Purification and properties of a low-molecular-weight phytase from Cladosporium sp. FP-1

A fungus producing high levels of phytase was isolated from air and identified as Cladosporium sp. The phytase production was stimulated by phytate in the medium used. The maximum production of phytase (108 U/ml) occurred in a medium containing 1.0 g of phytate per 100 ml. The phytase was purified to electrophoretic homogeneity by ion-exchange chromatography and gel filtration. Based on SDS-PAGE analysis, the molecular weight of the purified phytase was calculated to be approximately 32.6 kDa, and the narrow protein band indicated that this phytase is not glycosylated. The phytase has an optimum pH of 3.5, and an optimum temperature of 40 degrees C. The phytase activity was stimulated by 2-mercaptoethanol and dithiothreitol, and inhibited by Ba2+, Pb2+, iodoacetate, p-chloromercuribenzoate and phenylmethylsulfonyl fluoride. The phytase displayed high affinity for phytate and the Km was 15.2+/-3.1 microM. NMR analyses (1D and 2D) indicated that the end hydrolysis product of phytate was myo-inositol 1,2,5-triphosphate.     

Purification and characterization of phytase from Klebsiella pneumoniae 9-3B

Phytase, an enzyme that catalyzes the hydrolysis of phytate, was purified from Klebsiella pneumoniae 9-3B. The isolate was preferentially selected in a medium which contains phytate as a sole carbon and phosphate source. Phytic acid was utilized for growth and consequently stimulated phytase production. Phytase production was detected throughout growth and the highest phytase production was observed at the onset of stationary phase. The purification scheme including ion exchange chromatography and gel filtration resulted in a 240 and 2077 fold purification of the enzyme with 2% and 15% recovery of the total activity for liberation of inorganic phosphate and inositol, respectively. The purified phytase was a monomeric protein with an estimated molecular weight of 45kDa based on size exclusion chromatography and SDS-PAGE analyses. The phytase has an optimum pH of 4.0 and optimum temperature of 50°C. The phytase activity was slightly stimulated by Ca(2+) and EDTA and inhibited by Zn(2+) and Fe(2+). The phytase exhibited broad substrate specificity and the K(m) value for phytate was 0.04mM. The enzyme completely hydrolyzed myo-inositol hexakisphosphate (phytate) to myo-inositol and inorganic phosphate. The properties of the enzyme prove that it is a good candidate for the hydrolysis of phytate for industrial applications.     

一种耐热性植酸酶的分离纯化及其酶学性质研究

采用半固态发酵方式培养泡盛曲霉(Aspergillus awamori)AS3.324,通过有机膜超滤、阴离子交换层析、凝胶层析后,得到纯化的植酸酶。酶学性质表明,其反应最适温度为50-55℃,最适pH为5.5,在37℃下以植酸钠为底物的Km值为1.03 nmol/L,Vmax为2.13μmol/(L.min)。EDTA基本不影响植酸酶活性;Ca2+,Mg2+,Mn2+对植酸酶活性有轻微的抑制作用;Fe2+,Zn2+对酶促反应有显著的抑制作用。对该酶的耐热性研究表明,经较高温度条件处理后,仍有较高残余酶活性,与当今商品化的植酸酶相比,有较强的耐热性。泡盛曲霉植酸酶作为动物饲料添加剂具有广泛的应用前景。     

构巢曲霉产植酸酶的酶学特性分析

从构巢曲霉AnP-16菌株发酵液中纯化单一组分的耐热耐酸植酸酶,并对酶学特性进行分析,为其在食品工业中的应用提供理论依据。通过硫酸铵盐析、离子交换层析和疏水层析纯化植酸酶,SDS-PAGE电泳测定其分子量。结果表明,从该菌株发酵液中纯化到了单一组分的植酸酶,纯化倍数60.8倍、回收率41.6%,酶分子量约52 kDa。植酸酶最适作用温度和pH分别为55 ℃和pH4.0,在pH3.0~6.0范围酶活性较高,pH2.0~7.0下孵育3 h,仍能保持80%以上活性。植酸酶耐热性好,70 ℃孵育1 h仍能保持81%活性。Hg2+、Mn2+、Fe2+、Cu2+和Zn2+ 5 mmol/L浓度下对酶活性有明显抑制作用,但Ca2+和Mg2+在1和5 mmol/L浓度时均增强酶活性;有机溶剂甲醇和乙醇在2%浓度有激活作用;SDS抑制酶活性,但其它表面活性剂(Triton X-100和Tween 80)和有机溶剂(丙酮和异戊醇)对酶活性无明显影响。植酸酶有宽泛的底物特异性,但对植酸钠的催化活性最强,其K_m值为0.576 mmol/L。基于植酸酶耐酸耐热及宽广的底物催化活性,其有望应用于粮油食品加工领域,提高食品的营养价值。     

