河南省番茄灰霉病菌对嘧霉胺的抗药性检测

[目的]明确河南省番茄灰霉病菌对苯胺基嘧啶类杀菌剂嘧霉胺的抗药性状况。[方法]采用菌丝生长速率法测定从河南省不同保护地中采集、分离的101株番茄灰霉病菌对嘧霉胺的敏感性。[结果]高抗菌株8株(7.92%),中抗菌株52株(51.49%),低抗菌株22株(21.78%),敏感菌株19株(18.81%)。[结论]河南省番茄灰霉病菌已对嘧霉胺产生不同水平的抗药性,各地应合理更换或轮换使用与嘧霉胺无交互抗性的药剂,以确保番茄灰霉病的有效控制。     

稻瘟病菌对三环唑的抗药性研究

将从田间感病品种原丰早上分离的稻瘟病原始菌株，接种在经三环唑处理过的该品种上，待其发病后，逐代重复分离诱导菌株，并对原始菌株和诱导菌株对三环唑的敏感性以及三环唑对它们的持效期进行了测定，结果表明，随诱导代次的增加，菌株对药剂的敏感性渐次降低，药剂对菌株的持效期明显缩短，但其致病性无明显变化趋势。     

灰葡萄孢菌对啶菌恶唑的抗药性研究初报

以灰葡萄孢菌为试验耙标,通过室内药剂驯化、紫外灯诱变,获得抗性突变体,并对抗性突变体的适合度进行测定,以评价新型杀菌剂啶菌恶唑的抗性风险。结果表明,经药剂驯化10代,抗性突变体的EC50值与敏感菌株无明显差异。在诱导14代后,EC50有所上升,但是抗性突变体的生长速率、产孢量等明显低于敏感菌株。因此,初步推断啶菌恶唑是一种抗性风险较低的杀菌剂。     

季铵盐阳离子表面活性剂对三种灰葡萄孢菌的敏感性测定

采用菌丝生长速率法和琼胶平板表面萌发法测定了两种季铵盐阳离子表面活性剂对三种不同寄主的灰葡萄孢属病原菌菌丝生长及孢子萌发的影响,并分析了不同菌株对药剂的敏感性。结果表明:C8-10(双八、十烷基季铵盐)对灰葡萄孢属菌丝的毒力高于1227(十二烷基二甲基苄基氯化铵),而其对灰葡萄孢菌孢子萌发的抑制率却低于1227,两者对孢子萌发的抑制活性明显高于常规药剂腐霉利和嘧霉胺。两种季铵盐化合物对番茄灰霉病菌的毒力最高,其次是草莓灰霉病菌、烟草灰霉病菌。     

稻瘟病菌对烯肟菌胺的抗性诱导及交互抗药性

通过药剂驯化和紫外诱导2种方法诱导J1、H1、DL1和DL2稻瘟病菌菌株对烯肟菌胺的抗性,并测定其对烯肟菌胺与嘧菌酯、稻瘟灵的交互抗药性.结果经药剂驯化的J1、H1、DL1和DL2菌株均对烯肟菌胺产生了中等抗药性;经紫外诱导DL1和DL2菌株对烯肟菌胺产生了中等抗性,J1和H1未产生抗性仍为敏感菌株;经测定稻瘟病菌对烯肟菌胺与嘧菌酯、稻瘟灵的交互抗药性表明烯肟菌胺与嘧菌酯具有交互抗药性,与稻瘟灵无交互抗药性.     

乙醇发酵菌种的选育及发酵条件的初步探索试验

对5种燃料乙醇发酵菌种进行了选择,对较好菌种做了耐高温驯化及发酵条件的优化。结果表明,在以葡萄糖为单一碳源的发酵中,相对于休哈塔假丝酵母、嗜单宁管囊酵母、间型脉胞菌、好食脉孢菌,酿酒酵母种液制备时间最短(对数生长期在6～13 h),发酵制取乙醇得率最高;对酿酒酵母驯化后,得到一株可在42℃正常生长的菌株;经研究,该耐高温菌株的较优发酵条件为温度30℃、转速150 r/min、初始pH值5.0、接种量10%。该耐高温驯化后的酿酒酵母不能直接用于水解液发酵制乙醇,还需要经过驯化或菌种改良后方能使用。     

高产1,3-丙二醇的等离子体诱变菌株选育及发酵动力学分析

以肺炎克雷伯氏菌为出发菌株,利用大气压介质阻挡放电等离子体诱变,确立等离子体诱变的物理参数和最佳诱变条件,分别以不同浓度的底物和产物筛选培养基对诱变菌液进行驯化,并结合96孔板筛选单菌落,获得一株高产1,3-丙二醇的突变菌株,命名为Kp-M2,其间歇发酵1,3-丙二醇产量比野生菌提高43%.基于Logistic和Lue...     

