枯草芽孢杆菌HS18碱性蛋白酶分离纯化及其性质研究

从合浦珠母贝肠道中分离鉴定到酶菌株枯草芽孢杆菌（Bacillus subtilis）HS18,将枯草芽孢杆菌HS18的发酵液,经过硫酸铵盐析、DEAE-Sephadex A-50柱层析、Sephadex G-100柱层析3步纯化后得到了单一的酶蛋白,回收率为11.4%;经SDS-聚丙烯酰胺凝胶电泳测得酶蛋白分子量约为31ku;该蛋白酶最适反应温度为50℃,最适pH10.0;K＋及Na＋对酶有明显激活作用;丝氨酸蛋白酶特异性抑制剂（PMSF）能强烈抑制酶活性。表明所纯化到的蛋白酶为丝氨酸碱性蛋白酶。     

鸭肝谷氨酸脱氢酶的纯化与酶学性质研究

目的：获得鸭肝谷氨酸脱氢酶纯品并对其酶学性质进行研究。方法：采用丙酮脱脂、重金属离子沉淀、硫酸铵分级沉淀、DEAE-Sepharose 离子交换层析和Sephacryl S-200 凝胶层析方法,分离纯化鸭肝谷氨酸脱氢酶,用SDS- 聚丙烯酰胺凝胶电泳法进行纯度鉴定和酶相对分子质量测定。结果：从鸭肝中分离纯化获得电泳纯的谷氨酸脱氢酶,纯化倍数为60.93 倍,酶活力回收率为11.02%,比活力达24.37U/mg。酶相对分子质量为371.41,亚基相对分子质量为61.60。推测该酶由6 个相同亚基构成。该酶对NADH 的Km 为53.19μmol/L,最适pH 值为10.0,最适反应温度为35℃。该酶在pH8.0 左右较稳定;在40℃以下酶活力保持稳定。Zn2+、Li+ 和Cu2+ 对该酶具有显著的抑制作用。结论：分离纯化获得谷氨酸脱氢酶,该酶具有较高应用价值。     

绿色木霉β-葡萄糖苷酶的分离纯化及酶学性质

针对绿色木霉AS3.3711产生的β-葡萄糖苷酶组分,先后运用包括乙酸铵沉淀、透析、Sephadex G-150葡聚糖凝胶柱层析在内的一系列分离纯化技术对该纤维素酶进行纯化,得到β-葡萄糖苷酶纯化组分,并对该酶的酶学性质进行研究。纯化后酶液的蛋白质量浓度为8.12 mg/mL、酶活力为4.08 U/mL,纯化倍数达到18.48,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecylsulfate polyacrylamide gel electrophoresis,SDS-PAGE）测定分子质量为66.0 kD。绿色木霉β-葡萄糖苷酶在酸性条件下稳定性良好,最适pH值为5.0;在温度60～70 ℃能长时间保持较高酶活力,最适反应温度为60 ℃。金属离子中,Ca2+、Mg2+、K+对绿色木霉AS3.3711 β-葡萄糖苷酶活力起到促进作用,Ca2+促进作用最强;而Zn2+、Fe3+对该酶有抑制作用,Ag+、Cu2+、Hg2+重金属离子使β-葡萄糖苷酶几乎丧失了全部活性。     

苏云金芽孢杆菌精氨酸酶的纯化及酶学性质研究

从L-精氨酸诱导的苏云金芽孢杆菌SK20.001中分离纯化出SDS-PAGE电泳纯的精氨酸酶[EC3.5.3.1]。纯化酶的比活力为589.2U/mg,纯化过程酶的回收率为22.4%。酶经过聚丙烯酰胺凝胶电泳得到亚基的相对分子量约31000u。该酶的最适pH为9.8,最适温度为40℃,并且Mn2+显示出对酶有最强的激活作用。通过Lineweaver-Burk双倒数作图得到的精氨酸酶Km为15.2mmol/L。     

