内内葡聚糖苷水解酶的分离纯化和内源荧光性质

利用离子交换和分子筛层析技术从拟康氏木霉Ｓ－３８固态发酵液中提纯了两个内切葡聚糖苷酶组分。测定了一个组分的内源荧光性质。结果表明：该酶分子的内源荧光几乎都来自色氨酸。Ｎ－溴代琥珀酰亚胺（ＮＢＳ）修饰导致了酶活力的完全丧失,但是酶荧光残留了２５％,抑制剂纤维二糖与酶结合可使部分酶荧光得到保护,同时这种结合也可以保护一定的色氨酸荧光不被外来淬灭剂淬灭。     

油麻藤凝集素的荧光光谱研究

用化学修饰、内源荧光和荧光淬灭等方法研究了油麻藤凝集素（ＭＳＬ）的溶液构象变化和微环境的构象特征。研究发现ＭＳＬ分子中总共有９个色氨酸（Ｔｒｐ）残基,它们的荧光能被丙烯酰胺淬灭,但不易为ＫＩ接近而淬灭,ＭＳＬ经Ｎ－溴代琥珀酰亚胺（ＮＢＳ）修饰后,其内源性荧光发射谱发生相应变化,结果表明ＭＳＬ分子中部分Ｔｒｐ残基埋藏于分子内部,而位于分子表面的Ｔｒｐ残基可能处于分子的疏水袋中。     

油麻藤凝集素的荧光光谱研究

用化学修饰,内源荧光和荧光淬灭等方法研究了油麻藤集素（ＭＳＬ）的溶液的象变化和微环境的构象特征,研究发现ＭＳＬ分子中总共有９个色氨酸（Ｔｒｐ）残基,它们的荧光能被丙烯酰胺淬灭,但不易为ＫＩ接近而淬灭,ＭＳＬ经Ｎ－溴化琥珀酰亚胺（ＮＢＳ）修饰后,其内源性荧光发射谱发生相应变化,结果表明ＭＳＬ分子中部分Ｔｒｐ残基埋藏于分子内部,而位于分子表面的Ｔｒｐ残基可能处于分子的疏水袋中。     

胰蛋白酶与ANS的相互作用

利用荧光光谱法研究了在不同ｐＨ、压力及不同浓度的脲作用时荧光探针１,８－ＡＮＳ（１－ａｎｉｌｉｏｎｎａｐｈｔｈａｌｅｎｅ－８－ｓｕｌｆｏｎｉｃａｃｉｄ）与胰蛋白酶的相互作用．发现在低ｐＨ时ＡＮＳ可以结合到胰蛋白酶上,其中以ｐＨ２．０、３．０时结合最强．进一步的研究发现脲变性对胰蛋白酶结合ＡＮＳ的能力有很大的影响：１．５ｍｏｌ／Ｌ的脲即可使得胰蛋白酶结合ＡＮＳ的能力大大降低,但有趣的是即使高达４ｍｏｌ／Ｌ的脲对胰蛋白酶色氨酸残基荧光也无明显影响．另外,在ｐＨ猝变、脲变性、及逐渐改变压力时,胰蛋白酶色氨酸残基荧光和结合到胰蛋白酶分子上的ＡＮＳ的荧光的变化大不相同．上述结果暗示胰蛋白酶的色氨酸残基所在的区域和其结合ＡＮＳ的区域是两个不相同的区域．     

野花生豆凝集素的化学修饰与荧光光谱研究

野花生豆凝集素（ＣＭＬ）经ＳｅｐｈａｄｅｘＧ－２００测得分子量为１０３．ＯｋＤ．用对二甲基氨基苯甲醛（ＤＡＢ）为显色剂,测得每个ＣＭＬ分子含有５．９个色氨酸残基．在ｐＨ５．１,含８ｍｏｌ／Ｌ脲的醋酸缓冲液中,Ｎ－溴代丁二酰亚胺（ＮＢＳ）可修饰ＣＭＬ分子中的５．６个色氨酸（Ｔｒｐ）残基,同时使ＣＭＬ的凝血活性完全丧失．用焦碳酸二乙酯（ＤＥＰＣ）和Ｎ－乙酰顺丁烯酰胺（ＮＥＭ）分别修饰ＣＭＬ的组氨酸残基和半胱氨酸巯基后,ＣＭＬ的活性均无变化．ＣＭＬ在天然状态下荧光发射峰位于３３６ｎｍ处,用ＣＭＬ的专一性抑制糖Ｎ－乙酰半乳糖胺研究色氨酸的微环境,发现Ｎ－乙酰半乳糖胺可以淬灭ＣＭＬ中８８％的色氨酸残基萤光,Ｓｔｅｒｎ－Ｖｏｌｍｅｒ常数Ｋ＝１．７３Ｌ／ｍｏｌ．同时发现Ｎ－乙酰半乳糖胺能够保护ＣＭＬ,避免ＮＢＳ对ＣＭＬ的修饰作用,表明色氨酸可能是ＣＭＬ维待活性所必需,并直接参与和专一性抑制糖的结合,其微环境较为疏水．     

