纯化方式对麦麸酯酶农药检测效能的影响

为探讨麦麸酯酶应用于农残检测的可行性,采用固蓝B染色体系,以酶活抑制率为指标,研究了经Sephadex G-200凝胶过滤层析、双水相萃取、硫酸铵分段盐析3种方式纯化后的麦麸酯酶对6种农药的敏感性及最低检测限(LOD)。结果显示,经Sephadex G-200凝胶过滤层析、双水相萃取、硫酸铵分段盐析后的麦麸酯酶对6种农药的LOD范围分别是0.003～0.193 mg/L、0.005～0.116 mg/L和0.039～0.386 mg/L。其中,经凝胶过滤层析和双水相萃取后的麦麸酯酶对6种农药的敏感性相对于粗酶液均有不同程度的提高,而经硫酸铵分段盐析后的麦麸酯酶对6种农药的敏感性低于粗酶液。该研究为麦麸酯酶应用于农药残留检测奠定了一定的技术基础。     

家蝇中乙酰胆碱酯酶、羧酸酯酶和多功能氧化酶活性与抗药性的关系

本文对几种抗药性和敏感性家蝇品系的乙酰胆碱酯酶(AChE)、羧酸酯酶及多功能氧化酶(MFO)进行了测定.结果表明:①抗药性品系和敏感性品系的AChE活力差异不大, 有机磷抗性品系的AChE对对氧磷的不敏感性比敏感性家蝇明显增大.②某些抗药性家蝇的羧酸酯酶活力比敏感性家蝇大.③抗药性家蝇的MFO活力(O-脱甲基和环氧化)比敏感性家蝇均有不同程度的增高.④二氯苯醚菊酯抗性家蝇对有机磷有负交互抗性.     

两种体色生物型桃蚜对杀虫剂敏感性差异及其与酶活力的关系

为了明确不同体色生物型桃蚜Myzus persicae (Suizer)对药剂的敏感性差异,本文采用室内生物测定和酶活力测定法检测不同体色生物型桃蚜对杀虫剂的敏感性以及与3种解毒酶和1种靶标酶活力关系。结果表明,红色生物型的敏感性明显低于绿色生物型,其中对吡虫啉的敏感性差异最大,LC50分别为6796648和284597 mg/L,红色为绿色生物型2388倍;其次为毒死蜱,LC50分别为12295798和1936816 mg/L,红色为绿色生物型635倍;两种生物型桃蚜对阿维菌素敏感性差异最小,红、绿色生物型的LC50分别为311678 和290966 mg/L,红色为绿色生物型107倍。红色生物型体内3种解毒酶（羧酸酯酶、谷胱甘肽-S-转移酶和多功能氧化酶P450）的比活力均高于绿色生物型,红色型羧酸酯酶比活力为绿色型的31倍,谷胱甘肽-S-转移酶为41倍,多功能氧化酶P450为15倍,两种体色生物型3种解毒酶的活力差异均达显著水平。两种体色生物型体内乙酰胆碱酯酶比活力没有差异,说明乙酰胆碱酯酶比活力与敏感性关系不大。     

不同虫态烟蚜对杀虫剂敏感性及其与解毒酶活力关系

为更好地指导生产,本文研究了不同虫态烟蚜Myzus persicae对吡虫啉和丁硫克百威敏感性及其与羧酸酯酶、多功能氧化酶、谷胱甘肽S-转移酶比活力相关性。结果表明,3龄若虫对吡虫啉敏感性水平最低,4龄若虫对丁硫克百威敏感性水平最低。不同虫态烟蚜解毒酶活力随龄期逐渐增强,之后逐渐减弱,3龄若虫高于其他任何虫态,且多功能氧化酶和羧酸酯酶比活力与杀虫剂敏感性间存在强的负相关性。田间最佳防治虫态为1龄若虫、2龄若虫和成虫。     

杀虫剂不同施用方式对烟蚜抗药性发展和羧酸酯酶活力的影响

室内模拟田间杀虫剂施药方式,研究3种杀虫剂连用及其顺序轮用对烟蚜Myzus persicae(Sulzer)抗药性发展和羧酸酯酶活力的影响。结果表明,氧化乐果、灭多威、高效氟氯氰菊酯连续施药9次后,其抗性分别增长73.3,8.9,10.4倍;氧化乐果→灭多威→高效氟氯氰菊酯顺序轮用3次(施药9次)后,氧化乐果抗性指数增长了47.3倍,灭多威抗性指数增长了6.7倍,高效氟氯氰菊酯抗性指数增长了5.0倍。羧酸酯酶活力检测结果表明,3种杀虫剂连用9次后,烟蚜种群的α-NACarE活力分别增长了20.0,24.0和15.6倍,酶活力在0.6(OD600nm/aphid/min)以上的个体比例分别增加了73.4%,87.6%和43.8%;而3种杀虫剂顺序轮用3次(施药9次)后,烟蚜种群的α-NACarE活力增长了10.2倍,酶活力在0.6以上的个体比例增加了4.8%。结果证实杀虫剂轮换施用能延缓烟蚜抗药性的发展和烟蚜种群α-NACarE活力及高活力个体频率的增加。     

