对甜菜夜蛾高毒苏云金芽孢杆菌菌株的选育*

采用物理诱变——虫体传代模式,选育获得一株对甜菜夜蛾高毒菌株BtCZE 99985。通过摇瓶和40t发酵罐3年10批发酵试验,表明该菌株具有良好的发酵性能。摇瓶试验表明,与出发菌株93005、对照菌株HD-1-580、GC-91相比较,该菌株对甜菜夜蛾的毒效分别提高429％、655％、114％。40t发酵罐发酵试验表明,该菌株对甜菜夜蛾测定的LC50平均值为0．076μL/mL,比GC-91菌株(平均0．213μL/mL)的毒效提高180％。     

白刺链霉菌（Streptomyces albospinus）CT205产环己酰亚胺含量测定方法的建立

建立白刺链霉菌（Streptomyces albospinus）CT205代谢产环己酰亚胺含量的检测方法,为菌株CT205的开发利用提供技术支持。采用高效液相色谱法（HPLC）、生物活性法和分光光度法等三种检测手段分别测定菌株CT205发酵上清液及粗提物中活性物质环己酰亚胺(Cycloheximide)的含量。三种方法测定环己酰亚胺标准品均具有较好的线性关系,其中相关系数R²_HPLC法（0.999 2）>R²_{生物活性法}（0.993 7）≈R²_{分光光度法}（0.996 5）。HPLC法及生物活性法分别测定CT205发酵液及粗提物中活性物质含量分别为72.87、1 555.70及66.15、1 259.00μg/mL,HPLC法与生物活性法测定的发酵上清液中活性物质含量误差在+6.72 μg/mL。HPLC检测方法的准确性及灵敏度均高于生物活性法,适用于样品含量的准确测定,生物活性法适用于菌株诱变筛选及条件优化实验产生的大批量样品的测定及比较,分光光度法不适用检测杂质含量较多的样品。     

阿维菌素高产菌株的选育及阿维菌素B1的鉴定

自阿维链霉菌(Streptomyces avermitilis ATCC31272)中分离出了3种不同类型的菌株,其中只有产灰色孢子的菌株能产生阿维菌素(Avermectins),摇瓶发酵单位约100μg/mL。经高频电子流诱变和对发酵培养基的改进,选育出Sa-76菌株,其摇瓶发酵单位可达1000μg/mL。从其菌丝体中提取纯化了阿维菌素B₁晶体,其紫外吸收光谱、红外吸收光谱、核磁共振谱(¹HNMR和¹³CNMR)和质谱与国外报道的一致。Sa-76菌株又经2次亚硝基胍诱变,筛选出发酵单位2000μg/mL以上的Sa-76-8菌株。在此基础上,再次用亚硝基胍对Sa-76-8菌株进行了诱变,获得Sa-76-9菌株,结合发酵条件的优化,其发酵单位可高达3500～4000μg/mL。     

灭钉螺微生物的选育及抑螺效果观察

【目的】筛选出一种高效、持久、安全及价廉的灭螺微生物,对其进行鉴定并观察其抑螺功效。【方法】从钉螺孳生的土壤中筛选出4株灭螺活性较强的菌株(B8、B27、B36、B59),显微镜观察菌株形态和革兰氏染色均为G+杆菌。不同分离胶浓度的SDS-PAGE分析其灭螺活性成分;优势菌株经16s rRNA扩增后,PCR产物测序,序列比对,构建系统发育树鉴定其种属。【结果】灭螺结果表明,各试验组中发酵上清液各菌株间灭螺效果差异有统计学意义(χ2=21.286,P=0.002);细菌发酵液各菌株间差异也有统计学意义(χ2=17.298,P=0.008);菌体悬液各菌株间差异无统计学意义(χ2=7.579,P=0.271);此外,B59菌株的灭螺效果优于其它菌株,尤其是其发酵上清液浸泡48 h和72 h钉螺死亡率高达73.3%和96.7%。SDS-PAGE发现在B59细菌上清液中无蛋白带出现,推测其灭螺活性物质可能是其他成分;分子系统发育分析结果显示B59菌株位于Bacillus cereus (CP001746)分支上,一致性达100%。【结论】B59菌株的发酵上清液灭螺效果最好,其灭螺活性物质可能不是蛋白质,B59菌株被鉴定为Bacillus cereus。     

L－缬氨酸发酵供氧与补料过程控制的研究

高产缬氨酸的北京棒杆菌(corynebacterium pekinense)突变株125菌株,在2．6L自控发酵罐上分批培养结果表明,当发酵中后期DO为零时,产酸量较多。也可用kL。值为指标来调节过程的供氧强度,Kla=90．75h^-1时,产酸量最高。同时比较了恒速连续补加葡萄糖液,当F=3．75g／b时,产酸较高。由此获得了该菌株L一缅氨酸发酵的低供氧与恒速补糖的控制模式。总糖量为16．85％的发酵,可使产酸量达38．2g／L,转化率为22．67％。本文对有关试验结果,进行了发酵动力学的分析和讨论。     

