A Simple,Rapid, High Yield Isolation and Purification Procedure for Chloroperoxidase Isoenzymes

A simple four-step procedure has been developed for Isolation of chloroperoxldase from the mold Caldarlomyces fumago. Polyethyleneglycol precipitation of the contaminating pigment in the growth medium, followed by chromatography of the soluble enzyme fraction on a QAE-ZetaPrep-250 cartridge and ammonium sulfate precipitation affords Isolation of the chloroperoxldase. Extensive dialysis and chromatography on DE-53 cellulose allows the separation and further purification of chloroperoxldase A and B isoenzymes.     

酪氨酸酶在有机介质中的酶活性

研究了蘑菇酪氨酸酶在有机溶剂中催化邻苯二酚向邻苯醌的转化反应,结果表明酪氨酸酶在有机介质中能保持较高的活性,其活性主要受体系中的水活度控制.在适当控制酶分子上结合水的条件下,酶活性随温度升高而增大.无机盐的水合物可用于控制这种低水有机介质中的水活度.     

低水有机介质中的酶催化

酶不仅能在水溶液里催化化学反应,而且能在有机介质中显示催化活性．其中低水溶剂体系对有机合成最为有利．文章就低水溶剂体系中影响酶催化的三要素（水、溶剂和载体）以及酶在该体系表现出来的一些特殊性质进行了讨论,并列举了低水溶剂体系中的酶催化在有机合成,化学分析,和高分子化学等方面的应用．     

有机介质中酶促合成壬酸香草醇酯的研究

壬酸香草醇酯是与天然辣椒素酯结构最接近的一个化合物,本文研究了有机相中脂肪酶催化合成这个化合物的方法,为天然辣椒素酯的酶促合成探索方法。考察了酶、溶剂、酶的用量、底物浓度、溶剂水含量以及温度等因素对反应的影响,结果表明脂肪酶Novozym e 435的活性最好,最适条件为:在1 mL脱水丙酮中,香草醇与壬酸甲酯的浓度分别为50、75 mmol/L,酶量为20 mg,30℃下反应24 h,产率可达到60%以上,产物经硅胶柱层析纯化,并以1H NMR及MS进行了表征。     

一种简单、快速提取DNA的方法

随着分子生物学研究的迅速发展,提取ＤＮＡ已成为一项常规实验。用经典方法提取ＤＮＡ［１］,先用蛋白酶Ｋ、ＳＤＳ消化,然后用酚、氯仿抽提,乙醇沉淀,耗时较多,提取液需要多次转移,易引起交叉污染和ＤＮＡ丢失。本文利用硅藻（ｄｉａｔｏｍ）能够特异性吸附核酸的...     

A Rapid Method for Purification of Myelin Basic Protein

A rapid procedure for purification of myelin basic protein has been developed. White matter is delipidated with 2-butanol, and the residue is extracted at pH 7.5 and 8.5. Myelin basic protein is solubilized by extraction in acetate buffer, pH 4.5. The entire procedure requires less than 4 h, and gives homogeneous protein essentially free of protease activity. This procedure can be scaled down to process milligram amounts of white matter; thus it can be very useful for purification of myelin basic protein from very limited amounts of human white matter obtained during surgery.     

一种简捷的玉米大斑病病菌单孢分离方法

介绍一种利用普通光学显微镜直接分离玉米大斑病病菌单孢子的方法。该方法是通过敲打叶片先将孢子转移至水琼脂培养基上,然后利用自制的简易挑孢针在低倍镜下直接挑取大斑单孢子,从而达到分离纯化的目的。结果表明,此法虽然有一定的操作难度,但简便易行,污染少,高效快捷,为玉米大斑病病菌生理小种鉴定、DNA遗传多态性的进一步研究做了积极有益的基础工作。该方法值得推广到其他种类大型孢子真菌的单孢分离工作中。     

Purification and Characterization of a Thermo- and Organic Solvent-Tolerant Alkaline Protease from Bacillus sp. JER02

Bacillus sp. JER02 is a bacterial strain that can be grown in a medium containing organic solvents and produce a protease enzyme. JER02 protease was purified with a yield of 31.9% of total protein and 328.83-fold purification. K _m and V_max of this protease were established as 0.826 µM and 7.18 µmol/min, respectively. JER02 protease stability was stimulated about 80% by cyclohexane. It exhibited optimum temperature activity at 70°C. Furthermore, this enzyme was active in a wide range of pH (4-12) and showed maximum activity at pH 9.0. The nonionic detergents Tween-20 and Triton X-100 improved the protease activity by 30 and 20%, respectively. In addition, this enzyme was shown to be very stable in the presence of strong anionic surfactants and oxidizing agents, since it retained 77%, 93%, and 98% of its initial activity, after 1 hr of incubation at room temperature with sodium dodecyl sulfate (SDS), sodium perborate (1%, v/v) and H₂O₂ (1%, v/v), respectively. Overall, the unique properties of the Bacillus sp. JER02 protease suggested that this thermo- and detergent-stable, solvent-tolerant protease has great potential for industrial applications.     

