膜分离法纯化大蒜超氧化物歧化酶的条件研究

本文探讨了膜分离技术分离纯化大蒜超氧化物歧化酶(SOD )的工艺条件,研究了中空纤维超滤膜分离提纯大蒜SOD的工艺参数.通过单因素实验,分析了温度、压力、透过率对SOD活力回收率的影响;并通过正交实验确定出超滤膜分离法的最佳条件:温度32 ℃,压力0.15 MPa,透过率90%.在此基础上研究了纳滤膜对超滤液进行浓缩纯化的工艺条件,适宜的纳滤条件为:温度32 ℃,压力1.4 MPa.     

麦芽根中5′-磷酸二酯酶和5′-磷酸单酯酶的提取

建立一种高效快速的从麦芽根中提取5′-磷酸二酯酶和磷酸单酯酶的方法。通过将麦芽根粉碎后,取一定粒度的麦芽根加一定量的去离子水浸泡,过滤后得粗酶液。用紫外分光光度法测定5′-磷酸二酯酶和5′-磷酸单酯酶的酶活,研究粉碎粒度、料液比、抽提温度、抽提时间等因素对麦芽根中5′-磷酸二酯酶和5′-磷酸单酯酶提取的影响。将原料粉碎至100目,以去离子水为提取剂,于4℃提取20h,料液质量比为1:10,5′-磷酸二酯酶的酶活力可达160U/ml。该法简单、快速、准确、适应性强,为5′-磷酸二酯酶的提取与检测提供了有效的工具。     

饲用复合酶的浸提条件及稳定性

研究了黑曲霉Aspergillus niger 238发酵曲的最佳浸提条件。结果表明以5倍2％CaCl2溶液,于30℃,120r/min条件下浸提1．5h为佳。通过浓缩得到了浓缩酶液,对浓缩酶液的稳定性进行了初步研究。     

米黑毛霉(M.miehei)天冬氨酸蛋白酶的研究

用硫酸铵分部盐析及离子交换层析技术从米黑毛霉半固体培养物的浸提液中提纯了天冬氨酸蛋白酶,酶活力收率为19.5％,,比活力达3080SU/mg蛋白,提纯8.5倍。用聚丙烯酰胺凝胶电泳、SDS-凝胶电泳、等电聚焦、双向免疫扩散及免疫电泳等方法鉴定该酶均一。本文还报道了关于该酶生化性质、动力学性质及化学修饰的研究结果。该酶以天冬氨酸为其活性的必需基团,系一典型的天冬氨酸蛋白酶。     

木霉β-1,3-葡聚糖酶的分离纯化

目的:对木霉菌株LE02所产β-1,3-葡聚糖酶的分离纯化方法进行研究。方法:粗酶液分别用硫酸铵、乙醇和丙酮进行沉淀,再用DEAE-Sepharose CL-6B离子交换层析进一步分离纯化,并用SDS-PAGE法测其分子量。结果:硫酸铵分段盐析法沉淀酶蛋白的效果优于乙醇和丙酮沉淀;盐析得到的酶蛋白经透析浓缩后,再经DEAE-Sepharose CL-6B层析分离,可得到单一酶蛋白,总酶活回收率达78.71%,比酶活达到689.9U/mg,提高了53.74倍,经SDS-PAGE法测得该β-1,3-葡聚糖酶的分子量为80.137kDa。结论:采用硫酸铵分段盐析和离子交换层析法可获得电泳纯的β-1,3-葡聚糖酶,且酶活回收率高。     

黑曲霉纤维素酶的纯化及酶学性质研究

黑曲霉(Aspergillusniger)固态发酵后粗酶液经硫酸铵盐析,2次SephadexG-200柱层析后可提纯8倍左右.CMC酶最适作用温度为60℃,最适作用pH为3.5,30℃～70℃区间酶活力较稳定,在pH3.0～5.0范围内,50℃保温30min能保持80%的酶活力.CMC酶的Km、Vmax值分别为7.69%CMCg/ml、0.33mg/ml·     

