鉴定隶属于糖苷水解酶13家族(glycoside hydrolase family 13, GH13)的潮滩发光杆菌的β-半乳糖苷酶

【背景】糖苷水解酶13家族(glycoside hydrolase family 13, GH13)是已知最大的α-淀粉酶家族,不含有半乳糖苷酶。【目的】对海洋细菌潮滩发光杆菌(Photobacterium gaetbulicola)的一个蛋白BgalPg进行鉴定。【方法】通过保守位点分析和系统发育树确定BgalPg蛋白的家族分类;通过克隆、表达和纯化测定重组BgalPg蛋白的酶学性质并鉴定功能。【结果】BgalPg的蛋白序列新颖,与已知的碳水化物酶无同源性。序列分析结果表明该蛋白具有GH13家族的典型特性,并且隶属于GH13＿38亚家族。BgalPg对α-淀粉酶家族酶的相关底物均无催化活性,却能水解含有β-半乳糖苷键的底物p NP-β-Gal [(2.8±0.4) U/mg]和o NP-β-Gal [(1.4±0.3) U/mg],并且能水解乳糖[(0.40±0.01) U/mg],表现出典型的β-半乳糖苷酶活性。同时,该酶在pH 7.0–8.5稳定性好,60℃的半衰期为1.5 h。【结论】发现隶属于GH13家族的β-半乳糖苷酶。     

一种来源于青霉的新的α-半乳糖苷酶的分离纯化及其酶学性质

从丝状真菌中筛选到一株产α-半乳糖苷酶的菌株F63,对该菌株进行了形态观察和18SrDNA序列分析,该菌株属于青霉属。采用硫酸铵沉淀、阴离子交换层析和分子筛层析等方法分离纯化了该菌株的一种α-半乳糖苷酶。经过聚丙烯酰胺凝胶电泳,此酶蛋白的分子量约为82kDa。该α-半乳糖苷酶反应的最适pH为5.0,最适温度为45℃。此α-半乳糖苷酶的热稳定性在40℃以下,pH稳定性为pH5.0-6.0。与已报道的α-半乳糖苷酶的活性都受到Ag 的强烈抑制不同的是,该α-半乳糖苷酶受Ag 的抑制作用不显著。以pNPG为底物的Km值为1.4mmol/L和Vmax=1.556mmol/L.min-1.mg-1。该酶可以有效降解蜜二糖、棉子糖和水苏糖,但不能降解末端含α-半乳糖苷键的多糖。通过利用质谱技术对纯化的α-半乳糖苷酶进行鉴定以及内肽的N端测序证明该蛋白为一种新的α-半乳糖苷酶。     

粪便宏基因组来源低分子量碱性β-半乳糖苷酶的异源表达及酶学性质

【背景】β-半乳糖苷酶在食品加工、临床医疗及基因工程等领域有重要的应用价值,开发酶活性高、热稳定性强的β-半乳糖苷酶已成为研究热点。【目的】从西黑冠长臂猿（Nomascus concolor）粪便微生物宏基因组中挖掘新型β-半乳糖苷酶并进行酶学性质研究。【方法】以西黑冠长臂猿粪便微生物宏基因组DNA为模板扩增β-半乳糖苷酶基因GalNC1-8,构建重组表达质粒pEASY-E2/GalNC1-8,转化至大肠杆菌（Escherichia coli） BL21（DE3）异源表达,研究其酶学性质。【结果】获得GH35家族碱性β-半乳糖苷酶GalNC1-8,其分子量为28.18 kDa,最适温度为50°C,最适pH为8.0。将该酶在30-50°C下处理1 h,剩余酶活仍保持在80%以上;pH 7.0-9.0下处理1 h,剩余酶活大于54%。在含乙醇的反应体系中,其酶活性几乎不受影响;β-巯基乙醇、丙三醇、甲醇、Na⁺、K⁺和Li⁺对其酶活性有促进作用。在0.5-3.5 mol/L NaCl下处理1 h后,仍保留50%以上的酶活性。...     

