+/-0.08 mM and 98+/-0.15 micromol/min mg, respectively. The VR enzyme had a molecular mass of 37 kDa in SDS-PAGE, while in natural conditions its mass was 118 kDa. The K(m) value was >3.37+/-2.05 mM and its V(max) was 107.62+/-50.38 micromol/min mg for NADPH used as a cofactor. Both enzymatic activities were stable at pH 3.4, but in the presence of ethanol the CD activity decreased drastically while the VR activity was more stable. This is the first report that shows the presence of a CD and a VR enzyme in B. bruxellensis.     

牛背最长肌高铁肌红蛋白还原酶的分离及其酶学性质

以冷鲜牛背最长肌为原料,采用硫酸铵盐析、超滤法对原料肉中的高铁肌红蛋白还原酶进行粗分离,并研究温度、pH、金属离子对高铁肌红蛋白还原酶活性的影响.结果表明:在硫酸铵饱和度65％,超滤(＞50 kDa)条件下,牛背最长肌高铁肌红蛋白还原酶可得到分离与纯化.试验范围内,牛背最长肌高铁肌红蛋白还原酶的分子量为29.0～66....     

一种米曲霉蛋白酶的分离纯化及酶解特性研究

对米曲霉固态发酵所产蛋白酶分离纯化,采用硫酸铵盐析、DEAE-FF层析、Butyl-HP层析和Superdux 7510/300GL凝胶层析得到一种电泳纯的蛋白酶,SDS-PAGE显示分子量大小为27 ku左右。以酪蛋白为底物时,该蛋白酶Km=1.23 g·L-1,Vm=27.03μg·m L-1·min-1,最适反应条件为50℃,p H9.0。该蛋白酶对酪蛋白水解活性最高,而对牛血清蛋白的水解活性很低;对牛胰岛素B链上-Phe-Val-,-Cys-Gly-,-Glu-Ala-和-Arg-Gly-组成的肽键有较强的切割能力,酶切位点较多,对疏水性氨基酸具有较高的选择性,为米曲霉所产蛋白酶在食品上的应用提供有力的参考。      

Purification and characterization of a cationic peroxidase from artichoke leaves

Peroxidases are part of a large group of enzymes associated with cell wall biosynthesis, response to injury, disease, resistance and wound repair. Among peroxidase isoenzymes, a soluble cationic peroxidase (ALSP), not yet described, has been partially purified and characterized from artichoke leaves. The enzyme was shown to be a glycoprotein with a molecular weight of 51 000 and an isoelectric point of 9. The substrate specificity of the ALSP is characteristic of class III (guaiacol‐type) peroxidases. The ALSP was partially purified by ammonium sulfate precipitation, gel filtration, affinity chromatography, anionic exchange high‐performance liquid chromatography and isoelectrofocusing. The increase in specific activity was 43 times compared to the crude extract as estimated by the guaiacol assay. Three ALSP fragments were sequenced by tandem mass spectrometry de novo sequencing method. Copyright © 2007 Society of Chemical Industry     

Purification and characterization of a serine carboxypeptidase from Kluyveromyces marxianus

We purified a carboxypeptidase (CPY) from the yeast of Kluyveromyces marxianus. This enzyme was purified 170 times from a soluble extract of 100000 x g. Purification consisted in a fractionated precipitation with ammonium sulfate and two chromatographic steps consisting of anion exchange chromatography and hydrophobic interactions chromatography. The native enzyme depicted a molecular mass of 67 kDa by gel filtration. This serine carboxypeptidase depicted an optimal pH of 8.5 and was stable at a pH ranging from 6.0 to 9.0, its optimal temperature was of 45 degrees C and was unstable at temperatures above 55 degrees C; Michaelis constant and Vmax for N-benzoyl-l-tyrosine-p-nitroanilide were of 29 microM and 612 microM/min mg of protein, respectively. The enzyme was strongly inhibited by phenylmethylsufonyl fluoride (PMSF) and, to a lesser degree, by trans-epoxysuccinyl-l-leucylamido-(4-guanidine)-butane. This study indicated that K. marxianus carboxypeptidase could be an alternative to other animal-source carboxypeptidases in the industry.     

