一株Kluyveromyces lactis酵母胞内乳糖酶性质的研究

源自乳酸克鲁维酵母的β-半乳糖苷酶为胞内酶,其具有乳糖水解能力和半乳糖苷的转移作用。本实验运用单因素试验方法研究乳酸克鲁维酵母乳糖酶的性质。结果表明该酶在pH值为6.0~7.0和37℃~48℃间比较稳定,酶作用最适pH值在6.5,最适反应温度为43℃,Mn2+、Mg2+等对酶有明显的激活作用,而Zn2+、Cu2+等对酶活有抑制作用。该酶以ONPG底物的米氏常数为4.186mmol/L。     

乳糖酶提取与纯化的研究

用甲苯将培养所得的脆壁酵母菌体破碎,经丙酮处理,即得乳糖酶粗制品。产量230mg/1000ml发酵液,活力30,000 ONPG单位/mg酶干重,与美国印第安纳州埃尔哈特公司(ELKhart)曼尔斯(Miles)实验室生产的米曲霉乳糖酶(TAKAMINE BRAND FUNGALLACTASE 30,000)比较。对乳糖的水解率,差异不显著(P>0.05)。本制品适于实验室大量生产。酶粗制品经进一步盐析、透析和凝胶过滤,得到纯制品,产量 80mg/1000ml发酵液,活力55000 ONPG单位/mg酶干重,可作为生化制剂应用于生化分析中。     

高转苷活性乳糖酶编码基因的克隆与酶学特征研究

运用鸟枪克隆术,从环状芽胞杆菌B2301基因组中克隆出乳糖酶编码基因,其完整读框大小为5 133bp,编码1 710个氨基酸残基,不含典型细菌信号肽序列,与已有报道的β-半乳糖苷酶的最高一致性为93.6%。初步表达和纯化了此乳糖酶,重组乳糖酶在50℃(ONPG)/60℃(乳糖)和pH 6.0～6.5下表现出最高催化活性,Zn²⁺、Fe²⁺、Cu²⁺、EDTA和SDS对重组酶表现出不同程度的抑制作用;该酶在50和55℃下催化合成低聚半乳糖的V_max分别为2.21和2.47 g/(L·h),K_m分别为10.46和14.37 g/L。所获得的乳糖酶具有以乳糖为底物酶法合成低聚半乳糖的优良应用属性,可为后续针对此酶的高效表达与工业化应用奠定基础。     

固定化乳糖酶研究进展

乳糖酶水解乳糖为半乳糖和葡萄糖,可缓解乳糖不耐症.固定化乳糖酶极易与底物分离,可较长时间进行反复分批反应和装柱反应,提高酶稳定性,且利于过程控制,产物中无酶的残留,能简化提纯工艺;固定化乳糖酶与游离酶相比更适合多酶反应,使用效率更高,成本降低.文中阐述了国内外关于固定化乳糖酶载体、方法及其性质与应用等方面的研究进展.     

开菲酵母乳糖酶活性的研究

本研究分别以牛奶乳清和羊奶乳清为培养基,接种从开菲粒中分离出的纯种乳糖发酵酵母(开菲酵母Kluyvcromyces kefir)进行发酵,收集酵母菌体,溶菌,测定其中的乳糖酶活性,并研究温度及pH环境对乳糖酶活性的影响。结果表明:在发酵40～45小时时,乳糖酶的活性最高(羊奶乳清:6.964ONPG单位/ml发酵液,牛奶乳清:4.182 ONPG单位/ml发酵液)。进一步研究得出,此乳糖酶的最适pH为6.5,最适温度为45℃。     

乳酸克鲁维酵母乳糖酶性质的研究

采用单因素试验方法研究了乳酸克鲁维酵母乳糖酶的性质,该酶在pH值为6.0～8.4范围内比较稳定,酶作用最适pH值在6.4～6.6;最适反应温度为45℃,并具有良好的热稳定性;Mn2＋,Mg2＋,Na＋等对酶有明显的激活作用,而重金属离子如Zn2＋和Cu2＋等对酶活有抑制作用。实验测定得该酶以ONPG为底物的米氏常数为3.348mmol/L。     

