海藻糖合酶的分离纯化及部分酶学性质

用硫酸铵沉淀、凝胶过滤层析、离子交换层析对一株芽孢杆菌SH-110产生的海藻糖合酶粗酶液进行纯化,用PAGE电泳逐步分析纯化的结果,用SDS-PAGE电泳证明该酶的分子量为62.4 kD。酶反应最适pH值为7.0,最适作用温度为35℃,金属离子Zn2+、K+、Na+对酶活性有激活作用,Ba2+、Ca2+、Mn2+对酶活性有抑制作用。酶底物专一性初步分析结果表明,该酶确实是以麦芽糖为专一性底物来转化生成海藻糖的海藻糖合酶的一种新酶。     

酸性蛋白酶AP-10分离纯化及其酶学性质

采用活性炭吸附脱色、硫酸铵分级沉淀、脱盐和凝胶层析等技术对酸性蛋白酶AP-10浓缩液进行了分离纯化,并对其酶学性质进行了研究。结果表明,分离纯化后得到的酸性蛋白酶AP-10纯组分比酶活为5 800 U·mg^-1,纯化倍数为6倍;最适反应温度为40℃,热稳定范围为30～45℃;最适反应pH值为3.0,pH值稳定范围为3.5～6.5;Mn²⁺对酸性蛋白酶AP-10具有强烈的激活作用,Fe²⁺对酸性蛋白酶AP-10具有强烈的抑制作用。为拓展酸性蛋白酶AP-10的应用及开发高品质产品奠定了基础。     

壳聚糖降解酶的分离纯化及性质研究

青霉菌（Penicillium sp菌株）发酵液经20%-70%硫酸铵分级沉淀，DEAE-Cellulose离子交换层析，SephadexG-100凝胶过滤层析，得到聚丙烯酸胺凝胶电泳均一的壳聚糖酶，该壳聚糖酶具有很强的底物专一性。     

乙醇脱氢酶的初步分离及其酶学性质的研究

以硫酸铵为沉淀剂,采用盐析法对乙醇脱氢酶（ADH）进行了初步的分离纯化,ADH比活力从粗酶液的0.464 U·mg^-1提高到1.198 U·mg^-1,纯化倍数为2.582。研究了ADH的基本酶学性质,其最适作用pH值为7.0-10.0,pH值为8.0时酶活力达到最大,pH值为7.0时酶较为稳定;最适作用温度为37℃,温度为30-40℃时酶活力较为稳定,温度超过45℃后酶活力急剧下降。     

聚乙烯醇脱氢酶的分离纯化及其酶学性质

采用(NH4)2SO4分级沉淀及Q-Sepharose HP柱层析两步纯化,首次从可高效降解聚乙烯醇(PVA)的混合菌系发酵产物中分离纯化获得了纯蛋白聚乙烯醇脱氢酶(PVADH),并对其酶学性质进行了研究. 结果表明,PVADH分子量为134.3 kDa,最适作用温度为35℃,最适作用pH值为7.5;PVADH以仲醇为底物时酶活性普遍高于以其他醇类为底物时,PVADH与PVA反应产物中检测到羰基化合物,证实PVADH对PVA有降解作用.     

绳状青霉β-葡萄糖苷酶的分离纯化及其酶学性质研究

从绳状青霉（Penicillium funiculosum）深层发酵液中分离得到一种未知的β-葡萄糖苷酶,并对其酶学性质进行初步分析.粗酶液经硫酸铵沉淀、DEAE-Sephadex A-25离子交换层析、Sephadex G-50凝胶过滤层析等步骤,获得了一种凝胶电泳均一的β-葡萄糖苷酶,经SDS-PAGE测得其分子量为19 kDa,其最适反应温度为60℃、最适反应pH值为4.5.     

单环刺螠纤溶酶UFE-Ⅱ的分离纯化及其酶学性质

目的分离纯化单一组分的单环刺螠纤溶酶UFE-Ⅱ,并分析其酶学性质。方法单环刺螠体腔液经离心、超滤、离子交换层析、凝胶过滤等方法进行分离纯化,得到单一洗脱峰酶组分,经Native-PAGE、SDS-PAGE和飞行质谱分析,测定其纯度和相对分子质量;并以酪蛋白为底物,Folin-酚试剂法测定酶活力,对其酶学性质进行分析。结果分离纯化的UFE-Ⅱ具有水解纤维蛋白的活性,其水解酪蛋白的比活力达到375.6U/mg,纯化倍数为10.3倍,回收率为14.0%。UFE-Ⅱ为单一组分,纯度达99%以上,相对分子质量为24329。UFE-Ⅱ的最适反应温度约为45℃;最适反应pH值为7.0;Ca2+、Mn2+和Fe2+是该酶的强激活剂;Fe3+、Cu2+和Pb2+对该酶活力具有一定的抑制作用;SBTI和PMSF能完全抑制酶活力,表明该酶为丝氨酸蛋白酶;糜蛋白酶抑制剂可部分抑制酶活力,亮抑酶肽、抑蛋白酶肽、苯甲脒可较弱地抑制酶活力。结论从单环刺螠体内成功分离纯化出纤溶酶UFE-Ⅱ,并分析了其酶学性质,该酶具有进一步开发利用价值。     