乳制品中明串珠菌的分离鉴定及其生物学特性研究

对内蒙古自治区呼伦贝尔市牧区98个乳样中的明串珠菌进行了分离及生物学特性研究。共分离到明串珠菌3株，分别属于乳明串珠菌(EW21)，肠膜明串珠菌葡聚糖亚种(EW24—2)和肠膜明串珠菌肠膜亚种(CB8—1)。它们均可在pH=4．8的环境中生长，且菌株EW24—2和CB8—1可利用蔗糖生成葡聚糖。     

麻鸭脂肪氧合酶的分离纯化及其性质研究

采用硫酸铵沉淀、离子交换层析、凝胶色谱等方法对麻鸭脂肪氧合酶(Shelduck Lipoxygenase)进行了纯化。结果显示,麻鸭LOX粗提液经60%⁸0%硫酸铵沉淀、DEAE离子交换层析、Superdex75凝胶过滤、SOURCE15Q离子交换层析后可达电泳纯,麻鸭LOX比活力为85714.3U/mg,纯化倍数98.64,经SDS PAGE分析麻鸭LOX的分子质量约为62 kDa。麻鸭LOX与大豆LOX的酶学性质差异较大,麻鸭LOX的最适温度为30℃,最适pH为6.0,且热稳定性和pH稳定性均优于大豆LOX。同时,Zn2+、Ca2+对麻鸭LOX的激活作用明显,而Mn2+对麻鸭LOX具有强烈的抑制作用。     

Purification and characterization of 2-aminoacetophenone reductase of newly isolated Burkholderia sp. YT

We found that a newly isolated Burkholderia sp. produced (R)-2-amino-1-phenylethanol from 2-aminoacetophenone, showing the high stereospecificity. NADPH-dependent 2-aminoacetophenone reductase purified to homogeneity was a dimer with a molecular mass of 65,000. The purified enzyme did not reduce acetophenone and 1-phenyl-1-propanone. The purified enzyme converted 2-aminoacetophenone to only (R)-2-amino-1-phenylethanol.     

In situ dextran synthesis by Weissella confusa Ck15 and Leuconostoc pseudomesenteroides DSM 20193 and their effect on chickpea sourdough bread

This work evaluated, for the first time, the impact of in situ dextran (with different branching degree) produced by Weissella confusa Ck15 and Leuconostoc pseudomesenteroides DSM 20193 strains on the technological properties of chickpea–wheat sourdough bread prepared with three levels of chickpea flour (20, 30 and 40 g/100 g). In addition Lactiplantibacillus plantarum F8 strain (not dextran producing) and a control without sourdough fermentation were used. Specific volume, crumb hardness and moisture content of breads were evaluated during six days of storage. At the increase of chickpea flour from 20 to 40 g/100 g in the samples, the lowest decrease in bread volume (15%) occurred when W. confusa Ck15 was used. Moreover, these breads showed the lowest crumb hardness at each chickpea flour percentage, 46, 80 and 98 N. Hence, in situ dextran synthesis by W. confusa Ck15 might counteract negative effects caused by gluten-free chickpea flour on technological properties of bread.     

Purification and characterization of a glucoamylase from Rhizopus oryzae

Glucoamylase (EC 3.2.1.3) of Rhizopus oryzae NRRL 395 was purified approximately sevenfold by sequential ammonium sulfate fractionation, Biogel P-100 gel filtration, Q-Sepharose anion exchange and S-Sepharose cation exchange. The pH and temperature optima were 4·8 and 60°C, respectively. Enzyme was stable at temperatures up to 40°C and pH values between 3 and 8. The molecular weight was 67 000 daltons as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the pI was 8·7 as determined by chromatofocusing. The K_m for amylopectin and soluble starch were 0·98 and 1·34 mg/ml, respectively. The V_max for amylopectin and soluble starch were 782 and 136 μmoles of glucose produced per mg of protein per min, respectively. The enzyme activity was inhibited by Hg²⁺, Pb²⁺ and Cd²⁺, but not by EDTA.     