自絮凝酵母SPSC01在组合反应器系统中酒精连续发酵的研究

建立了一套由四级磁力搅拌发酵罐串联组成、总有效容积4000mL的小型组合生物反应器系统 ,其中一级罐作为种子培养罐。以脱胚脱皮玉米粉双酶法制备的糖化液为种子培养基和发酵底物 ,进行了自絮凝颗粒酵母酒精连续发酵的研究。种子罐培养基还原糖浓度为100g L ,添加 (NH₄)₂HPO₄ 和KH₂PO₄ 各 20g L ,以0.017h^-1 的恒定稀释速率流加 ,并溢流至后续酒精发酵系统。发酵底物初始还原糖浓度 220g/L ,添加 (NH₄)₂HPO₄ 15g/L和KH₂PO₄2 5g/L ,流加至第一级发酵罐 ,稀释速率分别为 0.017、0.025、0.033、0.040和0.05 0h^-1。实验数据表明 ,自絮凝颗粒酵母在各发酵罐中呈部分固定化状态 ,在稀释速率0.040h^-1 条件下 ,发酵系统呈一定的振荡行为 ,其他四个稀释速率实验组均能够达拟稳态。当稀释速率不超过 0 0 33h^-1 ,流出末级发酵罐的发酵液中酒精浓度可以达到 12 % (V/V)以上 ,残还原糖和残总糖分别在 0 11%和 0 35 % h^-1,流出末级发酵罐的发酵液中酒精浓度可以达到12%（V/V）以上,残还原糖和残总糖分别在0.11%和0.35%（W/V）以下。在稀释速率为0.033h^-1时,计算发酵系统酒精的设备生产强度指标为3.32（g·L^-1·h^-1）,与游离酵母细胞传统酒精发酵工艺相比,增加约1倍。     

基于PKS和NRPS基因的海洋来源抗病原菌活性菌株筛选及其代谢产物活性分析

基于聚酮合成酶基因(polyketide synthases gene,PKS)和非核糖体多肽合成酶基因(non ribosomal polypeptide synthase gene,NRPS),本研究从77株分离于北冰洋海泥的菌株中筛选出1株具有较高抗病原菌活性的菌株并对其进行了菌种鉴定。通过优化培养基组成和发酵条件提高了该菌株活性代谢产物的产量,并利用高分辨率质谱(high resolution mass spectrometry,HRMS)、核磁氢谱(¹H nuclear magnetic hydrogen,¹H NMR)和碳谱(¹³C NMR)对其主要代谢产物进行了结构鉴定。测定了该菌株主要代谢产物的抗菌谱及代谢产物对黄瓜枯萎病的影响。研究结果表明,该菌株为贝莱斯芽孢杆菌(Bacillus velezensis),其对植物具有一定的促生作用。当发酵条件为麦芽糖5g/L、胰蛋白胨10g/L、氯化钠10g/L、温度30℃、转速150r/min、发酵时间60h时,该菌株代谢产物的抑菌圈直径由(16.23±0.42)mm提高至(24.42±0.57)mm。菌株代谢产物含有大环内酯类化合物macrolactin A,其对多种病原细菌和真菌具有明显拮抗作用。黄瓜幼苗实验表明,该菌株代谢产物对黄瓜枯萎病具有防护作用,其作为生防菌剂具有一定的开发应用潜力。     

重组戊型肝炎病毒衣壳蛋白工程菌的高密度培养

在10L发酵罐中对戊型肝炎病毒衣壳蛋白在重组大肠杆菌中表达发酵工艺进行了研究,用分批培养方法探讨了不同培养基、培养基中磷酸盐浓度和Mg^2＋浓度等因素对菌体生长与重组蛋白表达的影响;用分批补料培养研究了不同的补料工艺对菌体生长与重组蛋白表达的影响,同时对重组菌诱导时期、诱导持续时间以及不同诱导温度表达包含体在尿素溶液中的溶解性进行了研究。结果表明,在优化后的培养基中,磷酸盐浓度、Mg^2＋浓度分别为80mmol/L 与20mmol/L时菌体生长与表达效果较好;分批补料培养中,37℃培养9h菌体达到对数期中期(约45OD600)为适宜诱导时期,加入终浓度为10mmol/L IPTG后诱导5h,OD600达到80以上,重组蛋白表达量达到29.74％,为最适收获菌体时间;37℃表达的包含体80％以上溶解在4mol/L的尿素溶液中,最终浓度达到14mg/mL; 10L发酵罐中确定的发酵工艺参数在30L发酵罐中进行了放大培养,10L发酵罐中确定的发酵工艺参数在30L发酵罐上具有可放大性与重复性, 可以应用于工业生产。     