米曲氨基酰化酶分离纯化及其保存稳定性

通过对米曲氨基酰化酶硫酸铵分级沉淀、疏水层析分离、SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)实验获得较高纯化倍数和较高活力的酶,并对酶保存稳定性进行研究.经分离纯化实验后,米曲氨基酰化酶的比活力由3.818 U/mg提高到106.738 U/mg,纯化倍数达28倍,活力回收率为22%.保存该酶的最适pH值为6.5～7.0,Co2 对酶有轻微的激活作用,Fe2 和CH3COO-对酶有明显地抑制作用,蛋白水解酶抑制剂1,4-二硫苏糖醇对酶的保存稳定性有较高提升.自制丙酮冻干粉保存稳定性良好,保存半年其酶活力仍能维持原酶活力的80%以上.     

芋头多酚氧化酶的分离纯化与酶学特性

以鲜芋头为原料,通过提取、30%～80%饱和度硫酸铵沉淀、透析、超滤,以及柱层析等步骤对芋头多酚氧化酶（polyphenol oxidase,PPO）进行逐级分离纯化,并探讨其酶学特性及抑制剂对其作用规律。结果表明：经DEAE-Sepharose、Superdex G-75色谱柱纯化后,得到了电泳纯PPO,比活力为27 471.26 U/mg,纯化倍数达6.37 倍。纯化后的PPO经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析,其分子质量约为24 kDa。PPO与底物结合能力顺序为：邻苯二酚＞间苯二酚＞间苯三酚＞焦性没食子酸＞L-酪氨酸。PPO的最适反应温度、pH值和反应时间分别为30 ℃、6.8和60 s。抗坏血酸和L-半胱氨酸对提取的PPO有较强的抑制作用,其抑制类型分别属于反竞争性抑制和竞争性抑制方式。结果表明,在芋头加工过程中通过控制温度、pH值或加入适当的抑制剂处理,可以有效减少褐变的发生。     

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）。     

嗜酸乳杆菌胆盐水解酶的分离纯化及酶学性质

对嗜酸乳杆菌La-XH1产生的胆盐水解酶进行分离纯化,并对其部分酶学性质进行研究。结果表明：嗜酸乳杆菌La-XH1胆盐水解酶的粗酶液经硫酸铵沉淀、DEAE-Sepharose CL-6B柱层析后的酶比活力分别为47.82 U/mg和115.85 U/mg,纯化倍数分别为4.46 倍和10.82 倍,酶的回收率分别为59.89%和25.11%;通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecylsulfate polyacrylamide gel electrophoresis,SDS-PAGE）电泳分析,该酶的分子质量约为43 kD,最适温度为40 ℃,最适pH值为6.0,Fe3+、Ca2+、Mg2+、Mn2+、Zn2+对该酶有激活作用,其中Fe3+的激活作用最强,Na+、K+对该酶几乎无作用,而Cu2+、Ba2+对该酶有很强的抑制作用。     

酸橙内生菌Bacillus thuringiensis Bt028几丁质酶的分离纯化及其酶学性质

为揭示酸橙内生菌Bacillus thuringiensis Bt028几丁质酶的基本酶学性质,采用离心、硫酸铵盐析、葡聚糖凝胶G-100层析及SDS-聚丙烯酰胺凝胶电泳等方法对菌株Bt028发酵液中的几丁质酶进行分离纯化鉴定,并考察该几丁质酶的最适温度、最适pH、催化动力学参数等性质。结果表明,Bt028菌株发酵液经离心、硫酸铵盐析、葡聚糖凝胶G-100层析分离纯化后获得电泳纯几丁质酶比活力为681.78 U/mg,纯化倍数为3.21,回收率15.52%。SDS-聚丙烯酰胺凝胶电泳结果表明,几丁质酶的分子质量为65 kDa。酶学性质研究结果表明,该酶的最适反应温度为60 ℃,最适反应pH为6.5,在温度低于60 ℃、pH5.5~7.5时有较好的稳定性;Mg2+、Ca2+、Hg2+、Co2+对该酶活力有抑制作用,而Cu2+和Fe3+有一定的促进作用;低浓度的甲醇、乙醇、正丙醇和二甲亚砜使酶的活性增加,但当浓度继续增大,反而会抑制酶的活性;丙酮对几丁质酶有激活作用,而甲醛对几丁质酶有抑制作用。在最适催化条件下,几丁质酶催化反应的米氏常数Km、最大反应速率Vmax、酶的转换数Kcat分别为29.533 mg/mL、108.696 μmoL/(L·min)和0.527/min。研究结果可为该酶的实际应用提供必要的工艺参数。     

