Purification and characterization of intracellular proteinase from Lactobacillus casei ssp. casei LLG

The intracellular proteinase of Lactobacillus casei ssp. casei LLG was isolated in the cytoplasmic fraction with 0.05 M Tris-HCl buffer (pH 7.5). The enzyme was purified by the fast protein liquid chromatography system equipped with ion-exchange and gel filtration chromatographies. This proteinase comprised a single monomeric form and had a molecular weight of about 55 kDa and an isoelectric point near pH 4.9. The optimum pH and temperature for the enzyme activity were determined to be pH 6.5 and 37 degrees C, respectively. The enzyme was inactivated by metal-chelating compounds (EDTA, 1,10-phenanthroline) and less affected by serine proteinase inhibitors (diisopropylfluorophosphate, phenylmethylsulfonyl fluoride). Proteinase activity was increased by Ca++, Mn++, and Co++, and inhibited by Cu++, Mg++, and Zn++. The activity of this enzyme to hydrolyze casein appeared to be more active on beta-casein than alphas1-casein and kappa-casein as monitored by polyacrylamide gel electrophoresis.     

嗜酸乳杆菌JQ-1胞壁蛋白酶的提取及分离纯化

对嗜酸乳杆菌JQ-1胞壁蛋白酶（CEP）的提取条件进行了研究,确定了胞壁蛋白酶的最佳提取条件,并对粗酶进行了分离纯化。采用溶菌酶结合非离子表面活性剂法对胞壁蛋白酶进行提取,得出其最佳提取条件为:菌粉与裂解液比例为1∶5（g/mL）,33℃下保温4.0h,离心取得的上清液即为粗酶液。通过聚乙二醇-20000浓缩,DEAE-Sephadex A-25和Sephadex G-100两步层析对粗酶液进行分离纯化,蛋白酶提纯倍数达到50.951倍,最后回收率为12.569%。且SDS-PAGE电泳检测蛋白酶为单体结构,分子量约为45ku。用纯化后的CEP水解β-酪蛋白,水解液的ACE抑制率为48%。      

干酪乳杆菌蛋白酶的性质研究

干酪乳杆菌(Lactobacilluscasei,Lc)6033经增菌培养、超声波破碎、高速冷冻离心后获得粗酶液,并经70%～80%饱和度范围的硫酸铵分级沉淀初步提纯,酶活力回收率(OD275)为9.71%,酶液最适pH为6.5左右、最适温度为40℃,Co2 、Mn2 能显著增强酶液活力;Cu2 、Fe3 、乙二胺四乙酸(EDTA)和苯甲基磺酰氟(PMSF)对酶液有较强的抑制作用;而Zn2 、碘乙酸和β-巯基乙醇的抑制作用较轻微。可见,酶液中可能含金属酶、丝氨酸酶及巯基酶等多种蛋白酶。     

Peptidase and proteinase activity ofLactococcus lactis,Lactobacillus casei andLactobacillus plantarum

The proteolytic system of several non-commercial strains of lactococci and lactobacilli that were isolated directly from traditional-Spanish, semi-hard, goats' milk cheese was studied. The aminopeptidase, X-prolyldipeptidyl aminopeptidase, dipeptidase and proteinase activity of these new strains was measured for the cytoplasmic, cell-wall/membrane and spontaneously released fractions. The aminopeptidase activity was exclusively intracellular and higher forLactobacillus casei subsp.casei than forLactococcus lactis subsp.lactis. Lactobacillus plantarum showed higher dipeptidase activity thanL. casei. The highest level of proteinase activity was recorded for the cell-wallmembrane fraction ofLactococcus lactis subsp.lactis IFPL 359, and was higher on β-casein than on α_s-casein for all the strains studied. These results suggest some different contribution of these strains to the proteolysis of cheese during ripening and they seem to complement each other when used together in the starter culture.     

