Purification and characterization of an extracellular beta-agarase from Bacillus sp. MK03

A new beta-agarase was purified from an agarolytic bacterium, Bacillus sp. MK03. The enzyme was purified 129-fold from the culture supernatant by ammonium sulfate precipitation, anion exchange and gel filtration column chromatographic methods. The purified enzyme appeared as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Estimation of the molecular mass by SDS-PAGE and gel filtration gave values of 92 kDa and 113 kDa, respectively. The N-terminal amino acid sequence of the enzyme showed no homology to those of other known agarases. The optimum pH and temperature for this enzyme were 7.6 and 40 degrees C, respectively. The predominant hydrolysis product of agarose by this enzyme was neoagarotetraose, indicating the cleavage of beta-1,4 linkage. This enzyme could hydrolyze neoagarohexaose to produce neoagarotetraose and neoagarobiose; it could not hydrolyze these products. The enzyme digested agarose by endo-type hydrolysis.     

Purification and characterization of an extracellular alpha-neoagarooligosaccharide hydrolase from Bacillus sp. MK03

An agarolytic bacterium was isolated from soil in Gifu prefecture, Japan, and identified as Bacillus sp. strain MK03. The strain secreted neoagarooligosaccharide hydroluse into the culture medium. The enzyme was purified 49.7-fold from the culture fluid by ammonium sulfate precipitation and anion-exchange and gel-filtration column chromatographic methods. The purified enzyme appeared as a single band on polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. Estimations of the molecular mass by gel filtration and SDS-PAGE gave values of 320 kDa and 42 kDa, respectively, indicating that the enzyme is octametric. The enzyme cleaved the alpha-1,3 linkage in neoagarobiose to produce 3,6-anhydro-L-galactose and D-galactose. It also selectively cleaved the alpha-1,3 linkage at the nonreducing end in neoagarotetraose or neoagarohexaose to give 3,6-anhydro-L-galactose and agarotriose or agaropentaose. The optimum temperature and pH for the enzyme were 30 degrees C and 6.1, respectively. The N-terminal amino acid sequence showed no homology to sequences of other known neoagarooligosaccharide hydrolases and agarases.     

Purification and characterization of a beta-glucosidase with beta-xylosidase activity from Aspergillus sojae

A beta-glucosidase (EC 3.2.1.21) was purified as an electrophoretically homogeneous protein from a solid culture of Aspergillus sojae. The molecular mass of the purified enzyme was estimated to be 250 kDa by gel filtration chromatography and 118 kDa by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). The isoelectric point of the enzyme was 3.80. The maximum velocity of rho-nitrophenyl beta-d-glucopyranoside degradation by the beta-glucosidase was attained at 60 degrees C and at pH 5.0. The purified enzyme was stable from pH 6.0 to 8.0, and up to 50 degrees C. The activity of the enzyme was significantly inhibited by Hg2+ and Cu2+, and stimulated by Mn2+ and Fe3+. The purified enzyme hydrolyzed beta-D-xylopyranosides as well as beta-D-glucopyranosides; the Km and Vmax values on rho-nitrophenyl beta-D-glucopyranoside were 0.14 mM and 16.7 micromol/min/mg protein, and on rho-nitrophenyl beta-D-xylopyranoside 0.51 mM and 12.2 micromol/min/mg protein, respectively.     

Purification and properties of a phytase from Candida krusei WZ-001

A phytase from Candida krusei WZ-001 isolated from soil was purified to electrophoretic homogeneity by ion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The phytase is composed of two different subunits with molecular masses of 116 kDa and 31 kDa on SDS-PAGE (or 120 kDa and 30 kDa on gel chromatography), with the larger subunit having a glycosylation rate of around 35%. The phytase has an optimum pH of 4.6, an optimum temperature of 40 degrees C and a pI value of 5.5. The phytase activity was stimulated by 2-mercapto-ethanol and dithiothreitol (DTT), and inhibited by Zn2+, Mg2+, iodoacetate, pI value of 5.5. The phytase activity was stimulated by 2-mercapto-ethanol ethanol and dithiothreitol (DTT), and inhibited by Zn2+, Mg2+, iodoacetate, p-chroloromercuribenzoate (pCMB) and phenylmethylsulfonyl fluoride (PMSF). The phytase displayed a broad substrate specificity and the K(m) for phytate was 0.03 mM. Phytate was sequentially hydrolyzed by the phytase. Furthermore, 1D and 2D NMR analyses and bioassay of myoinositol indicated that the end hydrolysis product of phytate was myoinositol 2-monophosphate.     