Effect of Different Supports on the Reaction Rate of Rhizomucor Miehei Lipase in Organic Media

Five different aluminas, a silica and a zirconia support were used to adsorb lipase (E.C. 3.1.1.3) from Rhizomucor miehei. The activity of the immobilised lipase was measured by esterification of dodecanol and decanoic acid in hexane. The immobilised lipase and the organic phase were pre-equilibrated separately to known water activities before mixing them to commence the reactions. The aluminas, which varied in pore sizes and surface areas, adsorbed similar amounts of enzyme. However, the esterification activities varied about 10-fold, increasing with increasing surface area. The silica and zirconia supports adsorbed about half as much lipase as the aluminas. The esterification reaction rates per unit quantity of enzyme adsorbed were compared with those for aluminas with similar surface areas; this specific rate was about 2 times higher for the zirconia, but the difference with silica was only small. There was no clear correlation between the esterification rates at fixed water activity and the amount of water adsorbed by the support used.     

A Simple and Gentle Method for Concentrating Protein Solutions

A gentle method for concentrating very dilute protein solutions is described. The high capacity of aminohexylagarose in adsorbing different proteins is utilized to handle small or large amounts of protein with practically no losses in material or activity. To concentrate very dilute protein solutions as they occur during purification procedures e. g. of enzymes, a one-step non-inactivating nethod is needed that may easily be integrated into the purification programm.

In the course of the purification of a labile enzyme (1) we developed a simple chromatographic method which seems to work for a large variety of proteins. The procedure is applicable to very dilute protein solutions, to small samples as well as to large scale preparations, and it is relatively inexpensive. It appears to be a very gentle method since in all cases tested no loss of enzymic activity could be observed.     

A Simple Method for Staining Fungal Substrate Hyphae in Agar Media

Morphogenetic processes often occur in fungal cultures in agar medium. These processes are difficult to study by light microscopy because the hyphae or other structures fail to have sufficient contrast for detailed study and photography. To overcome this difficulty, we developed a method to stain hyphae inside the agar without affecting the medium itself.     

一种快速mRNA差异显示技术及用于分离辐射诱导转录子

介绍一种从不同类型细胞或不同生长状态细胞中分离差异表达基因的快速ｍＲＮＡ差异显示技术．其特点是不用同位素标记,操作简便,在普通琼脂糖凝胶电泳中就能分辨差异显示的ｃＤＮＡ带,便于ＤＮＡ回收和进一步重组克隆．用此方法成功地分离到电离辐射诱导转录子．     

Modified Proteases for Peptide Synthesis in Organic Media

We showed that modified proteases could catalyze synthesis of a wide variety of peptides of various lengths and structures both in solution and on solid phase in organic solvents. The following modified proteases were studied as catalysts for enzymatic peptide synthesis in polar organic solvents (acetonitrile, dimethylformamide, and ethanol): pepsin sorbed on celite, a noncovalent complex of subtilisin with sodium dodecylsulfate, and subtilisin or thermolysin covalently immobilized on a cryogel of polyvinyl alcohol. The use of the noncovalent complex of subtilisin with sodium dodecylsulfate and immobilized subtilisin is especially promising for the segment condensation of peptide fragments containing residues of trifunctional amino acids with unprotected ionogenic groups in side chains, such as Lys, Arg, His, Glu, and Asp.     

Esterification in Organic Media for Preparation of Optically Active Secondary Alcohols: Effects of Reaction Conditions

Esterification in an organic solvent for enantioselective preparation of optically active secondary alcohols was investigated using porcine pancreas lipase (PPL), (R,S)-2-octanol and dodecanoic acid in heptane. The influence of different reaction conditions on esterification rate and enantioselectivity was studied. Removal of water and immobilization of PPL both led to distinct improvement of the extent of ester formation and enantioselectivity of catalysis. Studies allowing continuous control of optical enrichment in the product (ester) and the remaining substrate (alcohol) were carried out in order to further optimize the reaction conditions. Optically pure (R)-and (S)-2-octanol were prepared.     