产酯酶微生物菌种的筛选研究

研究了产酯酶微生物的筛选,包括筛选模型、酶活力检测方法及菌株的分布。对30多份土样以及实验室保存的菌种进行了大量的筛选,以添加三醋酸甘油酯、乳酸乙酯酯类物质对土样等样品富集,采用添加显色剂溴甲酚紫的快速简便平板显色法,观察水解变色圈直径和菌落直径的大小进行初筛。获得两者直径之比相对大的菌株174株,采用平板打孔检测法和摇瓶发酵比色法测酶活力相结合进行复筛,最终得到酯酶活力较高的24株菌株。就初筛和复筛方法及结果加以比较分析,复筛菌株做不同底物的酶活力检测,建立了一个有效、简便及快速的微生物酯酶的筛选模型。并对酯酶产生菌的立体选择专一性进行了初步考察。     

棉蚜对有机磷杀虫剂抗性的生化机理

本文对有机磷抗性和感性棉蚜Aphis gossypii三个种群抗性生化机制进行了讲究.首先用解毒酶的抑制剂测定药剂的解毒途径.进一步测定乙酰胆碱酯酶活力及其敏感性和多功能氧化酶、谷胱甘肽s-转移酶、α-乙酸萘酯酶和α-乙酸萘酯羧酸酯酶等解毒酶的活力.结果表明,体内条件下,多功能氧化酶与抗性有关,但在离体条件下,在棉蚜匀浆液中有内源抑制剂存在.α-乙酸萘酯酶和α-乙酸萘酯羧酸酯酶活力的增加,乙酰胆碱酯酶对杀虫剂敏感性的降低也是造成棉蚜对有机磷产生抗性的原因.     

寄主对豆野螟的药剂敏感性和体内解毒酶活性的影响

比较取食不同寄主植物的豆野螟MarucatestulalisGeyer幼虫对3种药剂敏感性的差异,及其体内4种解毒酶活性之间的差异。结果表明:取食不同寄主植物的豆野螟对高效顺反氯氰菊酯和灭多威的敏感性差异显著,豇豆>四季豆>扁豆;对茚虫威的敏感性差异不显著。取食不同寄主植物的豆野螟体内4种酶活性都发生了变化,乙酰胆碱酯酶(AChE)羧酸酯酶(CarE)和多功能氧化酶(MFO)活性之间存在显著差异,豇豆>四季豆>扁豆;但谷胱甘肽-S转移酶(GST)活性之间不具有显著差异。推测:取食扁豆的豆野螟体内产生对高效顺反氯氰菊酯和灭多威具有一定解毒作用的物质;而取食豇豆的豆野螟产生的某种物质对茚虫威具有一定的解毒作用,4种酶在其对茚虫威的解毒过程中起一定的作用。     

枯草芽胞杆菌乙酰木聚糖酯酶的鉴定及诱导剂对酯酶活力的影响

以枯草芽胞杆菌CICC 20034为研究对象,对其分泌的高相对分子质量酯酶进行鉴定,并考察诱导剂对其活力的影响。结果表明:枯草芽胞杆菌CICC 20034可分泌一种相对分子质量为1.07×105的酯酶,经蛋白质质谱鉴定为乙酰木聚糖酯酶,单体分相对子质量为3.56×104。在发酵培养基中添加乙酸乙酯和木糖可以显著的促进乙酰木聚糖酯酶的活力,而三丁酸甘油酯和大分子诱导剂——木聚糖、玉米芯粉和壳聚糖对酯酶的活力几乎无促进作用。枯草芽胞杆菌CICC 20034以乙酸乙酯为诱导剂时最高比酶活为0.62 U/mL,为已知报道的野生细菌乙酰木聚糖酯酶的最高酯酶活力。     