用于高果糖浆制备的宛氏拟青霉嗜热嗜酸菊粉酶酶学特性分析

为实现酶法水解菊糖制备高果糖浆,从宛氏拟青霉（Paecilomyces variotii）XS27发酵液中分离纯化菊粉酶,并对其酶学特性进行研究。发酵液经过硫酸铵盐析、透析、DEAE-Sepharose Fast Flow层析、Sephacry S-100分子筛过滤层析,得到电泳纯的菊粉酶,比活力327.4 U/mg,纯化倍数37.85。十二烷基硫酸钠-聚丙烯酰氨凝胶电泳测得菊粉酶为单一亚基的酶蛋白,分子质量62.0 kDa。菊粉酶能在较宽的pH值范围（3.5～6.5）内保持高活性,最适作用pH 4.0。在温度40～65 ℃之间,酶活力较高,最适作用温度为60 ℃。薄层色谱分析显示菊粉酶水解菊糖最终产物为果糖。以菊糖为底物,酶的Km和Vmax分别为5.93 μmol/L和75.18 μmol/（L·min）。Mg2＋、Mn2＋、Ca2＋对酶有显著激活作用,Ba2＋、Ni2＋和Hg2＋对酶有一定抑制作用。β-巯基乙醇、二硫苏糖醇和乙二胺四乙酸对酶有抑制作用,表面活性剂（十二烷基硫酸钠、Tween 80和Trition X100）以及乙醇对酶活力没有影响。从宛氏拟青霉XS27发酵液中分离纯化的菊粉酶在强酸高热的环境下具有强活性和稳定性,对表面活性剂乙醇有高耐受性,适合于果葡糖浆的工业化生产。     

产黄青霉抗真菌几丁质酶的纯化及特性分析

为实现抗真菌性能的耐热新型几丁质酶在食品防腐及生物防治方面的应用，从产黄青霉Xch23中分离纯化几丁质酶（Chi-Pc76）并研究其应用特性。通过硫酸铵沉淀、DEAE-Cellulose A52离子交换层析和Sephadex G-100凝胶过滤层析纯化得到均一的Chi-Pc76。最终得到Chi-Pc76纯化倍数17.1 倍，回收率21.6%，比活力为584.8 U/mg。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测得Chi-Pc76分子质量61 kDa。纯化后的Chi-Pc76最佳反应条件为pH 6.0、温度55 ℃。Chi-Pc76在宽泛的pH 4.0～10.0之间稳定性强，其显著的热稳定性可达到70 ℃。Chi-Pc76对底物胶体几丁质具有高的底物特异性，对乙二醇几丁质、α-几丁质、β-几丁质有中等活性，而对其他受试底物无活性或痕量活性。以胶体几丁质为底物Km和Vmax值分别为0.25 mg/mL和20 μmol/（min·mg）。金属离子Ca2＋、Ba2＋、K＋、Na＋对酶有明显激活作用；十二烷基硫酸钠、吐温80、苯甲基磺酰氟和尿素对酶活力基本无影响，但Mn2＋、Co2＋、Fe2＋、Ag＋、Cu2＋、Zn2＋、Pb2＋、Hg2＋和β-巯基乙醇、二硫苏糖醇均对酶活力有抑制作用。Chi-Pc76对黑曲霉、黄曲霉、尖孢镰刀菌和橘青霉均有显著的抗真菌活性，对比文献为产黄青霉抗真菌几丁质酶。鉴于Chi-Pc76纯化简便、热稳定性好、宽广的pH值稳定性、高效降解几丁质和抗真菌等特点，产黄青霉Xch23几丁质酶在几丁质废料综合利用、生物转化药理活性产品、食品保鲜以及植物病原性真菌和昆虫幼虫生物防治等方面具有潜在价值。     

Transglutaminase from Streptoverticillium ladakanum and application to minced fish product

The transglutaminase (TGase) from Streptoverticillium ladakanum was purified to electrophoretic homogeneity after ammonium sulfate fractionation and Blue Sepharose Fast Flow chromatography. The molecular weight of the purified TGase was 30.5 kDa estimated by Superdex 75HR gel filtration, and 37.5 kDa by SDS-PAGE. This enzyme, with optima at pH at 6.0 and 50°C was very stable at pH 5.0–7.0. It was strongly inhibited by PCMB, PMSF, Pb²⁺, Zn²⁺ and Cu²⁺, but not affected by EDTA and Ca²⁺. This suggested that the purified TGase was calcium-independent and its active center contained cysteine. It catalyzed the crosslinking of fish myosin heavy chain and substantially increased the gel strength of mackerel surimi.     