微小毛霉凝乳酶的分离纯化研究

研究微小毛霉(HL-1)凝乳酶的分离纯化条件及方法。研究酶的最适浸提温度、酶的浸提pH值和最适浸提时间,探讨离子浓度、加水量对浸提效率的影响,利用高速冷冻离心法、有机溶剂沉淀法,膜分离法和层析法等对粗酶液进行了分离。利用光谱法对纯化样品进行检测。酶的最适浸提温度为30℃;最适pH为6.0;浸提10 h活力最高;1%的氯化钠有利于酶的分离,加水比例为15时有利于提取,在10 000 r/min下离心10min澄清效果最好,95%的酒精沉淀效果最好,利用0.2μm的微滤膜可除去发酵液中的菌体,8 000的超滤膜可拦截凝乳酶蛋白,S300的填料可有效分离凝乳酶,纯度达95%以上。     

麦芽根中5'-磷酸二酯酶和5'-磷酸单酯酶的提取

建立一种高效快速的从麦芽根中提取5'-磷酸二酯酶和磷酸单酯酶的方法.通过将麦芽根粉碎后,取一定粒度的麦芽根加一定量的去离子水浸泡,过滤后得粗酶液.用紫外分光光度法测定5'-磷酸二酯酶和5'-磷酸单酯酶的酶活,研究粉碎粒度、料液比、抽提温度、抽提时间等因素对麦芽根中5'-磷酸二酯酶和5'-磷酸单酯酶提取的影响.将原料粉碎至100目,以去离子水为提取剂,于4℃提取20h,料液质量比为1:10,5'-磷酸二酯酶的酶活力可达160U/ml.该法简单、快速、准确、适应性强,为5'-磷酸二酯酶的提取与检测提供了有效的工具.     

正交实验法优化黄柏中黄连素的酶碱法提取工艺

为了提高黄柏中黄连素的提取得率与效率,采用传统方法碱提法与酶解辅助提取相结合来优化黄柏中黄连素的提取工艺。以黄连素浸提率为评价指标,考察纤维素酶浓度、酶解时间、料液比及浸提时间对提取效率的影响。在单因素实验的基础上,通过正交实验分析各因素的作用大小以及它们之间的交互作用。优选出黄柏中黄连素的酶碱法提取最佳工艺条件为:纤维素酶浓度15‰、酶解时间1 h、料液比1∶7、浸提时间7 h,在该条件下黄连素的浸提率可达8.99%。该提取工艺浸提率高,成本低,稳定性好,可用于生产上黄柏中黄连素的提取。     

固定化5′-磷酸二酯酶及其在核苷酸生产中的应用

钛氯活化纤维素固定5’-磷酸二酯酶,使酶活回收率达到70%以上。用这种固定化酶在75℃、pH5.2的条件下降解0.5%浓度的酵母核酸(RNA),用酶量可减少到1.75u/ml底物,酶活利用率可提高6倍以上。核酸降解率可达70%以上。     

用试管-玻片培养技术研究铵对木麻黄根瘤形成的影响

用植物试管玻片培养技术研究NH_4~ 对细枝木麻黄及Frankia菌株Co01共生体系建立过程的影响。NH_4~ (100,150ppm(NH_4)_2SO_4)通过阻止菌株Cc01与其宿主细枝木麻黄根毛壁的亲和作用来影响结瘤。但NH_4~ 不能阻抑菌株Cc01中结瘤基因pel和cel的表达及纤维素酶和果胶酶活性,且菌丝一旦侵入宿主皮层细胞,并形成根瘤原基及前根瘤,则NH_4~ (250ppm(NH_4)_2SO_4)就不再阻止原基进一步发育为成熟的根瘤。但在这种情况下,NH_4~ 能抑制根瘤的固氮活性。     

长梗木霉纤维素酶的产生及提取

对长梗木霉(Trichoderma Longibrachiatum)ANU_3-958纤维素酶的产生及提取进行了研究。结果表明,固体曲培养144小时,固体曲与浸提液比例为1:7(W/V),采用硫酸铵分级沉淀法时所得纤维素酶活力最高。所得冻干纤维素酶粉经测定:羧甲基纤维素(CMC)酶活最高为2788.89IU/g,滤纸糖酶活(FPA)最高为79.44IU/g。相对固体曲得率平均为13.28%。     

Repetitive recycling of guanosine triphosphate cyclohydrolase I for synthesis of dihydroneopterin triphosphate

A procedure for enzymatic production of dihydroneopterin triphosphate is described that allows GTP cyclohydrolase I to be reused repetitively. The reaction takes place in an ultrafiltration cell, and the product is collected in the filtrate, whereas the enzyme remains in the cell to be reused with additional substrate. This is repeated until the enzyme activity drops below a desirable level. The purity of the dihydroneopterin triphosphate is satisfactory for utilization of this compound for studies on enzymes involved in the synthesis of tetrahydrobiopterin and drosopterin. A procedure for purification of dihydroneopterin triphosphate is described that uses C18-silica and silica cartridges.     