Characterization of Ca-activated cell-bound proteinase from Virgibacillus sp. SK37 isolated from fish sauce fermentation

Cell-bound proteinase from Virgibacillus sp. SK37 isolated from the first month of fish sauce fermentation was characterized. The enzyme showed the maximum activity at 65 °C, pH 7.0 and 9.5, using azocasein as a substrate. The enzyme required at least 10 mmol/l Ca²⁺ to effectively hydrolyze casein and the extent of casein degradation increased with Ca²⁺ concentration. Ethylenediaminetetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF) largely inhibited the activity, indicating a characteristic of Ca²⁺-activated serine proteinase. Among six synthetic substrates tested, the enzyme preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-AMC, indicating a subtilisin-like proteinase. Although activity towards actomyosin at 20 g/100 ml NaCl decreased to 63% compared to at 5 g/100 ml, the enzyme showed high stability at 25 g/100 ml NaCl, 30 °C. This was the first study to report biochemical characteristics of cell-bound proteinase from a moderately halophilic bacterium isolated from fish sauce.     

Accelerated ripening of dry fermented sausage by addition of a Lactobacillus proteinase

Accelerated ripening of dry sausage was investigated by adding two different concentrations of proteinase isolated from Lactobacillus paracasei ssp. paracasei NCD0151 to sausage mixtures. Sausages with proteinase showed a greater pH decrease and a greater increase in lactic acid formation, water loss, protein degradation and peptide formation than control, no enzyme, sausages. Changes were most pronounced in the sausages containing 12 Ug^-1 proteinase. Addition of proteinase caused a specific increase in glutamate, serine and lysine content. Sensory evaluation after 14 days of ripening showed a significant increase in flavour intensity, maturity flavour, acidity, bitter taste and hardness in the proteinase-containing sausages. Addition of proteinase induced changes earlier in the fermentation and ripening period than in the control, thereby indicating an acceleration of maturation.     

The peptide hydrolase system of Lactobacillus reuteri.

Peptide hydrolase system of Lactobacillus reuteri CRL 1098, a lactic acid bacteria of sourdough origin, was investigated. This microorganism has a broad range of peptidases consisting of an active aminopeptidase, X-Prolyl-dipeptidylaminopeptidase, dipeptidase and tripeptidase. Aminopeptidase, iminopeptidase and endopeptidase are most likely located in the cytoplasmic fraction showing no detectable association with the cell membrane, while dipeptidase and tripeptidase are mainly associated with the latter fraction. The peptidases are metalloenzymes activated by Co²⁺ and inhibited by Cu²⁺, Hg²⁺, Cd²⁺ and by metal-complexing reagents. The aminopeptidase activity inhibited by EDTA can be restored by Mn²⁺ while that of di- and tripeptidase treated with 1,10-phenantroline can be restored by Zn²⁺ and Co²⁺, respectively.     

不同生长阶段保加利亚乳杆菌关键蛋白酶基因表达变化规律

乳酸菌自身不能直接利用外源蛋白质,必须通过蛋白水解体系水解外源蛋白质,生成供菌体正常生长需要的短肽和游离氨基酸。该研究选择6株保加利亚乳杆菌(KLDS1.0207、1.0501、1.1007、1.9201、1.9203、1.0208)进行脱脂乳发酵,在mRNA转录水平上,应用实时荧光定量PCR技术,探讨保加利亚乳杆菌的蛋白水解体系中关键蛋白酶基因(prtB、oppD、pepC、pepF、pepQ、pepX、pepT)在菌体不同生长阶段表达的动态变化。结果表明,所有菌株生长曲线均呈S型。随着菌体在脱脂乳中的不断生长,蛋白水解活力极显著增强(P<0.01),7个目的基因相对表达量呈现总体上调。6株菌的prtB和oppD基因在对数期的相对表达量极显著高于对照组(4h)(P<0.01)。对数期的胞内肽酶基因(pepC、pepF、pepQ、pepX、pepT)的表达量与对照组(4 h)相比,呈极显著上调(P<0.01),但菌株KLDS 1.0207的pepQ基因在对数期下调,而稳定期上调。由此可见,蛋白水解活力较高的菌株,其生长和产酸特性也较高;发现在脱脂乳中培养保加利亚乳杆菌,其关键蛋白酶基因的表达量呈时间依赖性,且菌株间各蛋白酶基因表达量有较大差异。     

Peptidase and proteinase activity ofLactococcus lactis, Lactobacillus casei andLactobacillus plantarum