木薯块根中β-葡萄糖苷酶的分离纯化及酶学性质

研究了木薯块根中β-葡萄糖苷酶的分离纯化及酶学性质。以缓冲液从木薯块根中获得粗提酶液,粗酶酶活力为9.37 U/g木薯干重;再分别通过丙酮沉淀、离子交换层析和凝胶过滤层析进行纯化,β-葡萄糖苷酶酶活力为1.14 U/g木薯干重,经纯化β-葡萄糖苷酶纯度提高了14.62倍,总活力回收率为12.14%,电泳测得其分子量约70 kDa。该酶米氏常数K_m为3.60 mmol/L,V_max为12.36μmol/(min·mg protein);其最适pH为7.0,pH在6.0～8.0之间有较好的稳定性;在40℃以内有良好稳定性,在4℃存放30 d酶活力剩余81.78%。Mn²⁺和K⁺对酶有一定的促进作用,Al³⁺、Cu²⁺、Mg²⁺、Zn²⁺、Ca²⁺、Ba²⁺、Na⁺、尿素和SDS对酶没有显著影响(P>0.05),而Fe³⁺、F...     

一株嗜热菌木糖异构酶的纯化与性质研究

研究了1株嗜热菌(Anoxybacillus flavithermus)所产木糖异构酶的分离纯化以及酶学性质。结果表明,经硫酸铵沉淀、Sephadex G-75凝胶过滤、纤维素DE-52弱阴离子交换柱和Q Sepharose Fast Flow强阴离子交换层析得到的木糖异构酶,分子量约为181 ku,由4个相同分子量的亚基组成。酶反应的最适温度为80℃,最适为pH为7.0且最适pH范围宽泛,pH6.0~11.0酶反应活性能保持80%左右。该酶热稳定性及耐碱性能良好,70℃保温1 h后酶活仍能保持近80%左右;pH5.0~8.0保温1 h后酶活仍能保持近80%以上,甚至pH12.0保温1 h后酶活性仍能保持40%左右。Mn2+和Co2+对酶活性有明显促进作用,Zn2+、Cu2+以及Al3+对酶活性有一定程度的抑制。     

牛源二肽基肽酶-IV的高效制备及性质

以高效制备二肽基肽酶IV(dipeptidyl peptidase-IV,DPP-IV)为目的,以牛肾为原料,经酸沉淀、硫酸铵盐析和凝胶过滤层析获得高纯度DPP-IV。还原条件下,纯化蛋白的分子质量为106 kDa,纯化倍数为169.9,得率为9.5%。对该蛋白进行质谱鉴定得到12个肽段,共517个氨基酸残基,与牛DPP-IV氨基酸序列的匹配度为100%。对其性质研究发现,DPP-IV为糖蛋白且以二聚体形式(210 kDa)存在,等电点为5.8。DPP-IV的二级结构具有典型的β-折叠构象,热变性温度为(72.7±0.3)℃。金属离子Zn²⁺、Fe²⁺及Fe³⁺对DPP-IV活力有明显的抑制作用,1 mmol/L的Zn²⁺对DPP-IV力抑制率高达98%。     

Ribulose-1,5-bisphosphate carboxylase/oxygenase from an ammonia-oxidizing bacterium, Nitrosomonas sp. K1: purification and properties

The ammonia-oxidizing chemoautotrophic Nitrosomonas sp. strain K1 exhibited marked ribulose-1,5-bisphosphate carboxylase (RubisCO) activity. The RubisCO [EC 4.1.1.39] was purified as an electrophoretically homogeneous protein. The molecular mass of the enzyme was estimated to be about 460 kDa by gel filtration, and it consists of two subunits [large (L): 52.2 kDa; small (S): 13.3 kDa] as demonstrated by SDS-PAGE. This confirmed that the enzyme has an L(8)S(8) structure. The K(m) values of the enzyme for RuBP, NaHCO3, and Mg2+ were estimated to be 0.112, 0.415, and 1.063 mM, respectively. The optimum pH and temperature for its activity were approximately 7.0 and 45 degrees C. The enzyme was stable up to 45 degrees C and in a pH range from 7.0-9.0 (4 degrees C, 48 h). The enzyme activity was inhibited by Cu2+, Hg2+, N-ethylmaleimide, p-chloromercuribenzoate, and SDS (0.1 mM). The activity was also inhibited by ammonium sulfate at high concentrations (38-303 mM) but the stability of the enzyme showed no inhibition at the same ammonium sulfate concentrations. The N-terminal amino acid sequences of the large and small subunits are AIKTYQAGVKEYRQTYW QPDYVPL and AIQAYHLTKKYETFSYLPQM, respectively.     