一株产低温β-半乳糖苷酶微杆菌的筛选鉴定、产酶条件及其酶学特性

【背景】低温β-半乳糖苷酶能在低温下仍保持较高的乳糖水解活性,筛选酶学特性适合在牛乳体系中高效水解乳糖的β-半乳糖苷酶生产菌株,是低乳糖牛乳加工产业关注的焦点。【目的】对天山中国一号冰川沉积物中分离的一株产低温β-半乳糖苷酶菌株的产酶条件和酶学特性进行研究。【方法】结合X-Gal平板法初筛和测定粗酶液酶活复筛,获得产低温β-半乳糖苷酶的菌株。通过形态学、生理生化试验及16S rRNA基因测序分析对筛选菌株进行鉴定,单因素摇瓶实验优化菌株的产酶条件,硫酸铵分级沉淀初步纯化β-半乳糖苷酶并对其酶学特性进行分析。【结果】通过形态学、生理生化特征和16S rRNA基因鉴定,确定菌株LW106为微杆菌属(Microbacterium)菌株;该菌株最适产酶温度为25°C,最佳产酶碳源为可溶性淀粉,培养基初始pH为7.0,接种量为3%;对初步纯化的低温β-半乳糖苷酶酶学性质的研究表明,LW106所产β-半乳糖苷酶的最适pH为6.0,最适反应温度为35°C,4°C时酶活为最大酶活的78%,4°C和pH 7.0时的稳定性最好,10 mmol/L的Na+对酶活性基本没有抑制作用,Ca~(2+)对酶活性具有一定的激活作用。【结论】菌株LW106所产低温β-半乳糖苷酶的酶学特性表明该酶在乳品低温加工领域具有进一步研究和应用的价值。     

滇金丝猴粪便微生物来源的β-半乳糖苷酶基因的表达及酶学性质

【目的】对滇金丝猴粪便微生物来源的β-半乳糖苷酶进行异源表达和纯化,并研究其酶学性质。【方法】从滇金丝猴粪便微生物的宏基因组中克隆出一个β-半乳糖苷酶基因galRBM20_1,对该基因进行异源表达和酶学性质分析。构建含有T7强启动子的pEASY-E2-galRBM20_1质粒,转化至大肠杆菌BL21(DE3),经IPTG诱导表达后进行酶学性质研究。【结果】滇金丝猴粪便来源的β-半乳糖苷酶(galRBM20_1)最适pH为5.0,在pH 4–7之间能保留70%及其以上的活性。最适温度为45°C,在37°C和45°C下耐受1 h,酶活不变。特别的是,该酶具有良好的Na Cl稳定性,经1–5 mol/L的Na Cl作用1 h后,相对酶活均能超过初始酶活:当NaCl的作用浓度为4 mol/L时,β-半乳糖苷酶相对酶活最高(146%);当NaCl的作用浓度为5mol/L时,β-半乳糖苷酶的相对酶活仍达到135%。【结论】本研究从滇金丝猴粪便微生物的宏基因库中克隆得到β-半乳糖苷酶基因galRBM20_1,并成功在大肠杆菌BL21(DE3)表达,首次从动物胃肠道宏基因组中获得具有耐盐和转糖基产Galactooligosaccharides(GOS)性能的β-半乳糖苷酶。该酶具有良好的耐盐性,和较广的pH作用范围,使其在食品、生物技术领域和环保方面的发展具有良好的应用价值。     

利用重组A蛋白偶联的琼脂糖凝胶纯化α-半乳糖苷酶单克隆抗体

目的:采用一种简单易行的纯化方法获得高纯度α-半乳糖苷酶单克隆抗体。方法:使用重组A蛋白偶联的琼脂糖凝胶捕获和纯化小鼠腹水中的α-半乳糖苷酶单克隆抗体。结果:获得了高纯度、高效价的α-半乳糖苷酶单克隆抗体。结论:应用重组A蛋白偶联的琼脂糖凝胶纯化α-半乳糖苷酶单克隆抗体是一种简单、有效的方法。     

微生物源α-半乳糖苷酶的研究进展

介绍了微生物源α-半乳糖苷酶的生理生化特性、合成调控机制的研究进展情况及其在食品、饲料、医药工业等领域的一些应用。Α-半乳糖苷酶均是糖蛋白,不同来源的α-半乳糖苷酶的作用基质特异性差别较大,作用基质特异性差别是由蛋白质部分N-末端氨基酸序列决定的。不同微生物来源的α-半乳糖苷酶其最佳作用条件、pH稳定性及耐热性差异较大。微生物α-半乳糖苷酶是一种诱导酶,其合成受多个基因的调控,高浓度的葡萄糖能抑制其合成。     

阴道乳杆菌耐热β-半乳糖苷酶的鉴定

利用改良的MRS培养基,从鸡粪样本中分离到多株产β-半乳糖苷酶的乳酸菌菌株。酶学性质分析发现,菌株1-1产生的β-半乳糖苷酶在37℃~60℃相对稳定,37℃酶活力达到183.9NLU/g菌体干重。进一步分析16S rRNA基因序列,确定菌株1-1为阴道乳杆菌(Lactobacillus vaginalis)。扩增分析β-半乳糖苷酶编码基因lacL和lacM,结果发现LacL亚基有642个氨基酸,LacM亚基有321个氨基酸,与罗伊氏乳杆菌MM2-3相应蛋白的相似性分别为86%和84%。     

