Effect of Bacillus thuringiensis on Habrobracon hebetor during combined biological control of Plodia interpunctella

Abstract  The suitability of combining microbial pesticides and an insect parasitoid for pest management of stored cereal in China was evaluated using laboratory assays. For this purpose, interactions between Bacillus thuringiensis ( Bt ), Bt -intoxicated host larvae and the parasitoid Habrobracon hebetor (Say) (Hymenoptera: Braconidae) were tested against Plodia interpunctella Hübner (Lepidoptera: Pyralidae) . Bt or H. hebetor alone caused 41.67% and 35.35% P. interpunctella larval mortality respectively. The Bt –parasitoid combined treatment significantly increased mortality of P. interpunctella (86%). Progeny development of H. hebetor was dependent upon its susceptibility to Bt . Fewer parasitoids emerged from Bt –parasitoid combined treatment than in non- Bt treatments. However, since Bt did not prevent parasitoid development, a combined treatment with Bt and parasitoid release could produce better protection against P. interpunctella than either treatments when used singly, because their lethal effects were additive to each other.     

基于易错PCR技术的短小芽孢杆菌YZ02脂肪酶基因BpL的定向进化

利用易错PCR技术对短小芽胞杆菌(Bacillus pumilus)YZ02脂肪酶基因BpL进行两轮定向进化研究, 分别获得最佳突变株BpL1-7和BpL2-1369, 其脂肪酶活力比出发酶分别提高了2倍和6倍。序列分析表明, 突变体BpL2-1369有4个碱基发生了突变: T61C/C147T/A334G/T371A, 其中有3个碱基突变导致了氨基酸的改变。通过SWISS-MODEL数据库模拟脂肪酶的结构显示, 3个突变氨基酸分别位于第1个a螺旋的第3个氨基酸、第4和第5个b折叠之间的转角以及第5个b折叠的第1个氨基酸位置。将野生型脂肪酶基因BpL和进化后的基因BpL2-1369的高效表达产物经Ni-Agarose柱和Sephadex-G75纯化后, 酶学性质测定表明: 突变脂肪酶的比活力比野生型脂肪酶提高了1.31倍, Km值由8.24 mmol/L降低至7.17 mmol/L; 在pH>8.0时的稳定性较野生型脂肪酶有所提高。     

苏云金芽胞杆菌营养期杀虫蛋白Vip与Cry蛋白的协同作用

将构建的营养期杀虫蛋白基因vip3表达质粒pBMB2328和含杀虫晶体蛋白基因(crylAc10或crylCa)质粒同时电转化无质粒突变株BMB171并双抗筛选。经PCR特异引物扩增验证,分别得到含crylAc10和vip83、crylCa和vip83的双基因重组菌BMB2830-171和BMB2882-171。用单基因重组菌作对照,分别测定了营养期杀虫蛋白Vip83与杀虫晶体蛋白CrylAc10和Cry1Ca两组蛋白对3种重要鳞翅目害虫毒力。经统计分析,结果表明两组杀虫蛋白Vip83与CrylAc10和Vip83与CrylCa之间对棉铃虫均存在拮抗作用,对甜菜夜蛾协同作用不明显;但对小菜蛾前协同作用不明显,而后则有增效作用,其共毒系数为32．6。双基因的遗传稳定性检测表明这种正负协同关系具有一定的分子遗传稳定性,可为高效广谱工程菌的构建提供依据。     

Study of the Antifungal Ability of Bacillus subtilis Strain PY-1 in Vitro and Identification of its Antifungal Substance （Iturin A）

A Bacillus strain,denoted as PY-1,was isolated from the vascular bundle of cotton.Biochemical,physiological and 16S rDNA sequence analysis proved that it should belong to Bacillus subtilis.The PY-1strain showed strong ability against many common plant fungal pathogens in vitro.The antibiotics producedby this strain were stable in neutral and basic conditions,and not sensitive to high temperature.From theculture broth of PY-1 strain,five antifungal compounds were isolated by acidic precipitation,methanolextraction,gel filtration and reverse-phase HPLC.Advanced identification was performed by mass spec-trometry and nuclear magnetic resonance spectroscopy.These five antifungal compounds were proved to bethe isomers of iturin A:A2,A3,A4,A6 and A7.In fast atom bombardment mass spectrometry/mass spec-trometry collision-induced dissociation spectra,fragmentation ions from two prior linear acylium ions wereobserved,and the prior ion,Tyr-Asn-Gln-Pro-Asn-Ser-βAA-Asn-CO~ ,was first reported.     