RC-3产硫酸软骨素酶的条件优化及酶的分离纯化

研究了菌株RC-3产硫酸软骨素酶的最适发酵条件及酶的分离纯化,分别优化了碳源种类、碳源浓度、氮源种类、氮源浓度和NaCl浓度等条件,在最适发酵条件下对RC-3进行发酵培养,收集菌体进行细胞破碎制备粗酶液。粗酶液依次经过Q-Sepharose Fast Flow(QFF)阴离子交换层析与Sephacryl S-1000凝胶过滤层析和高效液相色谱(HPLC)进行逐级分离纯化,并利用聚丙烯酰胺凝胶电泳(PAGE)的方法验证纯化酶活力。结果表明,最优碳源为0.5%的岩藻多糖(Fucoidan)加乳糖、最优氮源为1.0%的牛肉膏、NaCl的最优浓度为0.3%,在上述条件下发酵48h,获得的菌体生物量最高,酶活力较优化前提高3倍,达1066.89 U/mL。粗酶经过QFF离子交换层析和Sephacryl S-1000葡聚糖凝胶纯化后获得相对较纯的酶,该酶经过HPLC检测表明主要有2个峰,并用聚丙烯酰胺凝胶电泳证实了此二峰均具有酶活力。     

Heat inactivation and kinetics of polyphenoloxidase from palmito (Euterpe edulis)

Polyphenoloxidase (PPO, EC 1.14.18.1)was extracted from palmito (Euterpe edulis Mart) using 0.1 M phosphate buffer, pH 7.5. Partial purification of the enzyme was achieved by a combination of (NH₄)₂SO₄precipitation (35–90% saturation) and Sephadex G-25 and DEAE-cellulose chromatography. The purified preparation gave five protein bands on polyacrylamide gel electrophoresis, three of them with PPO activity. The K_mvalues for chlorogenic acid, caffeic acid, catechol, 4-methylcatechol and catechin were 0.57, 0.59, 1.1, 2.0 and 6.25 mM , respectively. PPO has a molecular weight of 51 000 Da, maximum activity at pH 5.6 with chlorogenic acid as substrate, and was stable between pH 5.0 and 8.0. The enzyme was heat stable at 50–60°C and inactivated at 75°C. The heat stability of palmito PPO was found to be pH dependent; at 50°C and pH 4.0 the enzyme was fully inactivated after 30 min. The pH/activity studies showed two groups with pK values c 4.6 and 6.7 involved in PPO catalysis.     

Polyphenoloxidase from DeChaunac grapes

Polyphenoloxidase (PPO) from red grape cultivar, DeChaunac, grown in New York State was isolated and purified 17-fold by using Phenyl Sepharose CL-4B column. Disc gel electrophoresis revealed near homogeneity of three isoenzyme bands. The molecular weight of this enzyme ranged from 73 000 to 85 000. The temperature and pH optima of the purified enzyme were 20 °C and 6.0, respectively. Kinetic studies showed that the thermal inactivation of the PPO followed first-order kinetics, with the activation energy, Ea = 52.39 Kcal mol^?1. The substrate specificity showed a high degree of PPO activity toward o-diphenolic compounds with the highest affinity toward caffeic acid among substrates studied. The apparent K_m values for caffeic acid and 4-methylcatechol as substrates for the Dechaunac PPO were 16 mmol and 25 mmol, respectively. The most potent inhibitors of the PPO were D.L-dithiothreitol and sodium metasulphite at the concentration level of 0.5 mmol.     