重组α-半乳糖苷酶的制备工艺研究

α-半乳糖苷酶是B→O血型改造研究中的关键工具酶。在获得了可分泌表达α-半乳糖苷酶的基因工程毕赤酵母菌株的基础上,进行了工程菌株在5L发酵罐中的发酵。发酵液上清中α-半乳糖苷酶活性为80～150U／mL,蛋白浓度为3～4.5mg／mL,比活性约为20-30U／mg。发酵液采用超滤、阳离子交换层析、疏水层析和阴离子交换层析等纯化方法,建立起了规模化生产重组α-半乳糖苷酶的工艺。制备的重组酶纯度经鉴定达98％以上,符合新生物制品的纯度要求。制备的重组α-半乳糖苷酶可有效地将B型红细胞改造成O型红细胞,从而解决了应用此酶开展B→O血型改造研究的关键问题。     

微生物α-半乳糖苷酶的研究进展

α-半乳糖苷酶在多种生物内广泛存在,微生物是目前α-半乳糖苷酶的主要来源。微生物α-半乳糖苷酶可按照底物特异性或序列同源性分类,在古菌、细菌和真菌中均存在,其性质与来源和家族有关,催化机理大多为构型保留机制,目前主要应用于食品与饲料工业,还可用于生物质降解和医药领域。展望了微生物α-半乳糖苷酶的研究趋势。本文对相关研究者具有一定的参考意义。     

Heterologous expression of glycoside hydrolase family 2 and 42 β-galactosidases of lactic acid bacteria in Lactococcus lactis

This study characterized a glycoside hydrolase family 42 (GH42) β-galactosidase of Lactobacillus acidophilus (LacA) and compared lactose hydrolysis, hydrolysis of oNPG, pNPG and pNPG-analogues and galactooligosaccharides (GOSs) formation to GH2 β-galactosidases of Streptococcus thermophilus (LacZ type), Lactobacillus plantarum and Leuconostoc mesenteroides subsp. cremoris (both LacLM type). Beta-galactosidases were heterologously expressed in Lactococcus lactis using a p170 derived promoter; experiments were performed with L. lactis crude cell extract (CCE). The novel GH42 β-galactosidase of Lb. acidophilus had lower activity on lactose, oNPG and pNPG but higher relative activity on pNP analogues compared to GH2 β-galactosidases, and did not transgalactosylate at high lactose concentrations. Temperature and pH optima for lactose hydrolysis varied between GH2 β-galactosidases. oNPG and pNPG were the preferred substrates for hydrolysis; in comparison, activity on pNPG-analogues was less than 1.5%. GH2 β-galactosidases formed structurally similar GOS with varying preferences.     

根霉α-半乳糖苷酶的三相分离及其酶学性质

根霉Rhizopus sp. A01发酵豆渣产α-半乳糖苷酶,粗酶液依次经过三相分离、Sephadex G-100凝胶过滤获得了电泳纯的α-半乳糖苷酶,纯化了6.7倍,总酶活回收率达到46%;凝胶过滤和SDS-PAGE显示该酶为相对分子质量为87.6kDa的单体蛋白。该酶水解对硝基苯-α-D-吡喃半乳糖苷的最适pH值为5.0,最适温度为55℃,表观Km、kcat/Km分别为2.56mmol/L、47,400L/mol·s;能微弱水解蜜二糖和棉子糖,水解蜜二糖的速率是水解棉子糖速率的3.4倍;水解活性受多种     

Isolation of poly(3-hydroxybutyrate) (PHB)-degrading microorganisms and characterization of PHB-depolymerase from Arthrobacter sp. strain W6.

Microbial degraders of poly(3-hydroxybutyrate) (PHB) were isolated from soil. Arthrobacter sp. strain W6 used not only PHB as a carbon source, but also PHAs such as poly(3-hydroxybutyrate-co-[5%]3-hydroxyvalerate), poly(3-hydroxybutyrate-co-[14%]3-hydroxyvalerate), and poly(3-hydroxybutyrate-co-[22%]3-hydroxyvalerate). PHB-depolymerase was purified to homogeneity from the culture broth of Arthrobacter sp. strain W6 by a procedure involving DEAE- and butyl-Toyopearl column chromatographies. The Mr of the enzyme was estimated to be about 47,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 8.5 and 50 degrees C, and was inhibited by phenylmethylsulfonyl fluoride, Hg2+, Ag+, and Pb2+.     