The proteolytic system of several non-commercial strains of lactococci and lactobacilli that were isolated directly from traditional-Spanish, semi-hard, goats' milk cheese was studied. The aminopeptidase, X-prolyldipeptidyl aminopeptidase, dipeptidase and proteinase activity of these new strains was measured for the cytoplasmic, cell-wall/membrane and spontaneously released fractions. The aminopeptidase activity was exclusively intracellular and higher forLactobacillus casei subsp.casei than forLactococcus lactis subsp.lactis. Lactobacillus plantarum showed higher dipeptidase activity thanL. casei. The highest level of proteinase activity was recorded for the cell-wallmembrane fraction ofLactococcus lactis subsp.lactis IFPL 359, and was higher on -casein than on _s-casein for all the strains studied. These results suggest some different contribution of these strains to the proteolysis of cheese during ripening and they seem to complement each other when used together in the starter culture.

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Peptidase- und Proteinase-Aktivität vonLactococcus lactis, Lactobacillus casei undLactobacillus plantarum

Zusammenfassung Das proteolytische System von mehreren nicht kommerziellenLactococcus- undLactobacillus-Stämmen wurde direkt vom traditionellen spanischen halbfesten Ziegenmilchkäse isoliert und untersucht. Die Aktivität von Aminopeptidase, X-Prolyldipeptidylaminopeptidase, Dipeptidase and Proteinase dieser neuen Stämme wurde in cytoplasmatischen, Zellwand-Membran- und spontan freigesetzten Fraktionen gemessen. Die Aminopeptidase-Aktivität erfolgte ausschließlich intracellular und war höher fürLactobacillus casei subsp. casei als fürLactococcus lactis subsp.lactis. Lactobacillus plantarum zeigte höhere Dipeptidase Aktivität alsL. casei. Die höchsten Werte für die Proteinase-Aktivität wurden für die Zellwand-Membran-Fraktion vonLactococcus lactis subsp.lactis IFPL359 gemessen. Für alle untersuchten Stämme war die Aktivität höher bei -Casein als bei _s-Casein. Dieses Ergebnis weist auf den unterschiedlichen Einfluß dieser Stämme bei der Proteolyse von Käse während der Reifung hin. Die Stämme scheinen sich gegenseitig zu ergänzen, wenn sie gemeinsam in Starterkulturen verwendet werden.

     

Isolation and characterization of Lactobacillus strains involved in koumiss fermentation

Koumiss is a slightly alcoholic fermented milk beverage, originally obtained by using natural mix starters (lactic acid bacteria and yeast). Seven Lactobacillus strains from lyophilized koumiss were isolated and identified as L. salivarius, L. buchneri and L. plantarum. The process of lactic acid fermentation caused by koumiss strains was faster (9–13 h) than that with other lactobacilli. The conversion ratio of glucose to lactic acid ranged from 47% to 79% and was strain dependent. All strains were resistant to low pH. Three of the strains isolated were viable during prolonged cold storage in fermented milk (3 weeks at 4°C).     

鲅鱼副产物中产蛋白酶微生物的分离与表征

目的 以鲅鱼副产物为实验材料,定向筛选高产蛋白酶的野生菌株。方法 以脱脂牛奶培养基对鲅鱼副产物中的产蛋白酶微生物进行初筛,采用福林酚方法对各菌株产蛋白酶的活力进行测定,复筛出具有高产碱性蛋白酶和中性蛋白酶的野生菌株,并通过分子生物学方法(16S rDNA测序分析)对高产蛋白酶菌株进行物种鉴定。进一步通过测定高产菌发酵过程中的菌株浓度和蛋白酶活力,分析菌株随发酵时间的生长情况和产酶动力学。结果 采用选择性培养基初步筛选获得8株具有蛋白酶活性的菌株,进一步复筛发现两株菌Z3和Z8菌株产蛋白酶的能力较强[碱性蛋白酶活力分别为(393.87±19.69)、(358.87±17.94)U/mL;中性蛋白酶活力分别为(344.87±17.24)、(291.20±14.56)U/mL]。经分子生物学测序比对分析菌株Z3和Z8分别鉴定为枯草芽孢杆菌(Bacillussubtilis)和蜡样芽孢杆菌(Bacilluscereus)。对两株菌的生产情况和产酶能力进行研究,发现两株菌的产酶模式均为部分生长偶联型,枯草芽孢杆菌Z3菌株合成碱性蛋白酶和中性蛋白酶的能力优于蜡样芽孢杆菌Z8。结论 本研究从鲅鱼副产...     