Purification and characterization of a novel glutamyl aminopeptidase from chicken meat

A novel glutamyl aminopeptidase (aminopeptidase A, EC 3.4.11.7) was purified from chicken meat by ammonium sulfate fractionation, ethanol fractionation, heat treatment, and successive column chromatographies of DEAE-Sepharose CL-6B and Sephadex G-200. The purified enzyme migrated as a single band on SDS-PAGE. The molecular weight of this enzyme was found to be 55,000 and 550,000 by SDS-PAGE and Sephadex G-200 column chromatographies, respectively. This enzyme hydrolyzed Glu- and Asp-, but not Leu-, Arg-, and Ala-2-naphthylamide (-2NA) at all. The optimum pH and temperature for hydrolysis of Glu-2NA was 7.5. and 70°C, respectively. Reducing agents such as cysteine and dithiothreitol inhibited the activity of this enzyme at concentrations of 1 mM. However, the activation by Ca(2+) and the inhibition by amastatin were not observed.     

乳酸片球菌细菌素的分离纯化及理化性质

将乳酸片球菌接种于MRS培养基,37℃静置培养17h产生细菌素。根据细菌素在pH6.0时与菌体的吸附力最强,pH2.0时吸附力最弱,通过调节pH和离心来分离纯化细菌素,得率为22.80%,比活为4.526×104AU/mg;细菌素的理化性质实验表明,细菌素经120℃处理20min仍保留70.08%的活性;在pH2～9范围内20℃处理3h抑菌活性稳定,当pH≥9时活性逐渐降低;经胃蛋白酶、胰蛋白酶、木瓜蛋白酶和蛋白酶K处理后均无抑菌活性。实验表明,该细菌素耐热、耐酸、耐碱,对蛋白酶敏感,具有开发为安全、天然的食品防腐剂的良好前景。     

Purification and characterization of a dipeptidase from Lactobacillus curvatus DPC2024

A dipeptidase was purified to homogeneity from a cell-free extract of Lactobacillus curvatus DPC2024 by chromatography on diethylaminoethyl-sephacel, phenyl sepharose, chelating sepharose fast flow and MonoQ. The purified dipeptidase was a monomer of ∼52 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel filtration chromatography. The enzyme was optimally active at pH 8 and 50°C and retained ∼10% of its maximum activity after pre-heating for 10 min at 70°C. The enzyme was a metallopeptidase, strongly inhibited by 0.1 mM ethylenediaminetetraacetic acid and o-phenanthroline and reactivated by a number of divalent metal ions. The enzyme was also inhibited by p-chloromercuribenzoate and β-mercaptoethanol. The enzyme was a strict dipeptidase, capable of hydrolysing a range of dipeptides but not tri-, tetra- or pentapeptides, p-nitroanilide derivatives of amino acids nor N- and C-terminal-blocked dipeptides. The N-terminal amino acid sequence of the first 20 residues showed significant homology with dipeptidases from Lactobacillus delbrueckii subsp. lactis DSM 7290 and Lactococcus lactis subsp. cremoris MG1363.     

樟绒枝霉中一种低分子量木聚糖酶的纯化及性质研究

本文研究了樟绒枝霉（Malbranchea cinnamonmea）S168中一种低分子量木聚糖酶（McXyn25）的纯化和酶学性质。采用硫酸铵沉淀和DEAE-52阴离子柱层析两步纯化,得到电泳级纯酶,分子量为25ku。该酶的最适pH为8.0,在pH 4.5¹1.0范围内稳定;最适温度为65℃,在60℃以下具有较高的稳定性。McXyn25表现出严格的底物特异性,在纸浆漂白方面具有较好的应用前景。此外,McXyn25能够水解木聚糖产生低聚木糖,且无木糖产生,说明该酶适合应用于低聚木糖生产。