青霉素酶发酵液的预处理和酶学特性

研究了在发酵液中添加絮凝剂对发酵液进行预处理,对蜡状芽孢杆菌（Bacillus cereus）产生青霉素酶的影响与酶学特性。实验结果表明,发酵液膜处理的难易程度与芽孢释放程度成正相关,在发酵液中添加0．02g／mL自制絮凝剂进行预处理,过滤效率提高10倍,而酶活损失仅5％;酶学特性研究结果表明,该酶最佳反应温度55℃时,酶的最适反应pH值7．0,金属离子锌、锰、镁对酶有激活作用,其浓度为0．25moL／L;酶热稳定性研究结果表明,在75℃下保温30min时,酶活损失85％。该酶在过量青霉素底物下,酶促反应为0级反应。     

复合诱变原生质体选育耐热碱性蛋白酶高产菌

以地衣芽孢杆菌(Bacillus licheniformis)53号为原始菌株,在原生质体形成和再生的最佳条件下制备原生质体,对原生质体进行复合诱变,对大量再生突变株进行筛选,最终获得了高产、稳定、耐热的碱性蛋白酶产生菌53-G38-6,产酶活力由1104U/ml提高到22080U/ml。适宜的发酵条件:培养基(％)胰蛋白胨1,酵母膏0.5,玉米粉5,Na_2HP0_4·12H_20 0.4,KH_2P0_4 0.03,Na_2CO_3 0.1,自然pH。42℃旋转培养44～48h,得到的蛋白酶热稳定性强,60℃处理1h剩余酶活55％。酶反应最适条件:62℃,pH10.0,在pH9～10.5范围内稳定。     

Proteomic analysis of Bacillus cereus growing in liquid soil organic matter

Bacillus cereus is believed to be a soil bacterium, but studied solely in laboratory culture media. The aim of this study was to assess the physiology of B. cereus growing on soil organic matter by a proteomic approach. Cells were cultured to mid-exponential phase in soil extracted solubilized organic matter (SESOM), which mimics the nutrient composition of soil, and in Luria-Bertani broth as control. Silver staining of the two-dimensional gels revealed 234 proteins spots up-regulated when cells were growing in SESOM, with 201 protein spots down-regulated. Forty-three of these differentially expressed proteins were detected by Colloidal Coomassie staining and identified by matrix assisted laser desorption ionization-time of flight MS of tryptic digests. These differentially expressed proteins covered a range of functions, primarily amino acid, lipid, carbohydrate and nucleic acid metabolism. These results suggested growth on soil-associated carbohydrates, fatty acids and/or amino acids, concomitant with shifts in cellular structure.     

Engineering surface hydrophobicity improves activity of Bacillus thermocatenulatus lipase 2 enzyme

Bacillus thermocatenulatus lipase 2 (BTL2) is a promising industrial enzyme used in biodiesel production. Although BTL2 has high thermostability and good resistance to organic solvents, the activity of BTL2 is suboptimal for industrial processes. To improve BTL2 activity, we engineered BTL2 lipase by modulating hydrophobicity of its lid domain. Through site‐directed mutagenesis, we constructed three mutants, namely Y225F+S232A, S232A+T236V and Q185L, to cover all uncharged hydrophilic amino acids within the lid domain. Activities of these mutants were characterized. Our findings suggest that one mutant (Y225F+S232A) showed ～35% activity increase in catalyzing heterogeneous hydrolytic reactions relevant for industrial applications. A mathematical framework was established to account for different molecular events that contribute to the observed apparent catalytic activities. Increases in hydrophobicity of lid domains were associated with increased interfacial adsorption of lipases and lower molecular enzymatic activities. The measured apparent activities of lipases include contributions from both events. Lid hydrophobicity can thus result in different changes in lipase activities depending on the mutation site. Our work demonstrates the feasibility of increasing BTL2 activity by modulating the hydrophobicity of lid domains and provides some guidelines for further improving BTL2 activity.     

枯草芽胞杆菌异戊二烯酶ComQ的生物信息学分析及对生物膜形态的影响

[目的]枯草芽胞杆菌ComQ是一种类异戊二烯生物合成酶.利用生物信息学预测分析了ComQ的生物学特性,对comQ基因进行过表达和敲除,构建突变菌,孔板发酵培养验证生物膜形态变化.[方法]运用NCBI (National Center for Biotechnology Information)网站里的Protein数据...     

以环氧乙烷为活性基的多孔颗粒状固定化青霉素酰化酶的制备

报道了用以环氧乙烷为活性基的多孔颗粒状载体（Eupergit-C）制备固定由巨大芽孢杆菌（B．megaterium）产生的青霉素酰化酶的研究。用已二胺,赖氨酸对载体进行化学修饰后制备固定化酶,获得了较好的固定结果。用未修饰的载体制备固化酶,经24h固定反应,酶活力达176．5IU／g（wet）,酶活力总叫率达53．7％,酶蛋白的固定量为19＝7mg/g（dr）,酶蛋白的固定效率达87．5％。游离酶的酶浓度对制备固定化酶的活力无显影响。当加酶量从312IU/g（dry）上升到6250IU／g（dry）时,固定化酶活力从89IU／g(wet）上升到475IU／g（wet）,总收率和固定化效率分别从99％和99％下降到26．5％和32．5％,酶蛋白的固定量从6．9mg/g（dry）上升到112mg/g（dry）,酶蛋白的固定效率从99％下降至80．5％。以酶活力为155IU/g(wet）,酶蛋白固定量为22mg/g(dry）的固定化酶水解青霉素G钾盐,经过20批循环水解后,剩余酶活力为92．5％。