Purification and characterization of the extracellular proteinase of Pseudomonas fluorescens NCDO 2085

Pseudomonas fluorescens NCDO 2085 produced a single heat-stable extracellular proteinase in Na caseinate medium at 20 degrees C and pH 7.0. The proteinase was purified to electrophoretic homogeneity using chromatofocusing, gel filtration and ion-exchange chromatography. The purification procedure resulted in a 158-fold increase in the specific activity and a yield of 3.5% of the original activity. The enzyme is a metalloproteinase containing Zn and Ca, with an isoelectric point at 5.40 +/- 0.05 and a mol. wt of 40 200 +/- 2100. It is heat-stable having D-values at 74 and 140 degrees C of 1.6 and 1.0 min respectively; 40 and 70% of the original activity remained after HTST (74 degrees C/17 s) and ultra high temperature (140 degrees C/4 s) treatments respectively. The amino acid composition of the proteinase was determined and compared with those from other Pseudomonas spp.     

泡菜中优良乳酸菌筛选及特性的研究

为了获得适用于纯种强化发酵泡菜生产的优良菌种,采用平板筛选法和发酵筛选法从泡菜中分离、筛选乳酸菌,采用形态、生理生化和16S rDNA等特性的分析技术对优选菌种进行鉴定,并通过发酵实验进一步研究优选菌种的发酵特性。经筛选,获得一株乳酸产量最高的菌种LP-09,该菌种被鉴定为植物乳杆菌（Lactobacillus plantarum）。发酵实验结果表明,菌种LP-09的最适温度和初始p H分别为37℃和6.0,具有较强的耐盐性,在含有40 g/L Na Cl的培养基中生长正常,在含有100 g/L Na Cl的培养基中可缓慢发酵产酸;同时,菌种LP-09具有较强的亚硝酸盐降解能力,对200 mg/L亚硝酸盐的降解率可达99.3%以上。      

Purification and characterization of extracellular proteinase produced by Brevibacterium linens ATCC 9172

The micro-organism Brevibacterium linens ATCC 9172 produced five extracellular proteinases, as shown by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) with copolymerized gelatine. One of these was purified to homogeneity by ion-exchange chromatography and native preparative PAGE. The optimum pH and temperature for the proteinase were 8.0 and 50°C, respectively. The enzyme remained stable over a pH range from 6 to 10. The molecular weight estimated by SDS–PAGE was 56 kDa. Serine proteinase inhibitors 3,4-dichloroisocoumarin (3,4-DCI) and phenylmethylsulphonylfluoride (PMSF) inhibited, while Mg²⁺ and Ca²⁺ ions activated the proteinase.     

草鱼消化道酸性蛋白酶的提取及鉴定

从草鱼消化道中分离制取粗草鱼消化道酸性蛋白酶并分析鉴定了酶的部分性质和成分。粗草鱼酸性蛋白酶的比活力为2300U/g,最适作用温度和最适作用pH分别为37℃和pH2³,在50℃保温30min后损失约80%的酶活力。50μmol/L的胃抑酶素抑制79.64%的酶活力。底物-聚丙烯酰胺凝胶电泳（底物-PAGE）和SDS-底物-聚丙烯酰胺凝胶电泳（SDS-底物-PAGE）表明酸性蛋白酶的主要活性成分的分子质量约为28.5ku。      

产细菌素弯曲乳杆菌的分离鉴定及细菌素特性初步研究

从西班牙传统色拉米香肠中分离到一株产细菌素菌株RX-6,其发酵液在排除有机酸、过氧化氢及菌体细胞干扰后,抑菌活性基本无变化;经硫酸铵盐析及透析处理后,抑菌活性明显增强;蛋白酶K处理后,抑菌活性消失,表明起抑菌作用的是蛋白类物质。通过菌体形态观察、生理生化特征实验、16SrRNA序列比对及系统发育分析,鉴定菌株RX-6为弯曲乳杆菌（Lactobacillus curvatus）。抑菌特性研究结果显示该菌株所产细菌素具有很好的热稳定性和酸碱耐受性,在121℃20min的高温处理后仍能保持一定活性,活性pH范围为3¹0;使用安全性高,胃蛋白酶和胰蛋白酶可将其完全分解,而酸性蛋白酶可将其部分分解;抑菌谱较广,对受试的7株李斯特菌（Listeria spp.）、3株金黄色葡萄球菌（Staphylococcus aereu）、1株大肠杆菌（Escherichia coli）及1株假单胞菌（Pseudomonas spp.）等都有明显抑制作用,显示出作为天然食品生物防腐剂的巨大应用潜力。      