裂褶菌产内切β-1,3-葡聚糖酶的特性研究

对裂褶茵所产内切β-1,3-葡聚糖酶进行有效分离纯化并用电泳法对其纯度进行鉴定,进而研究其酶学特性.结果表明:经过DEAE-Sephadex A-50离子交换层析和Sephadex G-75凝胶过滤分离纯化得到电泳纯分子量约为45 kD的内切β-1,3-葡聚糖酶,其最适pH为5.0,最适温度为45℃;Fe<'2+>、B...     

Purification and characterization of an alkaline manganese peroxidase from Aspergillus terreus LD-1

We report that Aspergillus terreus LD-1 produces an extracellular ligninolytic enzyme, manganese peroxidase (MnP), that reacts under alkaline conditions. This MnP was purified 13.1-fold from the culture supernatant to elicit a single band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of this MnP was estimated as either 43 kDa by SDS-PAGE or 44 kDa by gel permeation chromatography, suggesting a monomeric structure. The optimum pH and temperature of this MnP are 12.5 and 37 degrees C, respectively. This MnP is stable in the pH range 11.0 to 12.5 and also up to 40 degrees C. The K(m) values of this MnP for hydrogen peroxide, 2,6-dimethoxyphenol (2,6-DMP) and Mn2+ were 320 microM, 20 microM and 33 microM at pH 12.5, respectively. The activity of the MnP is completely inhibited by Hg2+, Pb2+, Ag+ and lactate. On the other hand, the MnP is activated by oxalate, maleate and fumarate. Maleate at 5 mM increased the MnP activity 5-fold. EDTA at 1 mM inhibited the MnP activity completely, but this inhibition was not observed in the presence of 1 mM Fe2+.     

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。     

坎皮纳斯类芽孢杆菌木聚糖酶的纯化以及酶学性质

采用硫酸铵盐析、Sephadex G-50凝胶过滤、DEAE Sepharose Fast Flow离子交换层析对坎皮纳斯类芽孢杆菌（Paenibacillus campinasensis）xy-7发酵液进行分离纯化,获得纯化的木聚糖酶,纯化倍数为26.36,酶活回收率为5.13%。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）结果为单一条带,分子质量为24.5 ku。酶学性质研究结果表明,该酶的最适反应温度为60 ℃,最适pH值为8.0。K+、Fe2+对酶有激活作用,Zn2+、Cu2+对酶有强烈的抑制作用。以榉木木聚糖为底物时,米氏常数Km=4.733 mg/mL,最大酶反应速率Vm=315.85 μmol/（min·mg）。     

Purification and characterization of dibenzothiophene sulfone monooxygenase and FMN-dependent NADH oxidoreductase from the thermophilic bacterium Paenibacillus sp. strain A11-2

A dibenzothiophene (DBT) sulfone monooxygenase (TdsA), which catalyses the oxidative CS bond cleavage of DBT sulfone to produce 2-(2-hydroxyphenyl)benzenesulfinate (HPBS) was purified from the thermophilic DBT desulfurizing bacterium Paenibacillus sp. strain A11-2 by multistep chromatography. The molecular mass of the purified enzyme was determined to be 120 kDa by gel filtration and the subunit molecular mass was calculated to be 48 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) indicating a dimeric structure. The N-terminal amino acid sequence of the purified TdsA was determined to be MRQMHLAGFFAAGNTHH, which revealed no significant similarity to any other known amino acid sequences. The purified TdsA absolutely required an oxidoreductase for its activity. This oxidoreductase (TdsD) was also purified to homogeneity, and its molecular size was calculated to be 50 kDa and 25 kDa by gel filtration and SDS-PAGE, respectively. TdsD was completely FMN-dependent, and FAD could not act as a cofactor. The N-terminal amino acid sequence of the purified TdsD was determined to be TSQTAEQSIAPIVAQYRHPEQPISALFVNR, which showed significant similarity to kinesin-like protein (44% identity). The optimal temperatures for the activity of TdsA and TdsD were 45 degrees C and 55 degrees C, respectively. Both enzymes showed optimal activity at pH 5.5. TdsA was slightly inhibited by sulfate, but not by 2-hydroxybiphenyl (2-HBP), which is another end product of DBT. TdsA showed higher activity toward bulkier substrates than its mesophilic counterpart, DszA. These properties suggest the applicability of biodesulfurization to the processing of actual petroleum fractions.     