几种杀菌剂对芦荟黑斑病菌室内活性测定

通过供试的7种药剂对芦荟黑斑病菌体外抑制作用测试结果表明,所有药剂对其孢子萌发都有不同程度的抑制作用,其中异菌脲、咪鲜胺、嘧霉胺的抑制效果要优于氟硅唑、代森锰锌、百菌清、苯醚甲环唑;7种药剂对芦荟黑斑病菌菌丝体的抑制作用测定结果显示,所有药剂对菌丝生长都有显著的抑制作用,其中咪鲜胺锰络合物可湿性粉剂对菌丝体抑制效果最好,抑菌率达84.24%;异菌脲、氟硅唑、苯醚甲环唑次之。     

8种杀菌剂对橡胶炭疽菌的抑制活性

采用孢子萌发法,测定8种杀菌剂对橡胶炭疽病原菌Colletotrichum gloeosporioides Penz.Sace孢子萌发的抑制活性.结果表明:保护性杀菌剂对孢子萌发抑制效果较为理想,其中以百菌清抑制活性最高;内吸性杀菌剂对孢子萌发抑制效果不如保护性药剂,对菌株孢子萌发的抑制效果大小顺序为抑霉唑＞嘧菌酯＞咪鲜胺＞多菌灵＞戊唑醇.供试4个菌株对杀菌剂的敏感性存在较大的差异,其中以菌株RCl4最不敏感,菌株RC244最敏感.     

抗药性基因突变筛选高产菌株

本文通过洁霉素对盐酸林可霉素产生菌 98-1菌株孢子的致死浓度测定 ,采用诱变剂EMS的四种不同诱变剂量对菌株孢子进行诱变处理 ,诱变处理的孢子涂布在含洁霉素 ( 2 0 0 0 0ug/ml)的高氏平板上 ,获得了大量的洁霉素抗性基因突变株 ,然后从 90 0株洁霉素抗性基因突变株中通过初筛获得高于诱变出发菌株产素能力的菌株 5 6株 ,再进一步通过摇瓶复筛 ,并结合菌丝生长及摇瓶代谢情况 ,获得优于出发菌株的诱变菌株 4株。将这 4个菌株连同出发菌株连续三批次进行摇瓶发酵 ,结果 4个突变株的产素能力 (产量 )及摇瓶代谢和菌丝生长情况均优于出发菌株 ,试验筛选出最优菌株 0 2 -0 3 -40 2。将 0 2 -0 3 -40 2进行罐上发酵生产试验 ,结果试验罐比对照罐平均效价提高 1 7.5 6% ,平均产量提高 1 9.3 4%。本文建立了林可霉素高产菌株的洁霉素抗性基因突变诱变快速高效的筛选方法     

焦化废水融合子生物强化处理试验

通过融合子对焦化废水生物强化降解试验表明:驯化后的融合子对焦化废水降解能力比未经驯化的融合子明显提高,在活性污泥中投加融合子菌株后焦化废水的降解率提高了16.2%,改善降解条件有利于提高生物强化的降解效果,废水中投加葡萄糖、Fe3+和废水稀释都能促进生物降解,综合运用效果更好。     

The role of the conserved threonine in P450 BM3 oxygen activation: substrate-determined hydroxylation activity of the Thr268Ala mutant

The hydroxylation activity of the Thr268Ala mutant of P450(BM3) has been shown to occur to varying degrees with small alterations in the structure of a fatty-acid substrate. Ten substrates were investigated, including straight chain, branched chain and cis-cyclopropyl substituted fatty acids with a straight-chain length that varied between 12 and 16 carbon atoms. The efficacy of the hydroxylation activity appeared to be governed by the chain length of the substrate. Substrates possessing 14 to 15 carbons afforded the highest levels of activity, which were comparable with the wild-type enzyme. Outside of this window, straight-chain fatty acids showed reduced activity over the other substrate types. These results provide a cautionary tale concerning the loss of ferryl activity in such cytochrome P450 threonine to alanine mutants, as the nature of the substrate can determine the extent to which hydroxylation chemistry is abolished.     