Partial purification and characterisation of polyphenol oxidase and peroxidase from marula fruit (Sclerocarya birrea subsp. Caffra)

Marula fruit, native to sub-Saharan Africa, is of growing commercially importance. Polyphenol oxidase (PPO) and peroxidase from the fruit were partially purified by a combination of temperature induced phase separation in Triton X-114, DEAE-ion exchange and Sephadex G100 gel filtration. PPO activity was purified 58-fold with 75% recovery while the purification factor for peroxidase was 19% with 25% recovery. The enzymes were characterised for enzyme concentration–reaction rate relationship, thermal stability, pH activity and stability, molecular weight, isoelectric point (pI) and kinetic parameters. PPO and peroxidase shared the same molecular weight (71 kDa) and pI (5.43). Thermal deactivation curves were bi-phasic for both activities. Peroxidase displayed maximal activity at pH 4.0 with ABTS (2,2′-azino-(bis-3-ethylbenzthiazoline-6-sulfonic acid)) and a K_M of 1.77 mM for hydrogen peroxide. The pH optimum for PPO was 7.0 with catechol. Marula PPO had K_M values of 1.41, 1.43, 3.73 and 4.99 mM for catechin, 4-methylcatechol, 3,4-dihydroxyphenylpropanoic acid (DHPPA) and catechol, respectively.     

茶叶多酚氧化酶三相分离纯化及酶学性质研究

为研究一种快速高效的茶叶多酚氧化酶（polyphenol oxidase, PPO）制备方法,本试验采用三相分离法（three phase partitioning, TPP）获取茶叶PPO,并研究其酶学特性。结果表明,通过两次三相分离纯化后可获得酶比活力210.46 U/mg、纯化15.76倍、回收率8.04%的茶叶PPO,其最亲和底物为邻苯三酚（K_m=6.82 mmol/L, V_max=5.36×10?2 OD/min）,最适反应温度和pH分别为45 ℃和pH5.5,在25 ℃和pH4.5~7.0时具有较好的稳定性。对纯化后茶叶PPO抑制作用最显著的化合物为抗坏血酸（IC₅₀=2.42±0.18 mmol/L）,其次是草酸和EDTA,柠檬酸抑制效果最弱,而卤化物无显著影响（P>0.05）。表明三相分离法能够较好的保留茶叶PPO活性,且操作简便、耗时短、成本低,有利于工业化应用。     

Inhibition of Polyphenoloxidase by Sulfite

When polyphenoloxidase (PPO) was exposed to sulfite prior to susbstrate addition, inhibition was irreversible. Trials to regenerate PPO activity, using extensive dialysis, column chromatography, and addition of copper salts were not successful. Increased concentrations of sulfite and pH levels less than 5 enhanced the inhibition of PPO by sulfite. At pH 4, concentrations greater than 0.04 mg/mL completely inhibited 1,000 units of PPO activity almost instantaneously. This suggested that the HSO₃- molecule was the main component in the sulfite system inhibiting PPO. Column chromatography, extensive dialysis, and gel electrophoresis did not demonstrate ³⁵SO₂ bound to purified pear PPO protein. Formation of extra protein bands of sulfite inhibited purified pear PPO fractions on gel electrophoresis was demonstrated. This and other evidence suggested that the major mode of direct irreversible inhibition of PPO was modification of the protein structure, with retention of its molecular unity.     

Purification of Polyphenoloxidase from the Purple‐fleshed Potato (Solanum tuberosum Jasim) and its Secondary Structure

ABSTRACT: Polyphenoloxidase (PPO) was purified from purple‐fleshed potatoes (Solanum tuberosum Jasim) using membrane concentration, ammonium sulfate fractionation, Resource Q ion exchange chromatography, and Sephacryl S‐200 HR gel permeation chromatography. PPO was purified 78‐fold from a crude extract. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis results showed that the purified enzyme has a major subunit molecular weight of 40 kDa. To elucidate the secondary structure of the purified PPO, circular dichroism (CD) was performed. The CD spectrum of the purified enzyme showed that PPO contains 35% α‐helix, 30% β‐turn, and 35% random coil structure.     