绿僵菌几丁质酶的分离纯化及性质

从自然罹病死亡的金龟子体内分离到一株金龟子绿僵菌（Metarhizium anisopliae）,它在几丁质的诱导下能产生较高活性的几丁质酶.发酵液经硫酸铵盐析、DEAE纤维素柱层析、Phenyl Sepharose^TM 6 Fast Flow疏水柱层析等方法,得到电泳纯的几丁质酶.用SDS-PAGE测得该酶相对分子质量为61.5 kD,而经质谱分析为57.14 kD.最适反应温度为55 ℃,最适反应pH值为6.0,酶的等电点pI为4.02,其N末端序列为VIGPAAPL,用硫酸-酚法测得其含糖量为56.2%.水解几丁质的K_m为14.5 μmol•L^-1.该酶在45 ℃,pH值3.0～9.5较为稳定.Zn²⁺、Ca²⁺、Ba²⁺和Mn²⁺离子对几丁质酶活性有明显的促进作用,而Hg²⁺、Co²⁺和Fe²⁺离子完全抑制几丁质酶的活性.此酶还可被EDTA所抑制,表明金属离子为其活性所必需.PMSF试剂对几丁质酶的活力影响比较大,丝氨酸可能是酶活力的必需基团.     

海洋青霉菌壳聚糖酶的分离纯化与性能

以从福建崇武海边的海螺、贝壳及其附着物中经过筛选分离得到海洋青霉菌(marine Penicillium oxalicum)为出发菌株,对固态发酵海洋青霉菌所产壳聚糖酶进行分离提取工艺条件的探索.分别采用不同相对分子质量的PEG(相对分子质量分别为1000,4000,6000,10000,20000)、AB-8大孔树脂...     

核酸酶P1的纯化和酶学性质研究

桔青霉发酵液通过硫酸铵分级沉淀、凝胶层析和离子交换纤维素层析等生化分离步骤,得到的核酸酶P1比活力为711U/mg,纯化倍数1500。纯化的酶蛋白纯品经SDS 聚丙烯酰胺电泳,呈现一条单带。该酶催化反应的最适pH范围为6～8,最适温度在70℃,在60℃以下时酶活较为稳定.锌离子对此酶有明显的激活作用,而铜离子具有明显的抑制作用。     

一株青霉发酵产生木素过氧化物酶的工艺条件

研究了一株青霉菌P6 (Penicillium sp. P6)液体发酵产生木素过氧化物酶的工艺条件,考察了青霉菌P6在天然培养基稻米糠、玉米糠、麦糠和合成培养基GMY中的产酶情况,结果表明,麦糠是较理想的底物. 通过单因素方法研究了添加苯甲醇和MnSO4对酶活的影响;采用正交实验对MnSO4添加量、麦糠含量和培养基pH进行了研究,在实验范围内最佳酶活达到了2.69 U/mL. 进一步通过单双因素回归实验研究了麦糠和MnSO4的最佳含量,得到了最佳培养条件为孢子接种量1.1×106 mL-1,培养时间8 d,500 mL三角瓶装液量150 mL. 最佳培养基组成为麦糠50 g/L,初始pH 4.8, MnSO4 5.66 mmol/L.     

Production of lipase from Penicillium sp. using waste oils and Nopalea cochenillifera

The present communication deals with the production of lipase from Penicillium sp. using waste oils and palm cactus (Nopalea cochenillifera) especially as nutrient source of low cost. Two different waste oils were tested: waste frying oil from an industrial kitchen and waste lubricating oil (WLO) from a gas station. Using Doehlert experimental design and response surface methodology, the optimum conditions for lipase production were 96?h fermentation, WLO as the inductor, with specific activity of 0.22?UA?mg^?1. The enzyme was able to remain with more than 58% of its original activity until 30?min at 60°C. The kinetic constants were K_m?=?9.93?mM and V_max?=?2.58 UA min^?1 using p-nitrophenyl palmitate (p-NPP) as substrate. Results showed that Penicillium sp. was able to produce lipase from waste oils using N. cochenillifera, thus having biotechnological potential in waste oil biotransformation.     

