小球藻病毒PBCV-1特异性溶壁酶(Lysin)的溶壁活性

从PBCV\|1感染小球藻NC64A的细胞裂解液中提取了Lysin的粗制剂,酶活底物范围分析表明,几丁质酶、壳聚糖酶和β\|1,3\|葡萄糖苷酶是Lysin活性的主要组成部分,并与小球藻细胞壁的组成成分相吻合。其中几丁酯酶和壳聚糖酶,特别是几丁酯酶在裂解小球藻细胞壁的过程中发挥了重要的作用。Lysin粗制剂经FPLC分离纯化得到分子量分别为52kD、56kD的两个几丁质酶(Chil和Chi2)和一个分子量为36kD的壳聚糖酶。     

噬菌体溶壁酶研究进展

溶壁酶是噬菌体在感染末期表达的蛋白质,可水解细菌的细胞壁,使子代噬菌体释放出来。研究表明,溶壁酶在体外能高效地杀死细菌,同样对感染细菌的模型动物有很好的治疗作用。因此,溶壁酶是一种新型的抗菌物质,具有广阔的应用前景。溶壁酶通过水解细菌细胞壁肽聚糖上糖与肽间的酰胺键或肽内氨基酸残基间的连键,从而使细菌裂解。溶壁酶分子由结合功能域和催化功能域两部分组成,其晶体结构使之具有对细胞壁肽聚糖水解的高效性和特异性。对噬菌体溶壁酶的体内外抗菌作用、抗菌机理、晶体结构等最新研究成果及其应用前景进行了综述。     

真菌细胞溶壁酶的产酶培养和应用效果

从土样中分离的７１株木霉（Ｔｒｉｃｈｏｄｅｒｍａ ｓｐ．）筛选出一株分解几丁质和葡聚糖能力较强的菌株。该菌株在麸皮、稻草粉、硫酸铵和诱导物为主成份的固体培养基上,经过２５℃、８４ｈ的培养,产生较高的真菌细胞溶壁酶,能溶解酵母、毛霉、黑曲霉及食用菌等丝状菌丝体的细胞壁,形成原生质体,经选用酵母、毛霉等进行原生质体再生,效果较优。     

Flavobacterium johnsoniae昆布多糖酶的分离纯化及其催化性质

约氏黄杆菌Flavobacterium johnsoniae具有分泌裂解酵母细胞壁酶系的能力,经初步分析发现其发酵液中具有葡聚糖酶、几丁质酶和蛋白酶等活性。通过离子交换层析、疏水层析和凝胶过滤层析,从该菌发酵液中分离纯化到一种昆布多糖酶。该酶分子量为35 kD左右,其最适反应温度为50°C,最适反应pH为5.0。以昆布多糖和昆布寡糖为底物的反应表明,该酶以内切酶作用模式进行催化水解。     

小黑麦抗真菌蛋白组分的分离纯化和性质研究

以木霉为指示菌,小黑麦中饲237种子中的蛋白提取物经过分离纯化后,得到了3种主要的抗真菌蛋白组分,经酶活检测鉴定,分别是分子量为30.5 kD的ClassⅡ型几丁质酶,两种分子量为51kD和23 kD的β-1,3-葡聚糖酶。其中几丁质酶的最适反应pH为6.0,最适反应温度为37℃,测定的N末端氨基酸序列与大麦几丁质酶的有很高的同源性。在一定条件下,这3种蛋白组分都有较强的抗木霉活性,并且有明显的协同作用,同时它们对离体易感小麦叶片上白粉菌有很好的生长抑制作用。     

噬菌体裂解酶Lysin1902原核表达及其与ε-聚赖氨酸联用效果评价北大核心

【目的】研究裂解酶Lysin1902与ε-聚赖氨酸(ε-PL)对大肠杆菌O157:H7的协同抗菌作用。【方法】通过Mega构建进化树、使用在线工具等分析大肠杆菌噬菌体裂解酶Lysin1902氨基酸序列组成和结构等;原核表达并纯化Lysin1902;通过平板裂解实验检测Lysin1902对大肠杆菌O157:H7灭活菌株的裂解活性;用96孔板法检测Lysin1902或ε-PL的活菌裂解能力;棋盘法验证Lysin1902和ε-PL联用效果。【结果】体外成功表达并纯化了Lysin1902。Lysin1902对大肠杆菌O157:H7灭活菌株具有裂解活性,但不能有效裂解活的大肠杆菌。噬菌体裂解酶与ε-PL联用结果表明,加入Lysin1902后,ε-PL能够完全控制大肠杆菌O157:H7增殖的使用浓度由0.7mg/mL降低到0.1 mg/mL。【结论】体外原核表达并纯化Lysin1902,其单独使用对活的大肠杆菌O157:H7无裂解活性,但与ε-PL联用可显著提高ε-PL对大肠杆菌O157:H7的裂解能力。     