人脑神经元特异性烯醇化酶的纯化方法

采用改良的Grace层析方法,经一次DEAE-Sephadex A50柱层析即从人脑中纯化了神经元特异性烯醇化酶,比活力为92.1U/mg,纯化倍数为59.4.该酶纯化后,经SDS-聚丙烯酰胺凝胶电泳鉴定为单一蛋白质谱带.此外,还测定了其部分理化性质,其亚单位分子量为45000,等电点pI为4.7,氨基酸组成分析表明其为一种酸性蛋白质;对2-磷酸甘油酸的K_m值为5.6×10^-4mol/L.     

A Simple and Efficient Method for the Purification of Human Gamma Interferon

Abstract

A preparative, sequential chromatographic procedure has been developed for the purification of human gamma interferon (HuIFN-γ). The four steps in the procedure are Controlled Pore Glass-adsorption chromatography, Concanavalin-A affinity chromatography, Heparin-Sepharose affinity chromatography and gel-filtration.

By virtue of the development of a coordinated effluent-affluent buffer scheme, eluants can also serve as loading buffers for the succeeding column. Consequently, crude HuIFN-γ preparations can be purified rapidly (approximately one week), easily, and is amenable to a semi-automated process.

The procedure has also been shown to be efficient. Here, as an example, it is reported that an overall purification of greater than 75,000-fold can be achieved, yielding a specific activity of 5.2·107 units/mg, and a recovery of 95.5%. In addition, the peak fraction, representing 37.8% of the applied activity, had a specific activity of l.0·108 units/mg protein and represents a purification of more than 145,000-fold.

An SDS-PAGE analysis of one such fraction indicated that approximately 40% of the final material was HuIFN-γ.     

PEG蛋白质分离纯化的新方法

ＰＥＧ化蛋白质的分离纯化比较困难,本工作发现ＰＥＧ化蛋白质仍能被硫酸铵盐析,据此可以简单地将ＩＬ－２及ＰＥＧ化ＩＬ－２沉淀出来,而将有毒的活化ＰＥＧ等副产物留在溶液中。此方法效果理想而又十分简便。文献中未见报道。此外,实验还发现,在ＰＥＧ－ＩＬ－２与ＩＬ－２的混和液中加入一定量的ＰＥＧ后,二者之间溶解度差别增大,当用ＳｅｐｈａｃｒｙⅠＳ－２００柱分离时,先用含１０％ＰＥＧ,０．２５ｍｏｌ／ＬＮａＣｌ的缓冲液平衡洗脱ＰＥＧ－ＩＬ－２,再降低盐浓度洗下ＩＬ－２,即可使二者完全分开。过去要将ＩＬ－２与ＰＥＧ－ＩＬ－２分离开非常困难,本工作解决了这个问题,这点亦未见文献报道。     

重组人IL-4大肠杆菌表达与纯化

根据大肠杆菌密码子偏爱性优化并合成人白细胞介素4基因,以pET30a( )为载体构建了重组表达质粒pET30a( )/rhIL-4,将重组质粒转化大肠杆菌BL21(DE3)感受态细胞,诱导表达并超声破菌检测重组蛋白的表达形式。采用5L发酵罐培养工程菌,发酵液OD600为0.6时诱导3.5h收集菌体,检测目的蛋白的表达量。收集的菌体经压榨破菌获得包涵体,通过包涵体变性、层析、透析复性等方法对rhIL-4进行纯化。采用人红细胞白血病细胞(TF-1)测定纯化的rhIL-4的生物活性。测序表明目的基因已插入载体pET30a( )中,重组蛋白以包涵体形式表达,单位体积重组蛋白的表达量达200mg/L发酵液,建立了对包涵体形式表达的rhIL-4纯化方法,最终得率为40mg/L发酵液,纯度大于98%,回收率为20%以上。免疫印迹法检测诱导表达的重组蛋白和纯化的蛋白为IL-4,N端氨基酸序列测定结果与理论相符,生物活性检测纯化的蛋白比活性达2.5×106AU/mg。这为rhIL-4进一步产业化研究建立了基础。     

辣根过氧化物酶在一种新型有机介质中的催化反应

选择合适的酶反应介质体系,是酶应用于有机合成的一个重要环节。利用适宜分子量的聚乙二醇（ＰＥＧ）可以将辣根过氧化物酶（ＨＲＰ）分散在甲苯中,摸索了ＨＲＰ在聚乙二醇（ＰＥＧ）－甲苯互溶体系反应的适宜条件,即ＰＥＧ／甲苯的比例、含水量、ｐＨ值、底物浓度等对酶活性影响,结果发现ＰＥＧ含量越低,含水量越高,酶的活力越高;酶在此体系中的最适ｐＨ值为７．０,最适过氧化氢浓度为２０ｍｍｏｌ／Ｌ,愈创木酚的浓度为０