Purification and characterization of fenitrothion hydrolase from Burkholderia sp. NF100

The organophosphorus pesticide hydrolase was purified to homogeneity from Burkholderia sp. NF100 by detergent extraction of the cell membrane fraction, anion-exchange, chromatofocusing, and gel filtration chromatographies. The purified enzyme had a molecular mass of 55 kDa and a pI 5.8, and the hydrolase activity was strongly inhibited by EDTA, dithiothreitol (DTT), Hg2+ and 1,10-phenanthroline. The optimum pH and temperature for the enzyme activity were 8.0 and 40 degrees C, respectively. The enzyme hydrolyzed five organophosphorus pesticides.     

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。     

Purification and properties of beta-N-acetylglucosaminidase from Vibrio alginolyticus H-8

Beta-N-acetylglucosaminidase [EC 3.2.1.30] from Vibrio alginolyticus H-8 was purified by column chromatography on DEAE-Sepharose FF, phenyl-Sepharose HP, Superdex 200HR, and Mono Q. The molecular weight of the enzyme was estimated by SDS-gel electrophoresis (SDS-PAGE) to be 75 kDa. The pI was 4.6. The activity was inhibited by Ag+, Hg2+, iodoacetate, p-chloromercuribenzoate, and N-ethylmaleimide.     

中性植酸酶产生菌的筛选、鉴定及酶学性质

从土壤中分离到一株高产中性植酸酶的菌株,酶活力达12.52U/mL。对其进行形态、生理、生化、分子生物学鉴定,初步鉴定为放射型根瘤杆菌(Agrobacterium radiobacter)。酶学性质研究结果表明：该酶的最适反应温度为45℃;最适反应pH 值为7.0;65℃处理60min 酶活力保持80% 左右,有一定耐热性;在pH5.5～8.0 之间,稳定性较好;Ba2+ 对酶活力有一定的激活作用,Fe2+、Mg2+、Zn2+ 和Cu2+ 对酶活力均有一定程度的抑制作用,其中Fe2+ 的抑制作用最强。     

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。     

ICP-AES分析植酸酶对制麦过程中游离态金属离子含量的变化

运用电感耦合等离子体原子发射光谱法(ICP-AES)考察了外源添加植酸酶对大麦发芽过程中游离态K+、Na+、Ca2+、Mg2+、Zn2+含量的变化。实验发现:添加外源植酸酶能提高发芽过程中游离态金属离子的含量,K+、Na+、Ca2+、Mg2+、Zn2+含量同比可提高10.7%、23.4%、16.3%、8.0%、75.5%。通过比较金属离子含量的变化可以为制定合理的制麦工艺提供参考。     

Characterization of a highly thermostable extracellular lipase from Lactobacillus plantarum

After screening for the presence of lipase activity in lactobacilli isolated from "chouri?o", a traditional Portuguese dry fermented sausage, a strain of Lactobacillus plantarum (DSMZ 12028) was chosen for extracellular lipase characterisation and purification. Proteinase K did not significantly affect lipolytic activity, as opposed to trypsin, which completely eliminated this activity. Among NaCl, Ca2+, EDTA, BSA, glycerol, Mn2+ and Mg2+, only Mn2+ and Mg2+ stimulated the lipase. Purification by gel filtration chromatography and gel electrophoresis revealed four bands, between 98 and 45 kDa, all with lipolytic activity against olive oil.     

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。     

PURIFICATION AND CHARACTERIZATION OF CANOLA SEED (BRASSICA sp.) PHYTASE

Germinated Altex and Westar (Brassica napus) and Candle and Tobin (B. campestris) cultivars of Canola were screened for phytase activity. On the basis of this preliminary screening, 7-day germinated Altex seedlings were selected as a source for isolation and characterization of phytase. Partial purification of a crude extract (FI) by acetone precipitation resulted in an 8-fold increase in phytase activity. Ion-exchange chromatography of the partially purified preparation (FII) yielded two fractions (FIIIA and FIIIB) both of which demonstrated phytase and phosphatase activities. Further purification by gel filtration chromatography resulted in two fractions (FIVA1 and FIVA2) from fraction FIIIA and two fractions (FIVB1 and FIVB2) from fraction FIIIB. Fraction FIVB1 demonstrated both phytase and phosphatase activities, FIVA2 and FIVB2 demonstrated phosphatase activity but no phytase activity and FIVA1 showed phytase but no phosphatase activity. Fraction FIVB1, which showed highest phytase activity (5.3 IU/mg protein), had the following characteristics: temperature optimum of 50°C, pH optimum of 5.2, K_m of 0.36 mM and relative activity for pyrophosphate 232 times higher than for phytate.     