猪血纤维蛋白溶酶原的纯化及性质研究

 用偶联有L-赖氨酸的Sepharose 4B亲和柱对猪血纤维蛋白溶酶原进行分离纯化,所得酶原在酸性及SDS-聚丙烯酰胺凝胶电泳中显示单一蛋白质区带,在碱性条件下电泳及等电聚焦电泳表现出较明显的不均一性。还原及非还原SDS-聚丙烯酰胺凝胶电泳测得酶原分子量为88kD,经尿激酶激活后,进行还原SDS-凝胶电泳,出现两条新的蛋白质区带,分子量分别为63kD和26kD。酶原含中性糖1.35％,N-末端为异亮氨酸。尿激酶激活后产生的血纤维蛋白溶酶以对甲苯磺酰-L-精氨酸甲酯为底物,测得Km为4.2m mol/L,V_(max)为13.5 IU。6-氨基已酸对酶活性有双重影响。此外,还观察了胰蛋白酶对猪血纤维蛋白溶酶原的激活。     

豇豆几丁质酶部分酶学特性的研究

该文测定了纯化的豇豆几丁质酶部分酶学特性。结果表明,该酶在pH5-8,温度低于60℃的范围内稳定性较好,酶活力最适pH为65,最适温度为50℃。10mmol/L浓度的Hg2 、Mn2 、Mg2 、Co2 等金属离子对酶活力有一定抑制作用,其中Hg2 离子抑制率最高(6883%)。Km(胶状几丁质)值为1662mg/ml;以SDS-PAGE电泳和SephadexG-100柱层析两种方法分别测得分子量为34kD、325kD;IEF电泳测得等电点为83。     

黏质沙雷氏菌L15-2几丁质酶的分离纯化与性质研究

几丁质酶高产菌株黏质沙雷氏菌L15-2在含有1%胶体几丁质、0.3%K2HPO4、0.3%KH2PO4、0.05%MgSO4、0.05?Cl2、0.001?SO4的产酶培养基中37℃培养4 d后,粗酶液经过DEAE Sepharose Fast Flow和Seph-adex G-100,电泳检测得到电泳纯的几丁质酶。该酶的性质特征测定结果表明,该几丁质酶的分子量为41.314 kD;最适作用温度为50℃,最适作用pH为6.6;在60℃之前热稳定性较好,在pH 4~10范围内较稳定;各种金属离子对酶活力影响不同,Fe2 、Zn2 抑制作用明显,而Ba2 、Mn2 、Ca2 对酶活性有一定的促进作用。几丁质酶对致病真菌的抑制作用也很明显。     

天麻球茎几丁质酶和β-1,3-葡聚糖酶的初步研究

天麻(Gastrodia elata)是真菌寄生植物。密环菌侵入初生球茎并在其皮层被消化,营养物供次生球茎生长需要;密环菌不能侵染生长小的次生球茎。我们从初生球茎分离并纯化了几丁质酶和β—1,3—葡聚糖酶,分子量各为31.5 kD和94kD,得率各为0.8和0.4 mg/100 g鲜重。纯化几丁质酶的内切酶比活为208 nmol GlcNAc s~(-1)mg~(-1),外切酶比活为4.1 nmol GlcNAcs~(-1)mg~(-1);纯化葡聚糖酶比活为546 nmol Glc s~(-1)mg~(-1)。以相同鲜重计,初生球茎中二种酶的总活性各为次生球茎的34和56倍;这主要是由于次生球茎的酶比活性很低。二种酶对平板上培养的木霉菌丝的生长均有抑制作用,但抑菌活性均较天麻抗真菌蛋白(GAFP)低。我们认为这两种酶在天麻初生球茎消化密环菌菌丝的过程中起重要作用,而对天麻球茎阻止和限制密环菌侵染的抗菌作用贡献甚少,后者主要属于天麻抗真菌蛋白。     

Quantitative fluorometric analysis of plant and microbial chitosanases.

A quantitative fluorometric assay for chitosanase activity in bacterial and plant tissues was developed. The assay can be conducted with either finely milled preparations of chitosan in suspension or dissolved chitosan; activity is based on measurements of glucosamine (GlcN) or oligomers of GlcN. GlcN is detected fluorometrically after reaction with fluorescamine with detection in the nanomole range. Fluorescence measurements of chitosanase activity and radioassay of chitinase in commercial preparations of chitinase from Streptomyces griseus revealed that both activities were present. Specific activities for the S. griseus chitosanase using suspended and soluble chitosans were respectively 1.24 and 6.4 mumol GlcN.min-1.mg protein-1. Specific activity of the S. griseus chitinase was 0.98 mumol GlcN.min-1.mg protein-1. Sweet orange callus tissue was tested for chitosanase and chitinase activity. It was necessary to remove small amine-containing molecules from the callus preparations before chitosanase activity could be assayed. The specific activity for chitinase and chitosanase in desalted extracts of nonembryogenic Valencia sweet orange callus tissue was determined to be 18.6 and 89.4 nmol GlcN.min-1.mg protein-1, respectively.     