PURIFICATION AND KINETIC CHARACTERIZATION OF POLYPHENOL OXIDASE FROM TOMATO FRUITS (LYCOPERSICON ESCULENTUM CV. MUCHAMIEL)

Two different polyphenol oxidase (PPO) fractions, soluble and particulate, were purified from unripe tomato fruits (Lycopersicon esculentum M. cv. Muchamiel). The PPO present in the soluble fraction was purified fivefold with a 43.5% yield after ammonium sulfate fractionation. PPO in the particulate was purified 4.56‐fold with a 23% yield using the nonionic detergent Triton X‐114. A strong correlation between tomato fruit PPO activity and the physiological disorder blossom‐end rot (BER) was found, with a large increase of the PPO activity in the particulate fraction. Kinetic characterization, including kinetic parameters, pH and temperature profiles, substrate specificity and inhibitors showed similarities in both the soluble and the particulate enzyme(s). However, thermal stability of the particulate enzyme was significantly higher than stability of the soluble PPO, thus indicating possible structural differences. Cupric ions were activators, probably because of their ability to reactivate PPO partly denatured during purification.     

白鲢鱼背侧肌焦磷酸酶的纯化及酶学特性

通过粗分离、50%～80%饱和度硫酸铵分级沉降、DE-52阴离子交换柱层析等步骤,从白鲢鱼背侧肌中分离纯化出焦磷酸酶。通过变性凝胶电泳分析,该酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳图谱中仅有一个分子质量为40 kD的条带。酶学特性研究表明,白鲢鱼背侧肌焦磷酸酶可专一性地水解焦磷酸盐,反应初速率时间范围为0～15 min,最适反应温度为45 ℃,最适反应pH值为7.5。Mg2＋对该酶有明显的激活作用,在Mg2＋浓度为5 mmol/L时酶活力最高。5 mmol/L的Ca2＋、Zn2＋、乙二胺四乙酸（ethylenediaminetetraacetic acid,EDTA）-Na2、EDTA-Na4、KIO3和1 mmol/L的NaF均对该酶有强烈的抑制作用。该酶水解焦磷酸四钠的最大反应速率为0.051 U/mg,米氏常数为0.54 mmol/L。     

黑曲霉β-葡萄糖苷酶的分离纯化及酶学性质研究

利用硫酸铵盐析、季氨乙基-琼脂糖凝胶FF（Q-Sepharose FF）离子交换层析、苯基-琼脂糖凝胶6 FF（Phenyl-Sepharose 6 FF）疏水层析和丁基-琼脂糖凝胶HP（Butyl-Sepharose HP）疏水层析对黑曲霉来源β-葡萄糖苷酶进行分离纯化,采用十二烷基硫酸钠-聚丙酰胺凝胶电泳（SDS-PAGE）测定其分子质量,并对其酶学性质进行研究。结果表明,经分离纯化后得到分子质量约为116 kDa的β-葡萄糖苷酶,纯化倍数达到50.39倍,回收率为4.65%,比酶活为103.80 U/mg,该β-葡萄糖苷酶的最适反应温度为60 ℃,最适反应pH值为5.0,在温度30～50 ℃,pH 2.0～8.0之间具有较好的稳定性。     

Esterolytic and Lipolytic Activities of Lactobacillus Casei-subsp-Casei LLG

The estcrolytic and lipolytic enzymes were produced by cell lysis of Lactobacillus casei-subsp-casei LLG during the late logarithmic growth phase. The enzyme was purified to 67 fold by ion exchange chromatography and gel filtration chromatography using the FPLC system. Polyacrylamide gel electrophoresis and sodium dodecyl sulfate-poly-acrylamide gel electrophoresis using the “Phast” system of the purified enzyme showed a single protein band for butyrate-esterase (3.2 × 10⁵ Dalton), caproate esterase (1.1 × 10⁵ Dalton) and capryate esterase (4.0 × 10⁴ Dalton), respectively. The maximum lipolytic activity was observed at pH 7.2 and 37°C. The enzyme activity was inhibited by silver and mercury ions but magnesium and calcium stimulated lipolytic activity. The Km and Vmax values for esterase-lipase of the strain LLG were 76 μM/min/mg of protein, and 0.57 mM, respectively. This enzyme was stable at room temperature for at least 2 days.