华广虻肠道溶纤活性蛋白的纯化和性质(英文)

经过 75 %饱和度硫酸铵沉淀、SephadexG 75凝胶过滤层析、Lys Sepharose 4B亲和层析和电泳制备洗脱 ,从华广虻 (TabanusamaenusWalker)腹部组织匀浆液中分离纯化出分子量约为 6 7KD的溶纤活性蛋白TAFP。经纤维蛋白平板测定表明 ,TAFP不仅具有纤溶酶作用 ,还具有激活纤溶酶原的作用 ;通过三肽生色底物测定发现 ,TAFP能分解纤溶酶原激活剂的生色底物—ChromozymUK及S 2 2 88。还能水解胰蛋白酶专一底物Bz Phe Val Arg NA及CBZ Gly Pro Arg NA ,表明TAFP具有类胰蛋白酶活性 ,专一水解精氨酸形成的酰胺键 (或肽键 )。TAFP无胰凝乳蛋白酶活性     

五带虻溶纤活性蛋白的纯化和性质

五带虻Tabanus qutnquectnctus Rlcardo腹部匀浆液经硫酸铵沉淀、Sephadex G-75凝胶层析、Fibrin-Sepharose 4B亲和层析和电泳制备等方法纯化后,获得在SDS—PAGE图谱上呈现单一区带的溶纤活性蛋白。该蛋白质既具有纤溶酶作用,又具有激活纤溶酶原的作用,其分子量为40kD,等电点为4.5,最适作用pH为9.0,最适作用温度为28℃,37℃处理2h活性完全丧失,Ca²⁺、Mn²⁺、Cu²⁺、Zn²⁺、Hg²⁺和PMSF能抑制其活性。     

杭州虻纤溶酶的纯化及其生物活性分析

经过７５％硫酸铵沉淀、ＳｅｐｈａｄｅｘＧ－７５凝胶过滤层析、ＤＥＡＥ Ｓｅｐｈａｄｅｘ Ａ－２５离子交换层析、Ｂ ｅｎｚａｍｉｄｅ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析,从杭州虻（Ｔａｂａｎｕｓ ｈｏｎｇｃｈｏｗｅｎｓｉｓ）腹部匀浆液中分离纯化出分子量约为３６．５ｋＤ的纤溶酶ＴＨＦＥ。经纤维蛋白平板测定表明,ＴＨＦＥ既具有纤溶酶作用,又具有激活纤溶酶原的作用。ＴＨＦＥ能分解纤溶酶原激活剂的生色底既具有纤     

FIBRINOLYTIC PROTEASES FROM THE GUT OF LARVAE OF FLESH FLY, BOETTCHERISCA PEREGRINA (DIPTERA: SARCOPHAGAE): PURIFICATION AND CHARACTERIZATION

Abstract Three fibrinolytic proteases, which were designated as BPGFF'l, BPGFP2 and BPGFP3 individually, were purified from the gut extract of larvae of Boettcherisca peregrina fed on artificial diet containing fibrin-rich pig blood-coagulated block. BPGFP1 consists of two subunits with MW 32 000 and 30 000. Both BPGFP2 and BPGFP3 are monomer with MW 40 000 and 28 000, respectively. These three proteases am similar in substrate and inhibitor specificity. All of them possess high activities against fibrinolytic protease specific substrates such as fibrin, Chromzym P, Chromzym UK and S-2288. They also strongly hydrolyze trypsin-specific substrates Bz-Phe-Val Arg NA, cBz Gly-Pro-Arg NA, Bz-Pro-Phe-Arg NA and Bz-Val-Gly-Arg NA. PMSF, STI, LBTI and SBBI can inhibit activity of these proteases. Activities of these three fibrinolytic proteases were found to be maximal at alkaline range of pH 9.0 ˜ 10.0.     

棕尾别麻蝇幼虫肠道溶纤活性蛋白酶的分离纯化和性质

从以富含纤维蛋白的血凝块为食物的棕尾别麻蝇幼虫肠道浸提液中分离纯化出3种具有溶纤活性的蛋白酶,分别命名为BPGFP1,BPGFP2和BPGFP3。其中,BPGFP1由两个分子量分别为32000和30000的亚基组成。BPGFP2和BPGFP3均为单体,分子量分别为40000和28000。这三种蛋白酶具有相似的底物特异性和抑制剂特性。三种蛋白酶均能降解溶纤活性蛋白酶的特异底物纤维蛋白,Chromzym,P,Chromzym UK和S－2288。三种酶还能够强烈降解类胰蛋白酶专一底物Bz-Phe-Val Arg NA,cBz Gly-Pro-Arg NA,Bz-Pro－Phe-Arg NA和Bz-Val-Gly-Arg NA.PMSF,STI,LBTI和SBBI能够对三种蛋白酶活怀有极强的抑制作用。三种溶纤活性蛋白酶均在pH9.0-10.0范围内表现出较高活性。     