Candida sp.脂肪酶的纯化及其性质

采用简单的两步法-离子交换层析和疏水层析法,对Candida sp. 99-125脂肪酶进行了纯化,比活提高了10.0倍,达到27200 U/mg,回收率为35.5%. SDS-PAGE电泳分析显示该酶的分子量约为38 kDa. 酶学性质研究表明,该酶最适反应温度为40℃,最适反应pH值为8.5,在室温下具有良好的稳定性. 钙离子和Tween80能够促进提高脂肪酶的活性,而铁离子、铜离子和SDS对其有明显的抑制作用.     

Purification of the Penicillium citrinum Lipase Using AOT Reversed Micelles

This work describes the extraction and back-extraction of a lipase from crude extract of Penicillium citrinum using AOT reversed micelles in isooctane. The effect of pH, ionic strength, AOT concentration on the protein forward and backward transfer at 20°C was studied. The maximum protein forward extraction (32·0%) was achieved at pH 4·0 with a 50 mmol dm⁻³ acetate buffer containing 100 mmol dm⁻³ KCl and 100 mmol dm⁻³ AOT in isooctane. Proteins were back-extracted (82·7%) to a new aqueous phase containing 100 mmol dm⁻³ pH 8·0 phosphate buffer and 1000 mmol dm⁻³ KCl. No enzyme activity could be detected either in the micellar phase or in the aqueous phase after protein back-extraction. However, the lipolytic activity was recovered after hydrophobic interaction chromatography on a Phenyl Superose column. The yield obtained for the overall process was 68% for activity, 26·4% for protein recovery and the purification factor was 810-fold. A single protein band at 33000 Da was obtained for SDS–PAGE analysis for the recovered and purified enzyme. © 1997 SCI.     

A Novel Cyclodextrin Glycosyltransferase from Alkaliphilic Amphibacillus sp. NPST-10: Purification and Properties

Screening for cyclodextrin glycosyltransferase (CGTase)-producing alkaliphilic bacteria from samples collected from hyper saline soda lakes (Wadi Natrun Valley, Egypt), resulted in isolation of potent CGTase producing alkaliphilic bacterium, termed NPST-10. 16S rDNA sequence analysis identified the isolate as Amphibacillus sp. CGTase was purified to homogeneity up to 22.1 fold by starch adsorption and anion exchange chromatography with a yield of 44.7%. The purified enzyme was a monomeric protein with an estimated molecular weight of 92 kDa using SDS-PAGE. Catalytic activities of the enzyme were found to be 88.8 U mg(-1) protein, 20.0 U mg(-1) protein and 11.0 U mg(-1) protein for cyclization, coupling and hydrolytic activities, respectively. The enzyme was stable over a wide pH range from pH 5.0 to 11.0, with a maximal activity at pH 8.0. CGTase exhibited activity over a wide temperature range from 45 °C to 70 °C, with maximal activity at 50 °C and was stable at 30 °C to 55 °C for at least 1 h. Thermal stability of the purified enzyme could be significantly improved in the presence of CaCl(2). K(m) and V(max) values were estimated using soluble starch as a substrate to be 1.7 ± 0.15 mg/mL and 100 ± 2.0 μmol/min, respectively. CGTase was significantly inhibited in the presence of Co(2+), Zn(2+), Cu(2+), Hg(2+), Ba(2+), Cd(2+), and 2-mercaptoethanol. To the best of our knowledge, this is the first report of CGTase production by Amphibacillus sp. The achieved high conversion of insoluble raw corn starch into cyclodextrins (67.2%) with production of mainly β-CD (86.4%), makes Amphibacillus sp. NPST-10 desirable for the cyclodextrin production industry.     

青蒿中青蒿素的纯化工艺研究

建立了一种青蒿中青蒿素的纯化方法:采用中性氧化铝柱层析,考察不同洗脱溶剂的分离效果,并对重结晶溶剂进行选择.氧化铝柱层析纯化青蒿素的最佳操作条件为洗脱剂石油醚∶异丙醚(V/V)=70∶30,青蒿素含量提高到85%以上,回收率达90%;经过50%乙醇重结晶,青蒿素含量大于99.5%.该方法简便,产品纯度高,回收率高.     

杜仲活性成分的提取及分离纯化方法研究进展

总结了近年来杜仲活性成分的提取和分离纯化方法,对每种方法的优缺点和适用范围进行了小结;以开发杜仲活性成分为目的,提出了提取和分离纯化杜仲活性成分的几点建议。     

苏云金杆菌（B.t.）晶体毒素的分离与纯化

本文介绍了苏云金杆菌晶体毒素的分离、溶解及纯化技术。