耐热乳糖酶的纯化及理化性质研究

目的:对从栖热菌(Thermussp.A3)中分离得到的耐热乳糖酶进行纯化及部分理化性质的研究。方法:采用聚丙烯酰胺凝胶盘状电泳分离耐热乳糖酶,用5-溴-4-氯-3-吲哚-β-D半乳糖苷(x-gal)进行酶染显色,切下单一电泳带进行回收;分别用SDS-聚丙烯酰胺凝胶电泳法(SDS-PAGE)及非变性孔径梯度聚丙烯酰胺凝胶电泳法测定其分子质量,用聚丙烯酰胺管状等电聚焦电泳法测其等电点。结果表明:用SDS-PAGE法及梯度电泳法测定该耐热乳糖酶的分子质量分别为51.1kD和65.6kD;用等电聚焦电泳法测得其等电点约为7.2。结论:从栖热菌中提取的耐热乳糖酶的理化性质不同于其它来源的乳糖酶。     

乳链球菌乳糖酶基因的分子克隆

以LacDNA片段为探针，筛选了pUC19为载体构建的乳链球菌（S.lactis)6030菌株的质粒DNA文库，得到插入片段约5.0kb的阳性克隆，并对插入片段进行限制性酶切分析，用ONPG法测定了重组质粒在大肠杆菌细胞中乳糖酶的表达水平。     

乳糖酶及其在乳品工业中的应用

本文从高产乳糖酶菌株的选育、产酶培养基的优化、乳糖酶的获得方法等几方面对乳糖酶的研究进行了综述，同时论述其在乳品工业中的应用。     

黑曲霉乳糖酶冻干保护剂的研究

针对乳糖酶在冻干过程中的失活问题,研究了几种保护剂对乳糖酶真空冷冻干燥过程中的保护作用,使用单因素筛选和正交试验方法,确定了乳糖酶保护剂最优配方为1%甘露醇、2%乳糖、7%可溶性淀粉,对乳糖酶真空冷冻干燥过程中具有显著保护作用,残余酶活为73.55%。     

STABILITY AND PROPERTIES OF A THERMOSTABLE β-GALACTOSIDASE IMMOBILIZED ON CHTTIN

Thermostable β‐galactosidase from an E. coli transformant containing the enzyme gene from P. woesei was immobilized at pH 4.0 and a glutaraldehyde concentration of 10 mM on chitin isolated from shrimp Crangon crangon shells. These preparations had a specific activity of 43,000 U/g of chitin at 85C using ONPG as substrate. The optimum pH and temperature for immobilized β‐galactosidase activity were 5.2 and 93C. Immobilization shifts the optimum pH for the activity of the enzyme by 0.2 units towards the acid side. The immobilized enzyme is stable at temperatures close to the optimal value, and the residual activity for ONPG hydrolysis of the preparations incubated 5 h in 0.1 M phosphate citrate buffer (pH 5.4) at 90C and 100C was 70% and 40% of the initial value, respectively.     