巨尾阿丽蝇幼虫肠道蛋白酶的分离纯化及性质

巨尾阿丽蝇幼虫肠液ＳＤＳ－ＰＡＧＥ后,Ｘ光片显影呈现３条蛋白酶活性带。ＩＥＦ后出现２条蛋白酶活性带,等电点分别为ＰＨ８．５和ＰＨ７．７。肠液经硫铵沉淀,ＳｅｐｈａｄｅｘＧ－７５凝胶过滤,ＳｅｐｈａｄｅｘＤＥＡＥＡ－２５离子交换和ＳＢＢＩ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析,分离化出分子量约为１４ＫＤ的巨尾阿丽蝇蛋白酶。     

麻蝇幼虫肠道类胰蛋白酶的分离纯化及性质(英文)

麻蝇幼虫肠液经硫铵沉淀, ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ－２５离子交换层析, ＳＢＢＩ－Ｓｅｐｈａｒｏｓｅ ４Ｂ亲和层析,分离纯化出一种分子量为 １６ｋＤ的蛋白酶。底物及抑制剂的特异性表明,该酶为类胰蛋白酶。其能够强烈地降解蛋白酶非专一底物酪蛋白和 Ｈｉｄｅ ｐｏｗｄｅｒ ａｚｕｒｅ,以及类胰蛋白酶专一底物 Ｂｚ－Ｐｈｅ－Ｖａｌ－Ａｒｇ ＮＡ, Ｂｚ－Ｐｒｏ－Ｐｈｅ－Ａｒｇ ＮＡ和Ｂｚ－Ｖａｌ－Ｇｌｙ－Ａｒｇ ＮＡ．该酶又能被丝氨酸蛋白酶抑制剂ＰＭＳＦ,类胰蛋白酶抑制剂 ＳＢ－ＢＩ和Ｌｅｕｐｅｐｔｉｎ强烈地抑制。蛋白酶在酸性环境下极不稳定,在弱碱环境（ｐＨ８．５－９．５）中活性最高。     

PURIFICATION AND PARTIAL CHARACTERIZATION OF A GUT TRYPSIN-LIKE PROTEASE FROM LARVAE OF FLESH FLY BOETTCHERISCA PEREGRIN A (DIPTERA: SARCOPHAGAE)

Abstract A 16kD protease was purified from the gut extract of larvae of Boettcherisca peregrina , after ammonium sulfate precipitation, DEAE-Sephadex A-25 ion-exchange chromatography and SBBI-Sepharose 4B affinity chromatography. The results of substrate and inhibitor specificity indicated that the protease behaved as a trypsin-like protease. It possesses high activity against non-specific substrate casein and Hide powder azure, and against trypsin-specific substrates Bz-Phe-Val-Arg NA, Bz-Pro-Phe-Arg NA and Bz-Val-Gly-Arg NA. It can be strongly inhibited by PMSF, phenymethysulfonyl fluoride (serine protease inhibitor), SBBI, soybean Bowman-Birk inhibitor and Leupeptin (trypsin-specific inhibitor). Activity of this protease was found to be maximal at the alkaline range of pH 8. 5–9. 5.     

一种来源于链霉菌的纤溶酶的纯化及其基因的克隆

链霉菌C3662的发酵液上清经 80 %硫酸铵沉淀 ,DEAE Sepharose和CM Sepharose层析分离后纯化出一种纤溶酶。SDS PAGE显示为单一的条带 ,分子量约为 30kD。以 pIJ699为载体 ,S .lividansTK2 4为宿主菌 ,鸟枪法克隆纤溶酶基因 ,从 30 0 0个转化子中挑选到 1个具活性转化子 ,经亚克隆 ,序列测定得到一个 90 3bp的完整ORF ,其GC %为 68.33% ,密码子第三位GC %为 95.6% ,符合链霉菌基因的典型特征。与多种蛋白酶具有较高的同源性     