大豆C4途径与光系统Ⅱ光化学功能的相互关系

测定了不同发育时期大豆（Ｇｌｙｃｉｎｅ ｍａｘ（Ｌ）Ｍｅｒｒ．）“黑农４１”叶片的４种Ｃ４酶（ＰＥＰＣａｓｅ（磷酸烯醇式丙酮酸羧化酶）、ＮＡＤＰ－ＭＤＨ（ＮＡＤＰ苹果酸脱氢酶）、ＮＡＤＰ－ＭＥ（ＮＡＤＰ苹果酸酶）和ＰＰＤＫ（丙酮酸磷酸二激酶））活性、荧光动力学数值（Ｆｖ／Ｆｏ（ＰＳⅡ活性）、ｑＰ（光化学淬灭）、ｑＮ（非光化学淬灭、ΦＰＳⅡ（有效ＰＳⅡ光化学效率））和光合速率。结果表明在“黑农４１”     

叶绿体腺三磷酶中色氨酸残基的修饰与性质的研究

利用Ｎ－溴代琥珀酰亚胺化学修饰叶绿体腺三磷酶,测得该该酶含３．５个色氨酸残基,其中２个色氨酸残基与酶活性有关。光氧化腺三磷酶伴随吸氧量增加,酶活性逐渐降低,经ＮＢＳ修饰或光氧化的腺三磷酶,其免疫抗原减弱。表明腺三磷酶中色氨酸基参与酶的活性和抗原性。     

白茯苓凝集素的荧光光谱研究

白茯苓凝集素（ＳＬＬ）分子中含有４个色氨酸（Ｔｒｐ）残基,ＮＢＳ修饰测得这４个Ｔｒｐ残基位于分子表面。ＳＬＬ在天然状态下荧光发射峰位于３３５ｎｍ处,离子强度和温度对其荧光光谱均无明显的影响。ＮＢＳ修饰后的ＳＬＬ失去凝血活性,相应荧光光谱的强度减弱,荧光发射峰发生蓝移,提示ＳＬＬ的构象发生改变。用ＫＩ·ＣｓＣｌ和丙烯酰胺淬灭剂研究ＳＬＬ分子中Ｔｒｐ残基的微环境,发现丙烯酰胺和ＣｓＣｌ能淬灭分子中１００％和５０％的Ｔｒｐ残基的荧光,而ＫＩ完全不能淬灭ＳＬＬ分子中Ｔｒｐ残基的荧光,因此Ｔｒｐ残基周围存在阴离子区,或者Ｔｒｐ残基处于分子表面的疏水环境中。     

红花菜豆凝集素的荧光光谱学研究

利用荧光光谱方法研究了红花菜豆凝集素（Ｐｈａｓｅｏｌｕｓｃｏｃｃｉｎｅｕｓｖａｒ．ｒｕｂｒｏｎａｎｕｓｌｅｃｔｉｎ,简称ＰＣＬ）,结果表明ＰＣＬ分子各亚基中的两个色氨酸（Ｔｒｐ）残基分别位于ＰＣＬ分子表面和分子内。标记了ＤＮＳ的ＰＣＬ荧光偏振研究指出,致使ＰＣＬ在１０ｍｍｏｌ／ＬＳＤＳ条件下失活的主要原因可能是亚基解离。荧光偏振研究还表明,甲状腺球蛋白、甘露聚糖、海参多糖硫酸酯可与ＰＣＬ结合。荧光探针ｂｉｓ－ＡＮＳ与ＰＣＬ的结合可引起明显的荧光增强和发射谱蓝移,表明ＰＣＬ分子中存有疏水区域。结合了的ｂｉｓ－ＡＮＳ还可和ＰＣＬ中的Ｔｒｐ发生能量传递。     