耐高温β-半乳糖苷酶的分离纯化与酶学性质分析

为从嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)XG24 发酵液中纯化到β- 半乳糖苷酶,并对酶学性质进行研究,利用硫酸铵分级盐析、DEAE-Sepharose Fast Flow 阴离子交换层析和Sephadex G-75 分子筛凝胶过滤层析等方法进行分离纯化。结果表明：经过系列步骤纯化后,酶纯度提高了54.5 倍,回收率20.4%,酶比活力达32.7U/mg。以邻- 硝基酚-D- 半乳糖吡喃糖苷(ONPG)为底物,研究β- 半乳糖苷酶的酶学性质。最适pH6.5,最适作用温度65℃。此菌株产β- 半乳糖苷酶在70℃以下和pH4.0～8.0 范围内具有较好的稳定性;Mg2+、Mn2+、Fe2+和Co2+ 对此酶有明显激活作用,而Cu2+、Ag+、Hg2+ 几乎完全抑制酶活性。以ONPG 为底物酶的Km 值为4.32mmol/L。SDS-PAGE 和凝胶过滤层析测得酶蛋白为单肽链蛋白,表观分子质量64kD。因此,嗜热脂肪芽孢杆菌XG24 β- 半乳糖苷酶在乳制品工业中具有潜在的应用价值。     

马克斯克鲁维酵母乳糖酶水解和转移性质比较

主要对马克斯克鲁维酵母(Kluyveromyces marxianus)产β-D-半乳糖苷酶的水解性质和转移酶学性质进行了比较。对比发现温度、pH、金属离子以及葡萄糖和半乳糖浓度对该酶的两种性质有不同影响,同时还研究了酶的动力学特性,以ONPG为底物,测得马克斯克鲁维酵母β-D-半乳糖苷酶的Km为0.51mmol/L,Vmax为0.532μmol/(mg protein·min)。以乳糖为底物,Km为0.56mmol/L,Vmax为0.31μmol/(mg protein·min)。这对研究生产低聚半乳糖有重要作用。     

CHARACTERIZATION OF A CHEMICALLY CONJUGATED β-GALACTOSIDASE BIOREACTOR1

A chemically prepared conjugate of avidin and E. coli β-galactosidase was adsorbed to biotinylated controlled-pore glass beads and used in a fluidized-bed bioreactor to assess the feasibility of bioselective adsorption immobilization technology. Biotinylated 200 nm pore diameter porous glass beads were prepared by reaction of 3-aminopropyl-glass beads with sulfosuccinimidyl-6-(biotinamido) hexanoate. Avidin and biotinylated β-galactosidase were sequentially adsorbed to the matrix. The fluidized-bed bioreactor was characterized with respect to β-galactosidase activity using both a lactose solution and o-nitrophenyl β-D-galactopyranoside (ONPG) as substrates. A lactose solution (4.5%, pH 7) was assayed for lactose hydrolysis at various flow rates. The bioreactor was operated for three months at 65–75% lactose hydrolysis with no loss in enzyme activity. The biocatalyst was characterized by amino acid analysis to determine the amount of each of the two proteins adsorbed. Results indicated 162 μ protein/mg beads of which 36% was avidin and 64% was β-galactosidase corresponding to 1 mole of avidin per mole of β-galactosidase monomer. Biocatalyst activity using ONPG as the substrate was 430 μmoles/min/mg protein, yielding a specific activity of 672 μmoles/min/mg β-galactosidase. These results lead to the conclusion that biospecific adsorption of the β-galactosidase conjugate onto biotinylated glass beads via avidin results in a biocatalyst that is stable and retains a high specific activity.     

ISOLATION AND SOME PROPERTIES OF THE THERMOSTABLE β-GALACTOSIDASE OF PYROCOCCUS WOESEI EXPRESSED IN ESCHERICHIA COLI

The enzyme with β-galactosidase activity from E. coli BL21(DE3) transformant containing the gene encoding enzyme from Pyrococcus woesei (DSM 3773) was isolated using cell extraction in 0.01 M phosphate buffer (pH 7.2), protein thermopredpitation at 85C, precipitation at acetone/extract ratio of 1:1 (v/v) and gel filtration on Sephadex G-200. The increase in the enzyme specific activity was determined using ONPG as substrate. The activity increased from 2.9 × 10³ U/mg protein to 37 × 10³ U/mg. Thermoprecipitation removed 78% of E. coli protein and retained 92% of the cell extract activity. The acetone precipitation and gel filtration applied in the next purification steps led to homogeneous enzyme with specific activity of 37,700 U/mg protein. The isolated enzyme had a half-life of 23 h and 9 h during incubation at 85C and 100C, respectively, in 0.1 M citrate-phosphate buffer (pH 5.4).     