钜齿远蚓(Amynthas dancataa)纤溶酶的分离纯化及其若干性质

 以野生型钷齿远蚓为材料,组织匀浆后,经生理盐水抽提,硫酸铵分级沉淀,葡聚糖凝胶过滤和DEAE离子交换层析,得到两种纯的蚯蚓溶酶,具有强烈的溶解纤维蛋白的作用。它们都是糖蛋白,非寡聚酶,分子量分别为23,000、40,000。测定了一个酶的氨基酸组成,它对某些底物的作用,被一些抑制剂抑制的程度,说明它是练氨酸蛋白酶类、胰蛋白酶类酶     

棉花病菌Xanthomonas campestris pv.malvacearum的一种具特异肽键专一性的胞外蛋白酶的纯化与鉴定

棉花病原体Xanthomonas campestris pv.malvacearum在酪蛋白(脱脂奶)存在下生长时产生胞外蛋白酶活性,其中至少包含3种蛋白酶,表观分子量分别为29(蛋白酶-1)、38和43kD。 蛋白酶-1被纯化,其最适pH在5.5～7.5之间。抑制研究表明蛋白酶-1可被Phosphoramidone、EDTA及1,10-邻二氮杂菲抑制,然后用锌离子温育重新激活,说明这是一个金属蛋白酶。发现蛋白酶-1特异地裂解肽链的天冬氨酸残基或半胱氯酸残基的氨基端侧,这种高度的肽键专一性预示这个酶在蛋白质链顺序分析中及由较大蛋白质制备特定多肽方面可能十分有用。     

绿僵菌分解昆虫外壳蛋白酶MAP-21的纯化与特性

以蝉蜕为底物诱导绿僵菌产生分解昆虫外壳蛋白酶 。发酵液经超滤、Ultrogel AcA 54凝胶层析、制备IEF电泳,纯化了一种蛋白酶MAP-21,SDS-PAGE电泳后经银染色呈单带。该酶的Mr为27kD左右,pI为76。它的特异识别氨基酸为Arg,其活性可被PMSF和TLCK抑制,表明其活性中心有Ser和His残基。它还可被胰蛋白酶的典型抑制剂Leupeptin、Antipain及STI等所抑制,而胰凝乳蛋白酶抑制剂TPCK和胰凝乳弹性蛋白酶抑制剂TEI对其活性无影响。专一底物和抑制剂特性试验结果表明MAP-21是类胰蛋白酶。此外,该酶还可被EDTA所抑制,表明金属离子为其活性所必需。另外还研究了MAP-21的最适作用温度和pH,以及温度耐受性等特性。     

PURIFICATION AND PROPERTIES OF DETERGENT-COMPATIBLE EXTRACELLULAR ALKALINE PROTEASE FROM Scopulariopsis spp.

A fungal alkaline protease of Scopulariopsis spp. was purified to homogeneity with a recovery of 32.2% and 138.1 U/mg specific activity on lectin-agarose column. The apparent molecular mass was 15 ± 1 kD by sodium dodecyl sulfate polyacryalamide gel electrophoresis (SDS-PAGE). It was a homogenous monomeric glycoprotein as shown by a single band and confirmed by native PAGE and gelatin zymography. The enzyme was active and stable over pH range 8.0–12.0 with optimum activity at pH 9.0. The maximum activity was recorded at 50°C and remained unaltered at 50°C for 24 hr. The enzyme was stimulated by Co²⁺ and Mn²⁺ at 10 mM but was unaffected by Ba²⁺, Mg²⁺, Cu²⁺, Na⁺, K⁺, and Fe²⁺. Ca²⁺ and Fe³⁺ moderately reduced the activity (～18%); however, a reduction of about 40% was seen for Zn²⁺ and Hg²⁺. The enzyme activity was completely inhibited by 5 mM phenylmethylsulfonyl fluoride (PMSF) and partially by N-bromosuccinimide (NBS) and tocylchloride methylketone (TLCK). The serine, tryptophan, and histidine may therefore be at or near the active site of the enzyme. The protease was more active against gelatin compared to casein, fibrinogen, egg albumin, and bovine serum albumin (BSA). With casein as substrate, Km and Vmax were 4.3 mg/mL and 15.9 U/mL, respectively. An activation was observed with sodium dodecyl sulfate (SDS), Tween-80, and Triton X-100 at 2% (v/v); however, H₂O₂ and NaClO did not affect the protease activity. Storage stability was better for all the temperatures tested (?20, 4, and 28 ± 2°C) with a retention of more than 85% of initial activity after 40 days. The protease retained more than 50% activity after 24 hr of incubation at 28, 60, and 90°C in the presence (0.7%, w/v) of commercial enzymatic and nonenzymatic detergents. The Super Wheel–enzyme solution was able to completely remove blood staining, differing from the detergent solution alone. The stability at alkaline pH and high temperatures, broad substrate specificity, stability in the presence of surfactants and oxidizing and bleaching agents, and excellent compatibility with detergents clearly suggested the use of the enzyme in detergent formulations.     