超嗜热酯酶EST2在不同宿主中的异源高效表达研究

来源于超嗜热古菌Alicyclobacillus acidocaldarius的酯酶EST2是目前报道的活性最高的超嗜热酯酶,具有极大的工业应用价值。为促进EST2的生产应用,将其分别在大肠杆菌及毕赤酵母中进行异源表达,并就不同宿主对表达情况和重组酶酶学性质的影响进行了分析。在大肠杆菌和毕赤酵母中重组表达的EST2酶学性质基本一致:最适温度分别为75℃和77.5℃,最适pH均为8.0,比活力分别为4656.6 U/mg和4078.3 U/mg,70℃水浴保温4.5 h,残余活力均在70%以上。在摇瓶发酵的基础上,于5 L发酵罐中进行了重组大肠杆菌及毕赤酵母的高密度发酵。毕赤酵母高密度发酵120 h菌体干重达68 g/L,最大表达酶活力为959.6 U/ml。大肠杆菌高密度发酵25 h菌体干重达60.8 g/L,最大酶活力14825.6 U/ml,表达量是毕赤酵母的15.4倍,单位时间产量是酵母的74.2倍。结果表明大肠杆菌发酵周期短、表达量高,更适合进行嗜热酯酶EST2的高效生产,这为促进嗜热酯酶在工业生物技术产业的应用奠定了基础。     

Mammalian carboxylesterase (CES) releases GPI-anchored proteins from the cell surface upon lipid raft fluidization

Mammalian carboxylesterase (CES) is well known as a biotransformation enzyme for prodrugs and xenobiotics. Here, we purified CES as a GPI-anchored protein (GPI-AP)-releasing factor (GPIase) that releases such protein from the cell surface. All five isoforms of CES showed this activity to various degrees. When the serine residue of the catalytic triad for esterase was replaced by alanine, esterase activity was completely disrupted, while full GPIase activity remained, suggesting that these two activities are exhibited via different mechanisms. CES6, a new class of mammalian CES, exhibited the highest GPIase activity and released specific GPI-APs from the cell surface after lipid raft fluidization. The released product contained a GPI component, indicating that GPI-AP was released by cleavage in GPI. These results revealed for the first time that CES recognizes and catalyzes macromolecule GPI-AP as well as small molecules.     

Cloning, purification and characterization of a thermostable carboxylesterase from Anoxybacillus sp. PDF1

The gene encoding a carboxylesterase from Anoxybacillus sp., PDF1, was cloned and sequenced. The recombinant protein was expressed in Escherichia coli BL21, under the control of isopropyl-β-D-thiogalactopyranoside-inducible T7 promoter. The enzyme, designated as PDF1Est, was purified by heat shock and ion-exchange column chromatography. The molecular mass of the native protein, as determined by SDS-PAGE, was about 26 kDa. PDF1Est was active under a broad pH range (pH 5.0-10.0) and a broad temperature range (25-90 °C), and it had an optimum pH of 8.0 and an optimum temperature of 60 °C. The enzyme was thermostable carboxylesterase, and did not lose any activity after 30 min of incubation at 60 °C. The enzyme exhibited a high level of activity with p-nitrophenyl butyrate with apparent K(m), V(max), and K(cat) values of 0.348 ± 0.030 mM, 3725.8 U/mg, and 1500 ± 54.50/s, respectively. The effect of some chemicals on the esterase activity indicated that Anoxybacillus sp. PDF1 produce an carboxylesterase having serine residue in active site and -SH groups in specific sites, which are required for its activity.     

滇重楼种子水浸液对三种植物种子萌发和幼苗生长的影响

利用滇重楼(Paris polyphylla Smith var.yunnanensis(Franch.)Hand.-Mazz.)种子外种皮和胚乳的水浸液对白菜(Brassica pekinensis(Lour.)Rupr.)、绿豆(Vigna radiata(Linn.)Wilczak)、小麦(Triticum aestivum L.)种子进行处理,研究滇重楼种子水浸液对3种植物种子萌发、幼苗生长和保护酶活性的影响,并利用GC-MS方法对滇重楼种子内源抑制物的成分进行分析。结果显示,不同浓度滇重楼外种皮、胚乳水浸液对上述3种受体植物的发芽率、苗高、根长及鲜重均产生影响,其作用强度和水浸液的浓度有关,总体上表现出低促高抑的双重浓度效应。滇重楼种子水浸液对白菜的影响作用最强,对绿豆的影响作用最弱,且胚乳水浸液的影响较外种皮强。不同浓度滇重楼种子外种皮和胚乳水浸液均能影响3种植物幼苗体内保护酶的活性,随着水浸液浓度的升高,叶片中超氧化物歧化酶(SOD)、过氧化物酶(POD)活性总体增加,与对照相比差异显著。白菜、小麦过氧化氢酶(CAT)活性减少,与对照相比差异显著;绿豆过氧化氢酶(CAT)活性增加,但与对照相比无显著差异。利用GC-MS方法从胚乳和外种皮水浸液中分别检出8种和2种物质。研究结果表明滇重楼种子中存在内源抑制物质,可能是导致种子休眠的原因;种子水浸液可能通过影响植物幼苗保护酶的活性进而影响其正常生长;有机酸类物质可能是滇重楼种子内源抑制物之一。     