紫色红曲霉中新型酯酶ESM1的纯化及其酶学特性研究

该研究以白酒大曲中筛选出的一株高产酯酶菌株HQ为研究对象,采用分子生物学对其进行菌种鉴定。通过硫酸铵二级沉淀、丁基琼脂糖凝胶柱和Superdex G-200柱对其所产的酯酶进行纯化,并进行N-端序列、质谱和酶学性质分析。结果表明,菌株HQ被鉴定为紫红曲霉（Monascus purpureus）,经纯化获得一个分子质量为60 kDa的酯酶,命名为ESM1。酯酶ESM1具有一段酸性蛋白酶的肽段序列,最适温度为60 ℃、最适pH值为7.0,在温度为50 ℃的环境和中性环境中表现出良好的稳定性。Zn2+、Ca2+和Na+对ESM1有促进作用,其中Zn2+促进作用最高,Cu2+、Mg2+、Fe2+和Mn2+有抑制作用,其中Cu2+的抑制作用最强。     

醋酸菌中乙醛脱氢酶的分离纯化及酶学性质

以实验室筛选得到的醋酸菌(Acetobacter pomorum)为实验菌株发酵产酶,通过细胞破壁,采用(NH₄)₂SO₄沉淀、透析、DEAE-Sepharose 离子交换层析及 Superdex G-75凝胶过滤层析分离纯化得到乙醛脱氢酶的酶液,并考察其酶学性质。该酶分子质量为221.60 kDa,单个亚基分子质量约为54.41 kDa,为四聚体结构;纯酶液比活力20.25 U/mg,纯化倍数为10.16倍,乙醛脱氢酶(aldehyde dehydrogenase,ALDH)的回收率为6.53%。酶学性质研究表明,ALDH促进乙醛分解的最适温度为50 ℃,40~50 ℃相对酶活力稳定性好;该酶的最适pH为7.0,当pH在5.5~7.5内酶活力表现稳定;金属离子对酶活性的影响实验表明,Na+、K+、Zn2+、Ba2+对该酶酶有不同程度抑制作用,而Mg2+、Ca2+、Al3+、Li+、Cu2+具有促进作用;ALDH的最适底物为乙醛,相对偏好直链醛类。ALDH活性较大,为后期表达和深入研究其生物学功能提供理论和数据支持。     

鸭卵清溶菌酶的分离纯化及性质

通过硫酸铵的分级沉淀、CM-Sepharose阳离子交换层析、Superdex-200凝胶过滤层析等步骤,从鸭卵清中获得电泳纯的溶菌酶,该酶的比活力达到33687.26U/mg,纯化倍数为109.44,回收率为28.00%。测得该酶分子质量约为14.82kD,对溶壁微球菌的最适反应温度为50℃,最适pH值为7,且在50℃以下及pH5～9有较好的稳定性,同时在最适条件下测得其Km值为0.0864mg/mL。Fe2+、Mg2+、Mn2+等金属离子对该酶有较强的抑制作用,而Zn2+、Cu2+、Co2+对该酶有一定的激活作用。     

根霉胞内α-半乳糖苷酶的分离及其酶学性质

根霉Rhizopus sp.A01的菌丝体破碎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54.8倍,总酶活回收率达到27.3%,在SDS-PAGE上显示相对分子质量为85.6 ku的单一条带,凝胶过滤表明该酶表观相对分子质量为302 ku。该酶水解对硝基苯-α-D-吡喃半乳糖苷的最适pH值为4.5,最适温度为55℃,表观Km值为(0.242±0.027)mmol/L,表观kcat/Km值为4.089×105L/(mol.s);对蜜二糖和棉子糖有弱的水解作用,水解速度依次为138.3μmol/(h.mg)、19.7μmol/(h.mg)。水解活性受Fe2+和Fe3+的显著激活,但受Mn2+、Cu2+、Hg+和Mg2+等离子的强烈抑制。该酶活性在pH4.0～8.2保持稳定,在50℃时保温90 min,残余酶活达到了48%。