产胸苷磷酸化酶短乳杆菌融合菌株的构建及其特性

利用紫外加热灭活双亲原生质体技术对短乳杆菌原生质体进行融合,考察短乳杆菌原生质体制备、再生及融合的影响因素,并对短乳杆菌融合子产酶能力和遗传稳定性进行了研究。结果表明:短乳杆菌在含0.6%甘氨酸发酵培养液培养至对数生长期,2 mg/mL溶菌酶于37℃恒温水浴酶解脱壁2 h,原生质体制备率和再生率可达95%和48%;20 W紫外灯距离20 cm照射50 min,60℃处理短乳杆菌原生质体60 min,原生质体的灭活率几乎为100%;将双亲灭活的原生质体用40%PEG6000,30℃恒温融合10 min,筛选融合子F15并进行5次传代测试其胸苷磷酸化酶活均在1.500 U/mg湿菌体,较亲本酶活提高了50%。     

马奶酒样乳杆菌乳糖酶LacZ基因的克隆,原核表达及活性分析

从开菲尔粒(Kefir)中分离出1株马奶酒样乳杆菌(Lactobacillus kefiranofaciens ZW3),具有较高的乳糖酶活性,以此菌株为材料,从其基因组中克隆得到LacZ型乳糖酶基因,该基因全长2007 bp,编码669个氨基酸。随后将该基因插入原核表达载体pET-32a中转入大肠杆菌BL21(DE3)进行过量表达,获得了其重组蛋白,纯化后分析了该重组蛋白的乳糖水解活性特点。结果显示,此蛋白在50℃,pH 7.0时乳糖水解活力最高,并在30~55℃,pH 5.0~9.0的范围仍能保持50%以上的酶活力,具有良好的工业应用潜力。     

Molecular and functional characterization of Lactobacillus sanfranciscensis strains isolated from sourdoughs

Fifty isolates of Lactobacillus sanfranciscensis from Italian sourdoughs were identified and typed by a polyphasic approach which included genotypic and phenotypic criteria. Genotypic diversity was characterized by Ribosomal Intergenic Spacer Analysis (RISA) of PCR amplified 16S-23S rDNA spacer region, denaturing gradient gel electrophoresis (DGGE) of PCR amplified rpoB (beta subunit of RNA polymerase) gene, and rep-PCR (PCR amplification of repetitive bacterial DNA elements) analyses. The RISA analysis produced a unique electrophoretical profile of four bands (ranging from 300 to 600 bp) for all L. sanfranciscensis isolates. The DGGE analysis of rpoB gene allowed the subdivision of isolates in four clusters. The resolution found by using rep-PCR with primers BOXA1R and REP1R-I/REP2-I allowed the widening of the level of isolates heterogeneity. Phenotypic diversity was evaluated by Biolog System and characterization of several technological traits (e.g., acidification kinetics, proteinase and peptidase activities). L. sanfranciscensis isolates used a large varieties of carbon sources such as dextrin, D-fructose, L-fucose, alpha-D-glucose, maltose, palatinose, L-rhanmose, L- and D,L-lactic acids and L-methionine. The acidification activity and related quotient of fermentation, and the peptidase (PepN, PepV, PepT, PepI, PepX, PepQ and PepR) activities markedly varied among strains. The same was found concerning the capacity to liberate amino acids during sourdough fermentation. This study could be considered as an example of a computerized analysis of the genotypic and phenotypic traits to reliably and rapidly differentiate sourdough isolates. Although some L. sanfranciscensis isolates combined several technological traits, the association of more selected strains seemed to be a requisite to get optimal sourdough characteristics.     

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.