钙离子对江浙蝮蛇蛇毒中性磷脂酶A2溶液构象的研究

用荧光光谱方法研究了钙离子对江浙蝮蛇毒中性磷脂酶Ａ２（简称ＮＰＬＡ２）构象的影响。结果表明,Ｃａ２＋能使酶中唯一的色氨酸残基的荧光增强：只有在Ｃａ２＋存在时,底物卵磷脂才明显改变酶分子中Ｔｒｐ周围的环境,使其光谱的兰移达７ｎｍ,荧光增强约一倍：酶中唯一的Ｈｉｓ残基被修饰以后,则没有上述两种现象发生;结合在ＮＰＬＡ２上的ｂｉｓＡＮＳ的荧光强度,随Ｃａ２＋浓度的增加而增强,提示Ｃａ２＋对ｂｉｓ－ＡＮＳ结合区域的构象有明显影响。     

极大螺旋藻多糖IPⅡA、IPⅡB的理化特性及抗肿瘤活性研究

极大螺旋藻（Ｓｐｉｒｕｌｉｎａｍａｘｉｍａ）经碱性热水抽提,酶解和Ｓｅｖａｇ法结合脱蛋白,醇沉淀得胞内多糖ＩＰⅠ．ＩＰⅠ经ＤＥ－５２和ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－２５柱层析得到组分多糖ＩＰⅡＡ和ＩＰⅡＢ,糖含量分别为７４．９％和６８．４％,ＩＰⅡＡ分子量为１．３×１０６,ＩＰⅡＢ分子量为５．０×１０５．以完全酸水解、薄层层析、气相层析、红外光谱、核磁共振氢谱对二者进行化学结构分析,证明均为酸性杂多糖;ＩＰⅡＡ糖苷键为α型,ＩＰⅡＢ中同时存在α和β型糖苷键．离体条件下,ＩＰⅠ与血癌细胞ＨＬ６０和Ｕ９３７分别共同培养,半固体琼脂培养法检测,结果表明,ＩＰⅠ对二者的作用具有选择性,同时ＩＰⅠ的促进和抑制作用均表现出一般药物所具有的剂量效应．     

The brown-rot basidiomycete Fomitopsis palustris has the endo-glucanases capable of degrading microcrystalline cellulose

Two endoglucanases with processive cellulase activities, produced from Fomitopsis palustris grown on 2% microcrystalline cellulose (Avicel), were purified to homogeneity by anion-exchange and gel filtration column chromatography systems. SDS-PAGE analysis indicated that the molecular masses of the purified enzymes were 47 kDa and 35 kDa, respectively. The amino acid sequence analysis of the 47-kDa protein (EG47) showed a sequence similarity with fungal glycoside hydrolase family 5 endoglucanase from the white-rot fungus Phanerochaete chrysosporium. N-terminal and internal amino acid sequences of the 35-kDa protein (EG35), however, had no homology with any other glycosylhydrolases, although the enzyme had high specific activity against carboxymethyl cellulose, which is a typical substrate for endoglucanases. The initial rate of Avicel hydrolysis by EG35 was relatively fast for 48 h, and the amount of soluble reducing sugar released after 96 h was 100 microg/ml. Although EG47 also hydrolyzed Avicel, the hydrolysis rate was lower than that of EG35. Thin layer chromatography analysis of the hydrolysis products released from Avicel indicated that the main product was cellobiose, suggesting that the brown-rot fungus possesses processive EGs capable of degrading crystalline cellulose.     

Enzymatic degradation of carboxymethyl cellulose hydrolyzed by the endoglucanases Cel5A, Cel7B, and Cel45A from Humicola insolens and Cel7B, Cel12A and Cel45Acore from Trichoderma reesei.

Enzymatic hydrolysis of carboxymethyl cellulose (CMC) has been studied with purified endoglucanases Hi Cel5A (EG II), Hi Cel7B (EG I), and Hi Cel45A (EG V) from Humicola insolens, and Tr Cel7B (EG I), Tr Cel12A (EG III), and Tr Cel45Acore (EG V) from Trichoderma reesei. The CMC, with a degree of substitution (DS) of 0.7, was hydrolyzed with a single enzyme until no further hydrolysis was observed. The hydrolysates were analyzed for production of substituted and non-substituted oligosaccharides with size exclusion chromatography (SEC) and with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS). Production of reducing ends and of nonsubstituted oligosaccharides was determined as well. The two most effective endoglucanases for CMC hydrolysis were Hi Cel5A and Tr Cel7B. These enzymes degraded CMC to lower molar mass fragments compared with the other endoglucanases. The products had the highest DS determined by MALDI-TOF-MS. Thus, Hi Cel5A and Tr Cel7B were less inhibited by the substituents than the other endoglucanases. The endoglucanase with clearly the lowest activity on CMC was Tr Cel45Acore. It produced less than half of the amount of reducing ends compared to Tr Cel7B; furthermore, the products had significantly lower DS. By MALDI-TOF-MS, oligosaccharides with different degree of polymerization (DP) and with different number of substituents could be separated and identified. The average oligosaccharide DS as function of DP could be measured for each enzyme after hydrolysis. The combination of techniques for analysis of product formation gave information on average length of unsubstituted blocks of CMC.     