中国虻属二新种：双翅目：虻科

本文报道了虻属二新种:晨螫虻 Tahanus matutinimordicus sp.nov.;似冲绳虻 T.okinawanoides sp.nov.,作者对新种的形态特征进行了详细描述,并与近似种作了比较。     

蚯蚓纤溶酶分子生物学进展

蚯蚓纤溶酶是近年发现的一种新型的溶解血栓物质,属丝氨酸蛋白酶,不同种属的蚯蚓中均可分离到,具纤溶活性和溶栓活性。有较好的热稳定性,多为单体酶,多数兼有纤溶活性和纤溶酶原激活活性。不同种属的蚯蚓分离的纤溶酶性质上有一定差别。已获得多种纤溶酶的N端序列及部分核酸序列,相互之间及与某些蛋白酶之间有一定的同源性。纤溶酶通过降解目的蛋白的特定位点而起作用 。     

贵州虻属五新种（双翅目：虻科）

本文记述采自贵州的虻属五新种:似类柯虻Tabanus cordigeroides sp.nov.、贵州虻T.guizhouensis sp.nov.、似杭州虻T.hongchowoides sp,nov.、伪青腹虻T.Pseudoliuiuentris sp.nov.和亚岷山虻T.subminshanensis sp. nov.,并分别进行了分类讨论。     

甘薯凝集素的提取及性质研究

抗蔓割病甘薯的叶片组织浸取液,经硫酸铵分级沉淀,甲壳素柱层析及葡聚糖凝胶过滤得到一种在PAGE或SDS－PAGE上均呈现单一蛋白带的甘薯凝集素。该凝集素没有血型专一性及被测动物红细胞专一性,其凝集活性可被N－乙酰葡萄糖胺或岩藻糖所抑制。甘薯凝集素在75℃加热10min,即丧失全部凝集活性,其凝集活性依赖于Ca^2 和Mg^2 ,Mn^2 则无作用。经Sephadex G-100和SDS－PAGE测定,凝集素相对分子质量为63000,中性糖含量为6.21%,该凝集素对蔓割病菌有抑制作用,是一种酸性糖蛋白。     

雪松聚球藻金属硫蛋白的纯化及其性质的研究

在雪松聚球藻的培养基中逐渐增加氯化镉的浓度以诱导藻细胞内金属硫蛋白的合成,经Ｓｅｐｈａｄｅｘ Ｇ－５０,ＤＥＡＥ－ｃｅｌｌｕｌｏｓｅ和Ｓｅｐｈａｄｅｘ Ｇ－２５柱层析,获得的ＭＴ没有亚型,经ＳＤＳ－ＰＡＧＥ分析是高度均一的。每个蛋白分子约含５个Ｃｄ原子,１０个巯基,分子量约为７．６ＫＤ,等电位ｐＨ４．５左右,半胱氨酸含量约占总氨基酸量的１５．５％,还含有少量的芳香族氨基酸,ＭＴ的最大紫外吸收在２５     

PURIFICATION AND CHARACTERIZATION OF PULLULANASE FROM Lactococcus lactis

This paper describes a simple and efficient method of isolation of a plullulanase type I from amylolytic lactic acid bacteria (ALAB). Extracellular pullulanase type I was purified from a cell-free culture supernatant of Lactococcus lactis IBB 500 by using ammonium sulfate fractionation and dialysis (instead of ultrafiltration), and ion-exchange chromatography with CM Sepharose FF followed by gel filtration chromatography with Sephadex G-150 as the final step. A final purification factor of 14.36 was achieved. The molecular mass of the enzyme was estimated as 73.9 kD. The optimum temperature for the enzyme activity was 45°C and the optimum pH was 4.5. Pullulanase activity was increased by addition Co²⁺ and completely inhibited by Hg²⁺. The enzyme activity was specifically directed toward α-1,6 glycosidic linkages of pullulan giving maltotriose units. Enzymatic hydrolysis of starch and amylose produced a mixture of maltose and maltotriose.     

闽北虻属一新种记述

1992年在闽北邵武市开展吸血虻类调查,于武夷山自然保护区西侧二都林场采获一批近似黑额虻标本,经鉴定系虻属新种,定名武夷山虻Tabanus wugishaneisis。