Biochemical and hydrolytic properties of multiple thermostable α-galactosidases from Streptomyces griseoloalbus: Obvious existence of a novel galactose-tolerant enzyme

The multiple α-galactosidases from Streptomyces griseoloalbus—α-Gal I, α-Gal II and α-Gal III were purified to homogeneity by a two-step chromatographic process. The molecular masses and pI of the three enzymes were 72, 57 and 35 kDa, and 4.41, 5.6 and 6.13, respectively. α-Gal I showed N-terminal sequence homology to S. coelicolor A3(2) family 27 α-galactosidase. The optimum pH and temperature of the three α-galactosidases were 5.0, 6.5 and 5.5 and 65, 50 and 55 °C, respectively. α-Gal I was stable up to 65 °C and α-Gal II and α-Gal III up to 55 °C for 2 h. Based on the hydrolytic properties α-Gal I could be classified as a member of GH27 family and α-Gal II and α-Gal III as members of GH36 family. Metal cations like Hg²⁺, Ag²⁺ and Cu²⁺ inhibited enzyme activity while Mg²⁺ enhanced the activity of α-Gal I. Interestingly α-Gal I showed unusual tolerance to even higher concentrations of galactose, unlike the other two α-galactosidases, which were competitively inhibited by galactose. Melibiose was a competitive inhibitor of all three enzymes. Histidine, tryptophan and carboxylic residues were essential for catalytic action of the three α-galactosidases.     

Fabry's disease as an -galactosidosis: evidence for an -configuration in trihexosyl ceramide

Pure α-galactosidases, devoid of β-galactosidase activity, were purified from coffee beans, ficin (a crude extract from figs), rat and rabbit small intestine. With the exception of the coffee bean enzyme, all α-galactosidase preparations released galactose from ³H- or ¹⁴C-labeled trihexosyl ceramide obtained from patients with Fabry's disease. Galactose liberation was specifically inhibited by α-galactosides, such as melibiose and stachyose, while lactose had no effect. Our results corroborate the α-galactosidase deficiency reported in Fabry's disease and establish that the terminal galactosyl residue of the trihexosyl ceramide stored in this condition has an α-configuration.     

The α-galactosidase from Escherichia coli K12

Growth of Escherichia coli on melibiose requires the induced synthesis of α-galactoside permease and α-galactosidase. Hydrolysis of the chromogenic substrate p-nitrophenyl-σ-galactoside by whole bacteria is dependent on intact oxidative metabolism. The α-galactosidase from E. coli was isolated for the first time as a soluble enzyme. In cell-free extracts p-nitrophenyl-α-galactoside hydrolisis was observed only at high protein concentrations and the activity decreased exponentially with the square of the dilution. The reason for this behaviour was shown to be that, unlike other known α-galactosidases, the enzyme of E. coli requires NAD. For optimal activity the enzyme also requires Mn²⁺, a high concentration of 2-mercaptoethanol, and a pH of 8.1. The approximate molecular weight of the active from of α-galactosidase as determined by sedimentation in a sucrose gradient is 200 000. Due to the instability of the enzyme, its purification has not been achieved.     

蜗牛酶中一种β-葡萄糖苷酶的纯化及酶学性质的研究

通过DEAE-Sepharose离子交换层析和Sephadex G-100凝胶过滤层析的联用从中华白玉蜗牛消化酶中分离出1种具有人参皂苷Rb_1水解活性的β-葡萄糖苷酶.纯化后该酶在SDS-PAGE上呈单一蛋白质条带.反应最适pH为5.6,最适温度是80 ℃.pH稳定范围很广,在pH为4.0～11.0的溶液中和温度60 ℃以下保持长时间稳定状态,是一个耐碱和中等耐热的糖苷酶.Na~+、K~+、Li~+、Ca~(2+)、Mg~(2+)、EDTA、DTT和SDS不影响该酶活性,而Cu~(2+)、Ag~+和Fe~(3+)对该酶则具有明显的抑制作用.pNPG为底物的动力学参数Km和Vmax分别为0.182 mmol/L和0.189 μmol/(min·mg).     