杂色云芝组成型漆酶Ⅰ的纯化和底物专一性

采用合成培养基培养杂色云芝As5 4 8,从发酵液中纯化出一种组成型漆酶同功酶Ⅰ .经超滤浓缩 ,DEAE SephadexA 5 0离子交换层析 ,Bio gelP 10 0凝胶过滤纯化了该酶 .SDS PAGE分析发现 ,该酶分子量为 6 8kD ,薄层等电聚焦测得等电点为 3 5 .漆酶Ⅰ的底物范围较宽 ,以O2 为电子受体 ,可以氧化多种木素单体模型物 ,包括 2 ,6 二甲氧基酚 ,2 ,2′ 联氮 二 (3 乙基 苯并噻唑 6 磺酸 )(ABTS) ,愈创木酚 ,咖啡酸 ,阿魏酸和邻联茴香胺 .结果表明 ,该酶在木质素的生物降解中可能有重要的作用和应用价值 .     

粗毛栓菌木聚糖酶的纯化及性质

以麦草粉为基质培养粗毛栓菌Trametes gallica,浸提固态培养物得浸提液,后经超滤浓缩、硫酸铵盐析、Phenyl-Sepharose CL-4B疏水层析、DEAE Sepharose fast flow阴离子交换层析和Sephadex G-150分子筛层析等分离与纯化步骤,获得部分纯化的木聚糖酶,其回收率和纯化倍数分别为1.45%和15.6。进一步经活性-PAGE回收,获得三种SDS-PAGE电泳纯级的木聚糖酶同工酶组分:XⅠ、XⅡ和XⅢ(按等电点从大到小排列)。三种组分分子量均约为19.0kDa;等电点分别为:5.6、4.7和4.0;含糖量分别为:0.25%、0.63%和3.4%;XⅠ既能降解木聚糖,又能降解纤维素;XⅡ的最适作用pH值为5.0,最适作用温度45℃;Mg2+、Fe2+对XⅡ有激活作用;Mn2+和Co2+有抑制作用;测得XⅡ的Km值为0.75mg/mL,Vmax为5,000mmoL/min·mg。     

Cellulase Complex of the Fungus Chrysosporium lucknowense: Isolation and Characterization of Endoglucanases and Cellobiohydrolases

Using different chromatographic techniques, eight cellulolytic enzymes were isolated from the culture broth of a mutant strain of Chrysosporium lucknowense: six endoglucanases (EG: 25 kD, pI 4.0; 28 kD, pI 5.7; 44 kD, pI 6.0; 47 kD, pI 5.7; 51 kD, pI 4.8; 60 kD, pI 3.7) and two cellobiohydrolases (CBH I, 65 kD, pI 4.5; CBH II, 42 kD, pI 4.2). Some of the isolated cellulases were classified into known families of glycoside hydrolases: Cel6A (CBH II), Cel7A (CBH I), Cel12A (EG28), Cel45A (EG25). It was shown that EG44 and EG51 are two different forms of one enzyme. EG44 seems to be a catalytic module of an intact EG51 without a cellulose-binding module. All the enzymes had pH optimum of activity in the acidic range (at pH 4.5-6.0), whereas EG25 and EG47 retained 55-60% of the maximum activity at pH 8.5. Substrate specificity of the purified cellulases against carboxymethylcellulose (CMC), beta-glucan, Avicel, xylan, xyloglucan, laminarin, and p-nitrophenyl-beta-D-cellobioside was studied. EG44 and EG51 were characterized by the highest CMCase activity (59 and 52 U/mg protein). EG28 had the lowest CMCase activity (11 U/mg) amongst the endoglucanases; however, this enzyme displayed the highest activity against beta-glucan (125 U/mg). Only EG51 and CBH I were characterized by high adsorption ability on Avicel cellulose (98-99%). Kinetics of Avicel hydrolysis by the isolated cellulases in the presence of purified beta-glucosidase from Aspergillus japonicus was studied. The hydrolytic efficiency of cellulases (estimated as glucose yield after a 7-day reaction) decreased in the following order: CBH I, EG60, CBH II, EG51, EG47, EG25, EG28, EG44.     