Chitinolytic and chitosanolytic activities from crude cellulase extract produced by A. niger grown on apple pomace through Koji fermentation

Enzyme extracts of cellulase [filter paper cellulase (FPase) and carboxymethyl cellulase (CMCase)], chitinase, and chitosanase produced by Aspergillus niger NRRL-567 were evaluated. The interactive effects of initial moisture and different inducers for FP cellulase and CMCase production were optimized using response surface methodology. Higher enzyme activities [FPase 79.24+/- 4.22 IU/gram fermented substrate (gfs) and CMCase 124.04+/-7.78 IU/gfs] were achieved after 48 h fermentation in solid-state medium containing apple pomace supplemented with rice husk [1% (w/w)] under optimized conditions [pH 4.5, moisture 55% (v/w), and inducers veratryl alcohol (2 mM/kg), copper sulfate (1.5 mM/kg), and lactose 2% (w/w)] (p<0.05). Koji fermentation in trays was carried out and higher enzyme activities (FPase 96.67+/-4.18 IU/gfs and CMCase 146.50+/-11.92 IU/gfs) were achieved. The nonspecific chitinase and chitosanase activities of cellulase enzyme extract were analyzed using chitin and chitosan substrates with different physicochemical characteristics, such as degree of deacetylation, molecular weight, and viscosity. Higher chitinase and chitosanase activities of 70.28+/-3.34 IU/gfs and 60.18+/-3.82 to 64.20+/-4.12 IU/gfs, respectively, were achieved. Moreover, the enzyme was stable and retained 92-94% activity even after one month. Cellulase enzyme extract obtained from A. niger with chitinolytic and chitosanolytic activities could be potentially used for making low-molecular-weight chitin and chitosan oligomers, having promising applications in biomedicine, pharmaceuticals, food, and agricultural industries, and in biocontrol formulations.     

球孢白僵菌胞外壳聚糖酶的纯化和性质*

球孢白僵菌Beauveria bassiana 1316-V1的培养上清液经硫酸铵分级沉淀,Sephadex G-75凝胶过滤,Chitosan-bead亲和层析,第二次Sephadex G-75凝胶过滤, 得到电泳纯的一种胞外壳聚糖酶,比活力达到45u/mg 。此酶的分子量为36 kD; 最适酶反应温度为60℃;最适pH为4.0;最适离子强度为 0.25mol/L NaCl; 37℃以下,pH 2.0~5.0之间稳定性好; Cu2+、Hg2+、Pb2+、Ni2+ 对该酶有强烈抑制作用;Ag+、Mn2+也有较强抑制作用;Fe2+有轻微激活作用。该壳聚糖酶是一种糖蛋白,含糖约为12.6%。酶的最适底物为脱乙酰度为90%的胶体壳聚糖;也能轻微水解CMC、DEAE-Cellulose和胶体几丁质;但不能水解片状的壳聚糖和几丁质。     

Effect of chitin sources on production of chitinase and chitosanase byStreptomyces griseus HUT 6037

The advantage of usingStreptomyces griseus HUT 6037 in the production of chitinase or chitosanase is that the organism is capable of hydrolyzing amorphous or crystal-line chitin and chitosan according to the type of the substrate used. We investigated the effects of the enzyme induction time and chitin sources, CM-chitosan and deacetylated chitosan (degree of deacetylation 75–99%), on production of chitosanase. We found that this strain accumulated chitosanase when cells were grown in the culture medium containing chitosanaceous substrates instead of chitinaceous substrates. The highest chitosanase activity was obtained at 4 days of cultivation with 99% deacetylated chitosan. Soluble chitosan (53% deacetylated chitosan) was found to induce chitinase as well as chitosanase. The specific activities of chitinase and chitosanase were 0.91 and 1.33 U/mg protein at 3 and 5 days, respectively. From the study of the enzymatic digestibility of various degrees of deacetylated chitosan, it was found that (GlcN)₃, (GlcN)₄ and (GlcN)₅ were produced during the enzymatic hydrolysis reaction. The results of this study suggested that the sugar composition of (GlcN)₃ was homogeneous and those of (GlcN)₄ and (GlcN)₅ were heterogeneous.     