古树大理茶优势内生真菌1个新四氢-β-咔啉二酮哌嗪的分离鉴定

为挖掘古树大理茶优势内生真菌间座壳属菌株Diaporthe tectonigena的化学成分,该研究采用硅胶、大孔吸附树脂Diaion HP20、葡聚糖凝胶LH-20等柱层析方法,对该菌株的大米固态发酵提取物进行分离纯化,并通过HRMS、¹H-NMR、¹³C-NMR、HSQC、HMBC和COSY等波谱分析,对所得化合物进行结构鉴定。结果表明:(1)从该菌株大米固态发酵提取物中分离得到4个化合物,其中新化合物1鉴定为四氢-β-咔啉二酮哌嗪类生物碱,命名为tectonicgenazine A。(2)3个已知化合物分别鉴定为trans-cyclo-(D-tryptophanyl-L-tyrosyl)(2)、1H-吲哚-3-羧酸-2,3-二羟基丙酯(3)和N-羟乙基-2-乙酰基吡咯(4),其中化合物3为首次从自然界中分离所得。     

桑树内生洋葱伯克霍尔德氏菌对家蚕黑胸败血病的防治及抑菌作用

为了开发新型微生物农药用于家蚕病害防治,研究了桑树内生拮抗细菌洋葱伯克霍尔德氏菌(Burkholderia cepacia)Lu10-1菌株对家蚕黑胸败血病的防治与抑菌效果.结果表明:Lu10-1菌株发酵上清液对家蚕黑胸败血病的预防及治疗有效率分别达到41.2％和24.0％.Lu10-1菌株的抗菌粗提物对黑胸败血菌有较强的拮抗作用,抑菌圈直径为18.20 mm;抗菌粗提物对黑胸败血菌的最小抑菌浓度(MIC)和最低杀菌浓度(MBC)分别为1.56 mg·mL-1和3.13 mg·mL-1;经抗菌粗提物处理后黑胸败血茵未出现对数生长期,细胞膜的渗透性发生改变,胞内蛋白质发生渗漏,胞内分子量较大的蛋白质出现降解现象,直至菌体破裂,细胞内容物流出,形成空腔,最后消融.初步认为Lu10-1菌株分泌的抗菌物质用于防治家蚕黑胸败血病具有较好的开发前景.     

Effects of media composition of delta-endotoxin production and morphology of Bacillus thuringiensis in wild types and spontaneously mutated strains

Bacillus thuringiensis strains HD-73 and 4412, and two spontaneous mutants termed 4412aa-ind and 4412sph-cry were studied for the ability to produce crystals of different size and shape when grown in a rich medium and in an appropriate minimal medium defined during this study. Strain 4412aa-ind showed medium-dependent variation in the crystal phenotype. Scanning electron microscopy was utilized in order to show crystal variations in size and shape. B. thuringiensis strains 4412aa-ind and 4412sph-cry grown in rich and in minimal media produced differences in crystal morphology, and SDS-PAGE gel indicated that crystal variation was only at the morphological level and not in composition of the amino acids. A further nineteen B. thuringiensis strains were tested for the ability to sporulate in two simple defined media. Of these strains thirteen were able to complete sporulation with crystal production in one or both media. All strains grew and sporulated in a medium containing the usual twenty amino acids, and no vitamins or other growth factors were required.     

紫外线使苏云金芽孢杆菌伴孢晶体失活机理的研究

用电镜、电泳、生物鉴定等方法,研究了紫外线对苏云金芽孢杆菌库斯塔克变种的伴孢晶体的影响。结果表明,紫外线能破坏伴孢晶体的表面结构和形态,降低伴孢晶体在碱性液和蚕胃液中的溶解度,5小时以上的长时间照射,能导致伴孢晶体完全不溶,因而不能降解为具有杀虫活性的毒素蛋白而失活。     

Bacillus thuringiensis potency bioassays against Heliothis armigera, Earias insulana, and Spodoptera littoralis larvae based on standardized diets

Bacillus thuringiensis screening programs based on the official potency bioassay using third-instar larvae and on a neonate bioassay were developed for Heliothis armigera, Earias insulana, and Spondoptera littoralis. In these bioassays, the diets were standardized to be suitable, with minor modifications, for feeding of the three lepidopterans. The bioassay protocol was based on determination of the LC50 of the microbial standard HD-1-S-80 in the insects susceptible to B. thuringiensis var. kurstaki strains. This was followed by preliminary screening of B. thuringiensis strains at the LC50 of the B. thuringiensis standard. The B. thuringiensis strains causing 100% mortality at this LC50 in the larvae were selected for potency determinations. The neonate bioassay was suitable for accurate determinations of potencies also in S. littoralis--a representative of insects weakly susceptible to the HD-1 standard. The role of the official and the neonate bioassays in developing microbial control programs is discussed.     