Fractionation of transgenic corn for recovery of recombinant enzymes

Two germ-separation methods, dry-milling and density separation by flotation, were evaluated for recovering recombinant β-glucuronidase (rGUS) that accumulated primarily in the germ of transgenic corn. The dry-milling process consisted of (i) seed tempering, (ii) degerming with a horizontal-drum degermer/dehuller, (iii) particle size fractionation with standard sieves, (iv) germ and endosperm separation by roller milling and sifting, and (v) removal of hulls by aspiration. Sieves nos. 5, 6, and 7 retained the majority of germ, and subfractions from these sieves were pooled as a germ-rich fraction. Mass balances showed that the germ-rich fraction, which constituted 17% of the total dry-milled corn weight, contained 49% of rGUS activity and 64% of the total recoverable oil. Germ fractionation by flotation was tested as a proof-of-concept method aimed at separating corn fractions based on their difference in specific gravity (sp gr). The process consisted of impact-grinding of corn kernels followed by density separation using 1.15 or 1.3 specific gravity sodium nitrate solution. The oil-containing germ fraction floated, whereas the heavier endosperm fraction sedimented. The flotation method was simpler and resulted in higher enzyme recovery, that is, the germ-rich fraction was 20% (w/w) of the initial corn weight, and accounted for 80% of rGUS activity and 77% of total oil. The sodium nitrate solution did not have an adverse effect on the enzyme activity.     

Study on the activity and stability of urease immobilized onto nanoporous alumina membranes

Nanoporous alumina membranes were employed as substrate materials for urease immobilization. Anodic porous alumina was prepared by the two-step anodization of high purity aluminum. By controlling anodization conditions, the nanoporous structure with desired dimension was obtained. Urease immobilization onto nanoporous alumina membranes was performed by four different protocols. Effect of pore diameter, pore length and immobilization methods on the activity and stability of immobilized enzyme was discussed in detail. The results show that the enzymes immobilized onto porous alumina with big pore diameter possess high activity and poor stability as compared to small pore diameter. The effect of pore length is complicated, the activity of enzyme increases with the increasing pore length for big pore size; while for correspondingly small pore size, enzymatic activity slightly depends on pore length. The immobilization methods have a slight effect on enzymatic activity, whereas enzyme immobilization by chitosan coating and reticulation with glutaraldehyde exhibits a good long-term stability as compared to that only via physical adsorption.     

Endo‐α‐Mannosidase‐Catalyzed Transglycosylation

In order for facilitating the synthesis of oligosaccharides, transglycosylation reactions mediated by glycoside hydrolases have been studied in various contexts. In this study, we examined the transglycosylating activity of a Golgi endo ‐ α‐mannosidase. We prepared various glycosyl donors and acceptors, and recombinant human Golgi endo‐α‐mannosidase and its various mutants were expressed. The enzyme was able to mediate transglycosylation from α‐glycosyl‐fluorides. Systematic screening of various point mutants revealed that the E407D mutant had excellent transglycosylation activity and extremely low hydrolytic activity. Substrate specificity analysis revealed that minimum motif required for glycosyl acceptor is Manα1– 2Man. The synthetic utility of the enzyme was demonstrated by generation of a high‐mannose‐type undecasaccharide (Glc₁Man₉GlcNAc₂).     

Trp89 in the lid ofHumicola lanuginosa lipase is important for efficient hydrolysis of tributyrin

To determine whether Trp89 located in the lid of the lipase (EC 3.1.1.3) fromHumicola lanuginosa is important for the catalytic property of the enzyme, site-directed mutagenesis at Trp89 was carried out. The kinetic properties of wild type and mutated enzymes were studied with tributyrin as substrate. Lipase variants in which Trp89 was changed to Phe, Leu, Gly or Glu all showed less than 14% of the activity compared to that of the wild type lipase. The Trp89Glu mutant was the least active with only 1% of the activity seen with the wild type enzyme. All Trp mutants had the same binding affinity to the tributyrin substrate interface as did the wild type enzyme. Wild type lipase showed saturation kinetics against tributyrin when activities were measured with mixed emulsions containing different proportions of tributyrin and the nonionic alkyl polyoxyethylene ether surfactant, Triton DF-16. Wild type enzyme showed a V_max=6000±300 mmol·min⁻¹·g⁻¹ and an apparent K_m=16±2% (vol/vol) for tributyrin in Triton DF-16, while the mutants did not show saturation kinetics in an identical assay. The apparent K_m for tributyrin in Triton DF-16 was increased as the result of replacing Trp89 with other residues (Phe, Leu, Gly or Glu). The activities of all mutants were more sensitive to the presence of Triton DF-16 in the tributyrin substrate than was wild type lipase. The activity of the Trp89Glu mutant was decreased to 50% in the presence of 2 vol% Triton DF-16 compared to the activity seen with pure tributyrin as substrate. Wild type lipase and all mutants except Trp89Glu had the same affinity for the substrate interface formed by 15.6 vol% tributyrin in Triton DF-16. The Trp89Glu mutant showed a lower affinity than all the other lipase variants for the interface of 15.6 vol% tributyrin in Triton DF-16. The study showed that Trp89 located in the lid ofH. lanuginosa lipase is important for the efficient hydrolysis of tributyrin and that this residue plays a role in the catalytic steps after adsorption of the lipase to the substrate interface.     