Purification and characterization of a novel thermostable phytase from the thermophilic Geobacillus sp. TF16

A novel phytase from thermophilic Geobacillus sp. TF16 was puri?ed approximately 5-fold using ammonium sulfate precipitation and ion exchange chromatography, and determined as a single band 106.04 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Optimum temperature and optimum pH were found to be 85°C and 4.0, respectively. The enzyme is highly thermostable and V_max and K_m values were calculated as 526.28 U/mg and 1.31 mM, respectively. It was also found that the enzyme exhibited a broad substrate selectivity and resistance toward proteases and effectively hydrolyzed soymilk phytate. These results suggest that this study provides an alternative phytase enzyme with enhanced properties.     

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...     

牛胰脏组织蛋白酶L的纯化和酶学性质

新鲜牛胰脏匀浆物经酸化粗提后,再通过盐析、柱层析等步骤纯化,制备了0.58 mg纯酶,纯化倍数达530.54。经凝胶电泳分析,该酶有2 个亚基,分子质量为29.1 kD和18.9 kD。牛胰脏组织蛋白酶L的最适反应温度为50 ℃,最适反应pH值为6.5。巯基还原剂二硫苏糖醇、L-半胱氨酸均明显激活了该酶活性,10 μmol/L的N-（反式-环氧丁二酰基）-L-亮氨酸-4-胍基丁基酰胺（E-64）可完全抑制其活性。1 mmol/L的Zn2＋对酶活性有明显抑制作用。该纯化酶可水解苄氧羰基-苯丙氨酰-精氨酰-甲基香豆素（Z-Phe-Arg-MCA）,其Km值为3.52 μmol/L。     

Purification and characterization of a novel beta-agarase from an alkalophilic bacterium, Alteromonas sp. E-1

A novel beta-agarase (EC 3.2.1.81) was purified from an agar-degrading alkalophilic bacterium, Alteromonas sp. E-1 isolated from the soil. This enzyme was obtained from a cell-free extract after sonication and purified 40.9-fold through treatment with streptomycin, ammonium sulfate fractionation and successive chromatography on anion-exchange and gel filtration columns. The molecular weight was estimated to be 82 kDa by SDS-polyacrylamide gel electrophoresis and 180 kDa by Superdex 200 gel filtration. The enzyme was inhibited by Mn2+, Cu2+, Fe2+, Zn2+ and Hg2+, and activated by K+, Na+ and EDTA, and its optimum pH and temperature for agarose degradation were 7.5 and 40 degrees C, respectively. This beta-agarase hydrolyzed agarose with rapid reduction of viscosity, and neoagarobiose [O-3,6-anhydro-alpha-L-galactopyranosyl(1-->3)-D-galactose] was detected from the early stage of the reaction. Neoagarobiose as the final product was selectively released from agarose, neoagarohexaose and neoagarotetraose by the reaction with this beta-agarase. This observation was different from that of other beta-agarases which produced mixtures of neoagarobiose and neoagarotetraose as the final hydrolysis products. The N-terminal amino acid sequence of this beta-agarase shows no homology to those of other beta-agarases that were so far reported.     

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。     

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。     

海栖热袍菌α-葡萄糖苷酶基因aglA的克隆、表达及酶学性质

主要研究了在大肠杆菌中克隆和表达海栖热袍菌(Thermotoga maritima)的一个α-葡萄糖苷酶(TM1834).通过PCR方法克隆编码T.maritima的一个α-葡萄糖苷酶基因aglA,构建重组质粒pHsh-AglA,电击转化Escherichia coli JM109,通过热激诱导高效表达.SDS-PAGE检测出蛋白相对分子质量约55 000,经热处理,阴离子交换层析和疏水层析纯化后的α-葡萄糖苷酶最适反应温度为90℃,最适反应pH为7.5,在pH 6.5～8.5,温度65～100℃之间酶活仍达到50％以上.在辅助因子NAD+,Mn2+和还原剂DTT的存在条件下达到最高酶活.