Activated insecticidal crystal proteins from Bacillus thuringiensis serovars killed adult house flies

Insecticidal crystal proteins (ICP) from Bacillus thuringiensis serovar kurstaki HD-1 and HD-73 were activated by immobilized trypsin or chymotrypsin. The activated toxins (10 μ g or more) as well as unactivated ICP killed adult house flies but not larvae. Bacillus thuringiensis strain son diego did not kill house flies. In this experimental system, the average life span of the adult house fly was 8 days and the activated toxins reduced it to 2 days. The unactivated insecticidal crystal protein also reduced it to 4 days at the same concentration as the activated toxin.     

Use of oligonucleotide probes to study the relatedness of delta-endotoxin genes among Bacillus thuringiensis subspecies and strains

Fifteen Bacillus thuringiensis strains representing 13 serotypes were screened with five oligodeoxyribonucleotide probes specific for certain regions of two published sequences and one unpublished sequence of B. thuringiensis delta-endotoxin genes. Of the 15 cultures, 14 hybridized with at least one probe; the B. thuringiensis subsp. thompsoni strain alone did not hybridize. Two B. thuringiensis subsp. kurstaki strains of commercial interest, HD-1 and NRD-12, were found to be so closely related as to be indistinguishable with this technique; the same situation was found with strains from B. thuringiensis subspp. dendrolimus and sotto. Five strains were identified as probably containing only one endotoxin gene. A probe specific for the gene from the B. thuringiensis subsp. kurstaki HD-73 strain hybridized to only 3 of the 15 cultures tested. The hybridization data suggest that the DNA sequences coding for the C-terminal region of the endotoxin protein are as well conserved as those coding for the N-terminal toxic portion.     

Identification and characterization of a previously undescribed cyt gene in Bacillus thuringiensis subsp. israelensis.

Mosquitocidal Bacillus thuringiensis strains show as a common feature the presence of toxic proteins with cytolytic and hemolytic activities, Cyt1Aa1 being the characteristic cytolytic toxin of Bacillus thuringiensis subsp. israelensis. We have detected the presence of another cyt gene in this subspecies, highly homologous to cyt2An1, coding for the 29-kDa cytolytic toxin from B. thuringiensis subsp. kyushuensis. This gene, designated cyt2Ba1, maps upstream of cry4B coding for the 130-kDa crystal toxin, on the 72-MDa plasmid of strain 4Q2-72. Sequence analysis revealed, as a remarkable feature, a 5' mRNA stabilizing region similar to those described for some cry genes. PCR amplification and Southern analysis confirmed the presence of this gene in other mosquitocidal subspecies. Interestingly, anticoleopteran B. thuringiensis subsp. tenebrionis belonging to the morrisoni serovar also showed this gene. On the other hand, negative results were obtained with the anti-lepidopteran strains B. thuringiensis subsp. kurstaki HD-1 and subsp. aizawai HD-137. Western analysis failed to reveal Cyt2A-related polypeptides in B. thuringiensis subsp. israelensis 4Q2-72. However, B. thuringiensis subsp. israelensis 1884 and B. thuringiensis subsp. tenebrionis did show cross-reactive products, although in very small amounts.     

Use of oligonucleotide probes to study the relatedness of delta-endotoxin genes among Bacillus thuringiensis subspecies and strains.

Fifteen Bacillus thuringiensis strains representing 13 serotypes were screened with five oligodeoxyribonucleotide probes specific for certain regions of two published sequences and one unpublished sequence of B. thuringiensis delta-endotoxin genes. Of the 15 cultures, 14 hybridized with at least one probe; the B. thuringiensis subsp. thompsoni strain alone did not hybridize. Two B. thuringiensis subsp. kurstaki strains of commercial interest, HD-1 and NRD-12, were found to be so closely related as to be indistinguishable with this technique; the same situation was found with strains from B. thuringiensis subspp. dendrolimus and sotto. Five strains were identified as probably containing only one endotoxin gene. A probe specific for the gene from the B. thuringiensis subsp. kurstaki HD-73 strain hybridized to only 3 of the 15 cultures tested. The hybridization data suggest that the DNA sequences coding for the C-terminal region of the endotoxin protein are as well conserved as those coding for the N-terminal toxic portion.     

八株芽孢杆菌菌株的分类及固氮活性的测定

从8个省市的不同土壤分离了200个菌株,筛选出8株（JR₁,JR₂,JR₅,ZZ₃,ZZ₁₂,ZZ₁₆,M₁,K₄）能在无氮培养基上生长良好的典型的芽抱杆菌,其中6株（JR₁,JR₂,JR₄和ZZ₃,ZZ₁₂,ZZ