蔗糖对不同氮源培养下水稻根部氨同化相关酶活性的影响

糖、有机酸以及氨基酸影响碳-氮代谢过程中的相关酶的基因表达和活性。将蔗糖分别加入到含有相同氮素浓度的（NH4）2SO4（NH4^＋）或丙氨酸（Ala）作为氮源的营养液中培养水稻,测定幼苗根的谷氨酰胺合成酶（GS）、依赖于NADH的谷氨酸合酶（NADH-GOGAT）、磷酸烯醇式丙酮酸羧化酶（PEPC）和依赖于NADP的异柠檬酸脱氢酶（NADP-ICDH）的活性。结果显示,蔗糖诱导NH4^＋氮源中幼苗根的GS、NADH-GOGAT活性,抑制Ala氮源中幼苗根的这两种酶活性,蔗糖对PEPC和NADP-ICDH活性的影响也不同;未加蔗糖时,以Ala作为氮源的幼苗根的GS、NADH-GOGAT、PEPC和NADP-ICDH的活性明显高于以NH4^＋为氮源时的活性;生物量和蛋白质水平的变化与上述参数的变化基本一致。基于Ala碳骨架的存在,这些结果表明,碳／氮平衡是影响这些酶活性差别表达的主要原因。     

鼠肝DNA甲基化酶的纯化及物理化学性质的研究

以Ｗｉｓｔａｒ大鼠肝为材料,确立了一个简便的纯化鼠肝ＤＮＡ甲基化酶的程序,包括：细胞的超声破碎、去内源核酸、硫酸铵盐析、磷酸纤维素亲和层析、ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０柱层析及ＳｅｐｈａｄｅｘＧ－１５０凝胶过滤。用不同浓度聚丙烯酰胺凝胶电泳和孔梯度凝胶电泳检测,纯化后的酶已达电泳均一,且酶的比活力提高１１２倍。以聚丙烯酰胺孔梯度凝胶电泳测得其天然酶的分子量为３６５ｋＤ,以ＳＤＳ－聚丙烯酰胺凝胶电泳测得该酶有两种亚基,大亚基为９５ｋＤ,小亚基为８５ｋＤ,推测该酶由两个大亚基和两个小亚基组成。     

Enzyme recovery in high-solids enzymatic hydrolysis of steam-pretreated willow: Requirements for the enzyme composition

In order to reduce the total enzyme consumption in high-solids static hydrolysis of nonwashed steam-exploded willowSalix caprea by mixed cellulase ofTrichoderma reesei + Aspergillus foetidus, two different approaches were proposed. In the first case, the enzyme activity adsorbed on residual solids after extended hydrolysis was used for hydrolysis of the newly added substrate. The initial mixing of fresh and hydrolyzed substrates was sufficient for the adsorbed enzyme redistribution and conversion of the new substrate portion, and constant mechanical stirring was not required. Feeding of two additional portions of the exploded hardwood adjusted to pH 4 with dry caustic into the reactor with simultaneous replacement of accumulated sugars with fresh buffer (pH 4.5) resulted, on average, in a 90% conversion of cellulose at the final enzyme loading of 8 IFPU per g ODM substrate, an average sugar concentration of 12%, and a glucose/xylose ratio of 5 : 1. In the second approach, weakly adsorbed cellulase fractions were used for static high-solids hydrolysis followed by their ultrafiltration recovery from the resultant sugar syrup. In contrast to the initial cellulase mixture whose residual activity in a syrup did not exceed 5–10% at the end of hydrolysis (48 h), up to 60% of weakly adsorbed enzyme fraction could be separated from sugar syrups by ultrafiltration and then reused. Weakly adsorbed enzymes displayed a hydrolysis efficiency of not less than 80% per IFPU enzyme consumed in extended hydrolysis of pretreated willow as compared to the original enzyme mixture. An electrophoretic study of the weakly adsorbed enzyme fraction identifiedT. reesei cellobiohydrolase II as the predominant component, whereas clear domination ofT. reesei cellobiohydrolase I was found by electrophoresis of proteins tightly bound to residual hydrolysis solids. Deceased     