Effective production of chitinase and chitosanase byStreptomyces griseus HUT 6037 using colloidal chitin and various degrees of deacetylation of chitosan

The advantages of the organismStreptomyces griseus HUT 6037 is that the chitinase and chitosanase using chitinaceouse substrate are capable of hydrolyzing both amorphous and crystalline chitin and chitosan. We attempted to investigate the optimization of induction protocol for high-level production and secretion of chitosanase and the influence of chitin and partially deacetylated chitosan sources (75–99% deactylation). The maximum specific activity of chitinase has been found at 5 days cultivation with the 48 hours induction time using colloidal chitin as a carbon source. To investigate characteristic of chitosan activity according to substrate, we used chitosan with various degree of deacetylation as a carbon source and found that this strain accumulates chitosanase in the culture medium using chitosanaceous substrates rather than chitinaceous substrates. The highest chitosanase activity was also presented on 4 days with 99% deacetylated chitosan. The partially 53% deacetylated chitosan can secrete both chitinase and chitosanase which was defined as a soluble chitosan. The specific activities of chitinase and chitosanase were 0.89 at 3 days and 1.33 U/mg protein at 5 days, respectively. It indicate that chitosanase obtained fromS. griseus HUT 6037 can hydrolyze GlcNAc-GlcN and GlcN-GlcN linkages by exo-splitting manner. This activity increased with increasing degree of deacetylation of chitosan. It is the first attempt to investigate the effects of chitosanase on various degrees of deacetylations of chitosan byS. griseus HUT 6037. The highest specific activity of chitosanase was obtained with 99% deacetylated chitosan.     

萝卜块根中两个具溶菌酶活性的几丁质结合蛋白的纯化及其特性

通过亲和层析和羧甲基一纤维素离子交换层析从萝卜的块根中分离到两个具溶菌酶活性的酶组份：CBP1和CBP2。两者经SDS-PAGE均显示单一蛋白染色条带,其对应的分子量分别为26．9kD和24．8kD。两种蛋白除有溶菌酶活性外,还有几丁质酶活性,但无壳聚糖酶活性。各种类型的几丁质对CBP1和CBP2都有较强的吸附作用,而在还原／非还原的单向SDS-PAGE中却观察不到两者分子中存在二硫键。     

纤维素酶中具有壳聚糖水解酶活性成分的鉴定

在壳聚糖酶的研究过程中,目前已发现37种酶具有非专一性地降解壳聚糖的能力[1].对这些非专一性酶水解壳聚糖的机理有两种看法:一些人认为,由于这些酶大都来自商业酶制剂,未经过进一步的纯化,故有人认为其中所含的少量杂质可能是产生水解活力的原因;但也有人认为,在所有的酶制剂中都存在同一种杂质似乎是不可能的,因为这些酶来源于广泛的微生物、真菌、哺乳动物和植物等.众所周知,酶具有高度的专一性,即对所催化的反应和底物有严格的选择性,一种酶往往只能催化一种或一类反应;有如此多的不同种类的酶能非专一性地水解壳聚糖.因而探讨具有水解…     

Constitutive and induced resistance to pathogens in Arabidopsis thaliana depends on nitrogen supply

Knowledge about the induced pathogen resistance of plants is rapidly increasing, but little information exists on its dependence on abiotic growing conditions. Arabidopsis thaliana plants that had been cultivated under different nitrogen regimes were treated with BION^®, a chemical resistance elicitor. The activities of three enzyme classes functionally involved in resistance (chitinase, chitosanase and peroxidase) were quantified over 8 d following treatment as resistance markers. Constitutive levels of three markers and the induced level of peroxidase and chitinase activity were significantly lower under limiting nitrogen supply. Under such conditions the increase of chitosanase activity after resistance induction was severely delayed, although the induced maximum activity of chitosanase was not significantly affected. Total soluble protein content decreased during the first 12 h after resistance elicitation. Thereafter, the induced plants cultivated under high N conditions reached higher protein contents than controls, whereas N‐limited induced plants continuously had reduced protein contents. A plant's investment in resistance‐related compounds can be severely constrained under limiting nitrogen supply.     

几丁质酶和壳聚糖酶对部分乙酰化壳聚糖作用方式的比较

通过对几丁质酶和壳聚糖酶降解部分乙酰化壳聚糖的作用方式的比较,得到几丁质酶切断壳聚糖的ＧｌｃＮＡｃ－ ＧｌｃＮＡｃ 和ＧｌｃＮＡｃ－ ＧｌｃＮ 或ＧｌｃＮ－ ＧｌｃＮＡｃ 糖苷键,而壳聚糖酶切断壳聚糖的ＧｌｃＮ－ ＧｌｃＮ 和ＧｌｃＮ－ ＧｌｃＮＡｃ 或ＧｌｃＮＡｃ－ ＧｌｃＮ 糖苷键,为得到较高聚合度的壳寡糖提供理论基础。