Plasticity in biochemical characteristics of cotton aphid,Aphis gossypii Glover in response to different host plants and neonicotinoid pesticides

In the present investigation an effort was made to realise the role of feeding host plants on some enzymes’ activities. The results showed that the enzymes’ activities were changed in the aphids feeding on different host plants which assist in detoxification of their host metabolites. It is important when the aphids are exposed to insecticides. The results indicated that the measured enzyme activity has significant changes depending on the host plant. It is shown in this study that there are no significant differences between different host plants on esterase activity (p = 0.446); however, there is a significant difference between GSH activity (p = 0.047) but this relationship is not significant on MFO activity (p = 0.417). Among three strains of Ag-PP, Ag-MO and Ag-FA, strain Ag-PP was the most resistant strain against neonicotinoids, and the resistance mechanism was related to metabolic increase in carboxyl esterase activity. The results showed that strain of Ag-MO was the most susceptible strain against neonicotinoids. The result of this investigation also showed that the general esterases might play an important role in conferring or contributing to neonicotinoid resistance in the cotton aphids.     

Enhanced acylation activity of esterase BioH from Escherichia coli by directed evolution towards improved hydrolysis activity

Esterase BioH is a critical enzyme for Biotin synthesis in Escherichia coli, which has been previously found to be active in the acylation of secondary alcohols and amines. Directed evolution towards improved acylation activity requires a high-throughput screening method. The aim of this study is to explore whether the acylation activity of BioH can be improved by directed evolution of its hydrolysis activity. A colorimetric method based on p-nitrophenyl butyrate hydrolysis was adopted in the high-throughput determination of hydrolysis activity. The wild-type BioH showed a hydrolysis activity of 18 U/mg, and the specific activities for α-phenylethanol and α-phenylethylamine acylation were 12.8 U/mg and 3.5 U/mg, respectively. After two rounds of directed evolution, seven mutants with improved hydrolysis activity were obtained, among which, K213E, Q70L/M170T and M197L/K213E also showed improvement in acylation activity. To further improve the acylation activity, site mutations were generated in different combinations at the four hot spots Q70L, M170T, M197L and K213E. Among the resulting mutants, Q70L/M197L/K213E showed the highest activity in α-phenylethylamine acylation with a 120% improvement, while Q70L/K213E had the highest α-phenylethanol acylation activity, which was improved by 70%. The results demonstrated that directed evolution of the hydrolysis activity might be a possible approach to improve the acylation activity of the esterase BioH.     

Development of Bed Reactor Using Brick Dust Immobilized CIVI-Cellulase from Seeds of Cowpea (Vigna sinensis L)

Cellulase extracted from seeds of Cowpea (Vigna sinensis L var VITA-4) was partially purified and immobilized on brick dust as solid support via glutaraldehyde. The percentage retention of the enzyme activity on brick dust was nearly 85%. After immobilization specific activity of the enzyme increased from 0.275 to 0.557 U mg^?1 protein with about 2 fold enrichment. The optimum pH and temperature of soluble enzyme were determined as pH 4.6 and WC, respectively whereas immobilized enzyme showed at pH 5.0 and 37°C, respectively. The V_max values for soluble and immobilized enzyme were determined as 6.67 and 1.25 mg min^?1, respectively whereas K_m values were 4.35 and 4.76 mg ml^?1, respectively. The immobilized enzyme displayed higher thermal stability than soluble enzyme and retained about 50% of its initial activity after 12 reuses. Immobilized enzyme was packed in an indigenously designed double walled glass bed reactor for continuous production of reducing sugars.     