五步蛇蛇毒的分离纯化及综合利用

五步蛇蛇毒冻干粉经过SephadexG-75分子筛层析,使纤溶酶和类凝血酶初步分离;DEAE阴离子交换层析对2种酶进一步分离纯化,分别得到了纤溶酶和类凝血酶。2种酶在HPLC图谱上均呈单一峰,在SDS-PAGE图谱上均为单一条带,纤溶酶分子量大约为24．1kDa,类凝血酶分子量大约为14．4kDa,与以往报道相符。酶的总活力回收率大大提高,纤溶酶的活力回收率达23．9％,类凝血酶的活力回收率达34．5％。实现了对蛇毒的综合利用,为进一步开发利用蛇毒探索了一条有效的途径。     

苦荞种子胰蛋白酶抑制剂的分离纯化及部分性质研究

采用凝胶层析及离子交换层析等方法,从苦荞种子中分离出一组胰蛋白酶抑制剂（ＴＢＴＩ－Ⅰ、Ⅱ）．对其性质研究表明：两个组分均对胰蛋白酶有较强的抑制作用,对胰凝乳蛋白酶抑制作用较弱,其中ＴＢＴＩ－Ⅱ的抑制作用大于ＴＢＴＩ－Ⅰ,两者对胃蛋白酶、木瓜蛋白酶及枯草杆菌蛋白酶均无抑制作用．用ＳＤＳ－聚丙烯酰胺凝胶电泳和ＳｅｐｈａｄｅｘＧ－１００凝胶层析分别对纯化产物进行分析得出ＴＢＴＩ－Ⅰ和ＴＢＴＩ－Ⅱ的近似分子量分别为１５．０ｋＤ和１８．０ｋＤ．ＴＢＴＩ－Ⅰ、Ⅱ都具有较高的热稳定性,在１００℃处理１０ｍｉｎ后可保留８６％左右的抑制活性．ＴＢＴＩ在酸性环境下较为稳定,在ｐＨ２．０条件下保温１ｈ,仍保留７５％的抑制活性．用Ｌｉｎｅｖｅａｅｒ－Ｂｕｒｋ作图法得知,该抑制剂属竞争性抑制类型,ＴＢＴＩ－Ⅱ的Ｋｉ值为３．５９×１０－７ｍｏｌ／Ｌ（以ＢＡＰＮＡ为底物）,对胰蛋白酶的摩尔抑制比为１∶１．４．     

β-1,4-endoglucanases from Talaromyces amestolkiae: Production of glucooligosaccharides from different β-glucans

Abstract

This article presents the purification and characterization of two β-1,4-endoglucanases from Talaromyces amestolkiae. The cellulase activities secreted by this fungus were studied in the presence of different carbon sources, attaining the maximal levels in the presence of Avicel as carbon source. In these conditions, two glycosylated β-1,4-endoglucanases with molecular masses of 25,573?kDa (EG1) and 51,825?kDa (EG2), were purified. Both isoenzymes have acidic isoelectric points, 5.4 and 4.6, respectively. Their optimum pH and temperature, either in crudes or after purification, were in the range normally used for the simultaneous saccharification and fermentation in bioethanol production. In addition, the enzymatic hydrolysis of different β-glucans by both enzymes was studied. In the assayed conditions, both enzymes hydrolysed carboxymethylcellulose, a typical substrate for endoglucanases, although EG2 was much more efficient. However, EG1 was also able to hydrolyse lichenan and laminarin. These findings suggest the potential interest of EG2 for specific hydrolysis of cellulose, present in plant cell walls, to produce bioethanol, while the more promiscuous enzyme EG1 could be used for production of glucooligosaccharides.     

韭菜叶绿体超氧化物歧化酶纯化及性质研究

经硫酸铵沉淀、ＳｅｐｈａｄｅｘＧ－２００凝胶过滤和ＤＥＡＥ－Ｓｅｐｈａｃｅｌ层析３个步骤将韭菜叶绿体ＳＯＤ纯化到均一程度。鉴定该酶是Ｃｕ．Ｚｎ－ＳＯＤ,测得其分子量约３２０００Ｄ,亚基分子量约为１６２００Ｄ,Ｎ－末端氨基酸为Ａｌａ。该酶在紫外与可见光区的吸收峰分别在２６５ｎｍ和６７５ｎｍ。实验表明该酶热稳定性良好。