Improved purification of α-amylase isolated fromRhizomucor pusillus by affinity chromatography

A highly purified extracellular -amylase was isolated fromRhizomucor pusillus with minimum loss of enzymatic activity. The enzyme was purified from the mycelium-free liquid filtrate of the thermophilic moldRhizomucor pusillus. Maximum enzyme yields were attained after 5 days of growth on liquid starch-yeast extract at 45°C and pH 7.0. The crude enzyme preparation was first concentrated 80-fold by ultrafiltration. Purification was recently achieved with high-performance liquid chromatography and Waters Protein Pak 300 SW. Improved purification was then achieved with a dextrin-bound affinity column, with a 59-fold increase in specific activity from the crude enzyme preparation. This final enzyme preparation produced a single band on polyacrylamide gel electrophoresis. The molecular weight determined by SDS gel electrophoresis was 52,000 daltons.     

双水相萃取技术在白藜芦醇提纯工艺中的应用

论文研究了双水相萃取体系在提纯白藜芦醇工艺中的应用,用乙醇-硫酸胺-水双水相体系使虎杖提取液中的各物质按极性不同在油水两相中得到分离。双水相萃取时,白藜芦醇的含量远高于有机溶剂萃取,达34.29%。可见双水相技术可以完全代替有机溶剂萃取技术提纯白藜芦醇,产品纯度高,具有工艺简单,毒性小,成本低等优点。     

Enzyme regeneration during hydrolysis of steam-pretreated willow and requirement for cellulase complex composition

In order to reduce the total enzyme consumption in high-solids static hydrolysis of nonwashed steam-exploded willow Salix caprea by mixed cellulase of Trichoderma reesei + Aspergillus foetidus, two different approaches were proposed. In the first case, the enzyme activity adsorbed on residual solids after extended hydrolysis was used for hydrolysis of the newly added substrate. The initial mixing of fresh and hydrolyzed substrates was sufficient for the adsorbed enzyme redistribution and conversion of the new substrate portion, and permanent mechanical stirring was not required. Feeding of two additional portions of the exploded hardwood adjusted to pH 4 with dry caustic into the reactor with simultaneous replacement of accumulated sugars with fresh buffer (pH 4.5) resulted, on average, in a 90% conversion of cellulose at the final enzyme loading 8 IFPU per g ODM substrate, an average sugar concentration of 12%, and a glucose/xylose ratio of 5:1. In the second approach, weakly adsorbed cellulase fractions were used for static high-solids hydrolysis followed by their ultrafiltration recovery from the resultant sugar syrup. In contrast to the initial cellulase mixture whose residual activity in a syrup did not exceed 5-10% at the end of hydrolysis (48 h), up to 60% of weakly adsorbed enzyme fraction could be separated from sugar syrups by ultrafiltration and then reused. Weakly adsorbed enzymes displayed a hydrolysis efficiency of not less than 80% per IFPU enzyme consumed in extended hydrolysis of pretreated willow as compared to the original enzyme mixture. An electrophoretic study of the weakly adsorbed enzyme fraction identified T. reesei cellobiohydrolase II as the predominant component, whereas clear domination of T. reesei cellobiohydrolase I was found by electrophoresis of proteins tightly bound to hydrolysis residual solids.     

IL-1018-57-PE40高效表达、纯化及细胞活性之研究

以IL-10的功能短肽(40肽,即IL-10第18号至57号氨基酸)为导向部分与PE40(绿脓杆菌外毒素除去受体结合区后的剩余部分)融合分别构建了IL-101857PE40的胞质和胞周质表达质粒,其中,IL101857PE40在Rosettablue(DE3)中以高效胞质可溶形式表达,在BL21(DE3)pLysS中以胞周质分泌形式表达;表达宿主菌Rosettablue(DE3)超声波破碎后,依次通过硫酸铵盐析、疏水层析、铜离子亲和层析、阴离子交换层析纯化后,得96%重组毒素纯品;细胞活性实验、细胞ELISA和荧光标记实验表明,构建的IL101857PE40符合免疫毒素的作用机理。因此,该实验为PE免疫毒素的规模制备和纯化做了一定的有益的探索。