Interconversion of the A and B forms of ceramide trihexosidase from human plasma

The human plasma α-galactosidases which specifically hydrolyze galactosyl-(α1→4)galactosyl(β1→ 4)glucosylceramide consist of an A group with optimal enzymatic activity at pH 5.4, and a B group, which is characterized by optimal activity at pH 7.2. The relationship between the A and B groups of these α-galactosidases (ceramide trihexosidases) has been investigated with regard to their sialic acid content. Partial neuraminidase treatment of the most acidic (A-1) form of ceramide trihexosidase yields a complex mixture of 14 enzymatically active proteins separable by isoelectric focusing. Exposure to neuraminidase for a longer time causes an almost complete conversion of the A-1 form to a protein which has the same electrophoretic properties as the least acidic (B-V) form. Conversely, a crude kidney sialyltransferase preparation can be used to incorporate either CMP[1-¹⁴C]sialic acid or UDP-N-acetyl[1-¹⁴C]glucosamine into the B-V form of the enzyme. Sialyltransferase treatment causes the formation of a complex mixture of enzymatically active proteins, one of which has the same electrophoretic characteristics as the A-1 and A-2 forms of ceramide trihexosidase. On the basis of these studies it is suggested that the multiple forms of plasma ceramide trihexosidase are glycoproteins which differ primarily in their sialic acid content.     

土壤中高产蛋白酶菌株产酶条件及酶学性质

【背景】微生物蛋白酶已经成为工业用蛋白酶的主要来源,筛选具有特殊环境适应性的微生物成为生物酶资源的开发热点。【目的】通过对青藏高原土壤微生物产蛋白酶菌株的筛选、优化及相关特性研究,寻找新的蛋白酶资源,为高原菌种资源利用提供科学依据。【方法】采用形态学和分子生物学对筛选菌株进行菌种鉴定,利用单因素试验和正交试验对菌株进行发酵条件优化及酶学性质的探究。【结果】筛选出一株高产蛋白酶菌株XC2,经鉴定菌株XC2为枯草芽孢杆菌(Bacillus subtilis)。XC2最优产酶条件:可溶性淀粉4.0%,牛肉膏1.0%,K~+0.6%,培养温度34°C、初始pH 7.0、接种量2.0%的条件下200 r/min振荡培养13 h,所产蛋白酶活力最高为638.5 U/mL。XC2所产蛋白酶最适反应温度60°C,最适pH9.0;40-50°C、pH8.0-10.0条件下酶活稳定性较高;Mn~(2+)对酶活力有明显激活作用,而Zn~(2+)、Cu~(2+)、Fe~(2+)、Fe3+对酶活力有明显抑制作用。【结论】枯草芽孢杆菌XC2有较强的产碱性蛋白酶的能力,具有较好的应用前景。     

Purification and characterization of β-galactosidase from probiotic Pediococcus acidilactici and its use in milk lactose hydrolysis and galactooligosaccharide synthesis

β-galactosidase is a commercially important enzyme that was purified from probiotic Pediococcus acidilactici. The enzyme was extracted from cells using sonication and subsequently purified using ammonium sulphate fractionation and successive chromatographies on Sephadex G-100 and Q-Sepharose. The enzyme was purified 3.06-fold up to electrophoretic homogeneity with specific activity of 0.883 U/mg and yield of 28.26%. Molecular mass of β-galactosidase as estimated by SDS-PAGE and MALDI-TOF was 39.07 kDa. The enzyme is a heterodimer with subunit mass of 15.55 and 19.58 kDa. The purified enzyme was optimally active at pH 6.0 and stable in a pH range of 5.8–7.0 with more than 97% activity. Purified β-galactosidase was optimally active at 50 °C. Kinetic parameters K_m and V_max for purified enzyme were 400 µM and 1.22 × 10⁻¹ U respectively. Its inactivation by PMSF confirmed the presence of serine at the active site. The metal ions had different effects on enzyme. Ca²⁺, Mg²⁺ and Mn²⁺ slightly activated the enzyme whereas NH₄⁺, Co²⁺ and Fe³⁺ slightly decreased the enzyme activity. Thermodynamic parameters were calculated that suggested that β-galactosidase is less stable at higher temperature (60 °C). Purified enzyme effectively hydrolysed milk lactose with lactose hydrolysing rate of 0.047 min⁻¹ and t_1/2 of 14.74 min. This is better than other studied β-galactosidases. Both sonicated Pediococcus acidilactici cells and purified β-galactosidase synthesized galactooligosaccharides (GOSs) as studied by TLC at 30% and 50% of lactose concentration at 47.5 °C. These findings indicate the use of β-galactosidase from probiotic bacteria for producing delactosed milk for lactose intolerant population and prebiotic synthesis. pH and temperature optima and its activation by Ca²⁺ shows that it is suitable for milk processing.