Random mutagenesis improves the low-temperature activity of the tetrameric 3-isopropylmalate dehydrogenase from the hyperthermophile Sulfolobus tokodaii

In general, the enzymes of thermophilic organisms are more resistant to thermal denaturation than are those of mesophilic or psychrophilic organisms. Further, as is true for their mesophilic and psychrophilic counterparts, the activities of thermophilic enzymes are smaller at temperatures that are less than the optimal temperature. In an effort to characterize the properties that would improve its activity at temperatures less than the optimal, we subjected the thermostable Sulfolobus tokodaii (S. tokodaii) 3-isopropylmalate dehydrogenase to two rounds of random mutagenesis and selected for improved low-temperature activity using an in vivo recombinant Escherichia coli system. Five dehydrogenase mutants were purified and their catalytic properties and thermostabilities characterized. The mutations favorably affect the K(m) values for NAD (nicotinamide adenine dinucleotide) and/or the k(cat) values. The results of thermal stability measurements show that, although the mutations somewhat decrease the stability of the enzyme, the mutants are still very resistant to heat. The locations and properties of the mutations found for the S. tokodaii enzyme are compared with those found for the previously isolated low-temperature adapted mutants of the homologous Thermus thermophilus enzyme. However, there are few, if any, common properties that enhance the low-temperature activities of both enzymes; therefore, there may be many ways to improve the low-temperature catalytic activity of a thermostable enzyme.     

人细小病毒B19-VP1u保守区外C端氨基酸对sPLA2活性的影响

为探寻人细小病毒B19-VP1u保守区外氨基酸序列对sPLA2活性的影响,依次截短B19-VP1u保守区外C端的氨基酸序列,表达纯化截短突变的蛋白后分别检测其sPLA2活性。结果显示,截短7个氨基酸时,酶活性没有明显变化;截短15个氨基酸时,酶活性显著降低;截短23个氨基酸时,几乎没有酶活性。表明保守区外C端的第8～24个氨基酸之间的序列对酶活性有显著影响,推测其在维持酶的正确三维结构中起重要作用。     

Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis

Benzoylformate decarboxylase (BFD) from Pseudomonas putida wassubjected to directed molecular evolution to generate mutantswith increased carboligase activity which is a side reactionof the enzyme. After a single round of random mutagenesis mutantswere isolated which exhibited a 5-fold increased carboligaseactivity in aqueous buffer compared to the wild-type enzymewith a high enantiomeric excess of the product (S)-2-hydroxy-1-phenyl-propanone.From the same library, mutants with enhanced carboligase activityin water-miscible organic solvents have been isolated. The selectedmutants have been characterized by sequencing, revealing thatall mutants carry a mutation at Leu476, which is close to theactive site but does not directly interact with the active center.BFD-L476Q has a 5-fold higher carboligase activity than thewild-type enzyme. L476 was subjected to saturation mutagenesisyielding eight different mutants with up to 5-fold increasedcarboligase activity. Surprisingly, all L476 mutants catalyzethe formation of 2-hydroxy-1-phenyl-propanone with significantlyhigher enantioselectivity than the wild-type enzyme althoughenantioselectivity was not a selection parameter. Leu476 potentiallyplays the role of a gatekeeper of the active site of BFD, possiblyby controlling the release of the product. The biocatalyst couldbe significantly improved for its side reaction, the C–Cbond formation and for application under conditions that arenot optimized in nature.     

Alteration of the specificity of the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase A

The NADPH-dependent 2,5-diketo-D-gluconic acid (2,5-DKG) reductaseenzyme is a required component in some novel biosynthetic vitaminC production processes. This enzyme catalyzes the conversionof 2,5-DKG to 2-keto-L-gulonic acid, which is an immediate precursorto L-ascorbic acid. Forty unique site-directed mutations weremade at five residues in the cofactor-binding pocket of 2,5-DKGreductase A in an attempt to improve its ability to use NADHas a cofactor. NADH is more stable, less expensive and moreprevalent in the cell than is NADPH. To the best of our knowledge,this is the first focused attempt to alter the cofactor specificityof a member of the aldo–keto reductase superfamily byengineering improved activity with NADH into the enzyme. Activityof the mutants with NADH or NADPH was assayed using activity-stainednative polyacrylamide gels. Eight of the mutants at three differentsites were identified as having improved activity with NADH.These mutants were purified and subjected to a kinetic characterizationwith NADH as a cofactor. The best mutant obtained, R238H, producedan almost 7-fold improvement in catalysis with NADH comparedwith the wild-type enzyme. Surprisingly, most of this catalyticimprovement appeared to be due to an improvement in the apparentk_cat for the reaction rather than a large improvement in theaffinity of the enzyme for NADH.