耐高温β-半乳糖苷酶菌株筛选及酶特性研究

采用乳糖作为单一碳源、X-gal做显色剂,从奶牛场土壤中筛选到90株产β-半乳糖苷酶的菌株。根据菌落在初筛平板上菌落蓝色深浅和蓝色圈直径,复筛出10株酶活较高的菌株。以ONPG为底物,测定该10株菌发酵液β-半乳糖苷酶的水解活性及稳定性,复筛出1株革兰阳性好氧细菌,命名为菌株XG24。通过形态学、生理生化实验以及菌株16SrDNA序列分析,菌株XG24鉴定为嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)。所产酶最适反应温度和pH分别为65℃和6.5℃;该酶在实验所用温度40~75℃内酶活性稳定,70℃孵育1h,残留酶活仍有80%;在pH4.0~8.0之间酶活相对稳定。浓度为1mmol/L的Mg2+、Fe2+、Co2+以及Mn2+离子对酶有强烈的激活作用,但Hg2+、Cu2+及Ag+抑制酶活性。酶活还受到高浓度的乳糖水解产物葡萄糖和半乳糖一定的反馈抑制作用,其他糖对酶活性没有明显影响。这些酶的优良生化特性赋予了该酶在乳品工业中具有重要的应用价值。     

Microencapsulation of β-galactosidase with different biopolymers by a spray-drying process

The aim of this work was to investigate the possibility of producing microparticles containing β-galactosidase, using different biopolymers (arabic gum, chitosan, modified chitosan, calcium alginate and sodium alginate) as encapsulating agents by a spray-drying process. This study focused on the enzyme β-galactosidase, due to its importance in health and in food processing. Encapsulation of β-galactosidase can increase the applicability of this enzyme in different processes and applications. A series of β-galactosidase microparticles were prepared, and their physicochemical structures were analyzed by laser granulometry analysis, zeta potential analysis, and by scanning electron microscopy (SEM). Microparticles with a mean diameter around 3 μm have been observed, for all the biopolymers tested. The microparticles formed with chitosan or arabic gum presented a very rough surface; on the other hand, the particles formed with calcium or sodium alginate or modified chitosan presented a very smooth surface. The activity of the enzyme was studied by spectrophotometric methods using the substrate ONPG (O-nitrophenyl-β,d-galactopyranoside). The microencapsulated β-galactosidase activity decreases with all the biopolymers. The relative enzyme activity is 37, 20, 20 and 13%, for arabic gum, modified chitosan, calcium alginate and sodium alginate, respectively, when compared with the free enzyme activity. The enzyme microparticles formed with arabic gum shows the smallest decrease of Vmax, followed by the calcium alginate, sodium alginate, and modified chitosan.     

Immobilization of β-galactosidase from Cicer arietinum (gram chicken bean) and its catalytic actions

β-Galactosidase (β-d-galactosidase galactohydrolase EC 3.2.1.23) isolated and purified from Cicer arietinum (gram chicken bean) was immobilized on two kinds of modified resin D202 with glutaraldehyde. Both the immobilized enzymes had high protein-binding capacity and high yield of enzyme activity. Kinetics results showed that the enzyme activity attained its maximum at 57°C, pH 6 for the immobilized β-galactosidase I and 52°C, pH 6 for the immobilized β-galactosidase II, respectively. The operational pH range was increased. Kinetic constants (K_m, V_max and E_a) for the free and bound enzymes, with ONPG as substrate, were studied. Results showed that K_m and V_max of immobilized enzymes were decreased while E_a of them was increased. The effects of some compounds and organic solvents for the free and immobilized enzymes were discussed. Inhibitory constants for raffinose, lactose and d-galactose, which were all reversible inhibitors of the enzymes, were also obtained.