Metal ion effects on target sites of modification in metallocarboxypeptidase B

Native carboxypeptidase B and its Co2+-substituted derivative were oxidized by the active-site-directed agent m-chloroperbenzoic acid. The following results were obtained a) In the cobalt enzyme there was a decrease in both the peptidase and the esterase activities, whereas in the zinc enzyme only the peptidase activity decreased. Peptide or ester pseudo-substrates protected the cobalt enzyme but not the zinc enzyme against inactivation. b) Upon oxidation and formation of Co3+, cleavage of peptide bonds occurred in the cobalt enzyme but not in the zinc enzyme. Both enzymes retained their original metal content. c) Following oxidation of the enzymes, amino acid analysis revealed a modification of a methionyl residue in the zinc enzyme only; the cobalt enzyme, on the other hand, showed a modification of a histidyl residue. d) Peptide mapping of the enzymes after cleavage by cyanogen bromide indicated that two methionyl peptides were missing in the oxidized zinc enzyme. These peptides point to Met-64 as the site of modification. The peptide map of the oxidized cobalt enzyme was similar to that of the unmodified native (i.e., zinc) enzyme. These studies indicate that the specific metal ion present in the enzyme imposes certain structural and functional differences on the active site, leading to differing reactivities of specific amino acid residues and to a different alignment of the active-site-directed reagent in the two enzymes.     

土耳其斯坦叶螨多重抗性品系选育及解毒酶活性的变化

为探索土耳其斯坦叶螨的多重抗药性及其生化机理,在室内对敏感系(SS)土耳其斯坦叶螨分别用螺螨酯、甲氰菊酯和阿维菌素的混剂进行处理,选育出多重抗性品系(Mp-R).结果表明: 选育至15代,土耳其斯坦叶螨的抗性指数达35.74倍.对不同品系的解毒酶活性分析显示,Mp-R品系相对SS品系的羧酸酯酶(CarE)、谷胱甘肽-S-转移酶(GSTs)和多功能氧化酶(MFO)的比活力分别是SS品系的1.21、1.53、9.18倍.说明CarE、GSTs、MFO的活性升高可促进土耳其斯坦叶螨对3种杀虫剂多重抗性的形成;MFO的活性升高可能是土耳其斯坦叶螨对3种杀虫剂产生多重抗性的主要原因.测定Mp-R品系和单抗品系(Ip-R)的农药感性和解毒酶活力变化发现,3种杀虫剂的混合使用可能会延缓土耳其斯坦叶螨对甲氰菊酯的抗性形成,加快对阿维菌素的抗性形成.     

Further characterization of α-galactosidase I-glycoprotein lectin from Vicia faba seeds

The nature of the glucose/mannose specific lectin activity of α-galactosidase I from Vicia faba seeds has been examined. Gel filtration in the presence of high concentrations of glucose and SDS-PAGE failed to detect favin, a classical lectin which also occurs in the seed. A comparison of the haemagglutinating activities of the α-galactosidases from Vigna radiata and V. faba seeds strongly suggests that the catalytic site of the Vigna enzyme is also responsible for its agglutinating activity and that the catalytic and lectin sites are at different loci in the case of V. faba α-galactosidase I. The latter conclusion is supported by an investigation of the effects of glucose, mannose and galactose on the catalytic and lectin activities and by results obtained by demetallization of the V. faba enzyme. A single galactose-binding site and two mannose binding sites per subunit of enzyme I were detected by the method of equilibrium dialysis and the association constants for these monosaccharides measured. Mannose did not appear to affect the binding of galactose to the enzyme or vice versa. The removal of glycan chains from α-galactosidase I with endo-β-N-acetylglucosaminidase H released an active dimeric form of α-galactosidase. The possible involvement of lectin-glycoprotein interactions in the stabilization of the tetrameric form of the enzyme is considered.     

Purification and characterization of a keratinolytic metalloprotease from Chryseobacterium sp. kr6

The Chryseobacterium sp. kr6 strain has been described as a highly keratinolytic bacterium showing effective feather-degrading and de-hairing activities. A keratinase Q1 enzyme was purified from Chryseobacterium sp. kr6 culture by Phenyl Sepharose and Superose 12HR chromatography. This enzyme showed a specific activity of 967U/mg for keratin azure. Electrophoresis under denaturing conditions showed a monomeric protein with approximately 64kDa. The enzyme showed pH and temperature optima of 8.5 and 50 degrees C, respectively. The inhibitory effect of EDTA, EGTA and 1,10-phenanthroline characterized Q1 enzyme as a Zn-metalloprotease. Its activity was increased by three-fold in the presence of Ca(2+). ESI-MS/MS analysis of peptides generated from a tryptic digestion revealed sequence homology which may characterize the Q1 keratinase as a member of the M14 metalloprotease family, with a consensus glycosylation region similar to proteins from Chryseobacerium meningosepticum.