地衣芽孢杆菌原生质体电转化方法的研究

为了解决地衣芽孢杆菌(Bacillus licheniformis)工业菌株难以转化的问题,将原生质体制备、电穿孔和原生质体再生技术相结合,建立了一种地衣芽孢杆菌原生质体电击转化方法。在对菌体生长状态、溶菌酶作用时间、电转电压、渗透压保护剂等条件进行优化后,试验了将不同类型的表达载体,即游离型质粒p GJ103(3.3kb)和整合型质粒p AX01(9.3kb),分别转入两株地衣芽孢杆菌工业生产菌株B.licheniformis CICC 10181和B.licheniformis CICC 20204中。实验结果显示,对数生长期后期的菌体酶解40min后制备的地衣芽孢杆菌原生质体得率为96%,再生率达25%以上。原生质体与质粒DNA在最适电压0.6k V/mm下电击转化,并以0.5mol/L山梨醇或甘露醇作为渗透压保护剂进行再生培养后,最终游离型质粒的转化率可达0.88×102~1.1×102CFU/μg p GJ103,整合型质粒的转化率达到0.45×102~0.52×102CFU/μg p AX01。该方法为地衣芽孢杆菌野生工业菌株的遗传改造提供了一种新的、高效的转化手段。     

不同抑制剂对地衣芽孢杆菌生长影响的研究

报道了地衣芽孢杆菌对不同抑制剂的反应。结果表明,当ｐＨ在７．０时,链霉素浓度为５００μｇ／ｍＬ,青霉素浓度１０００μｇ／ｍＬ,头孢唑啉钠浓度为５００μｇ／ｍＬ时对地衣芽孢杆菌有明显抑制作用。这对正确使用地衣芽孢杆菌制剂（整肠生）提供了可靠的理论依据。     

地衣芽孢杆菌的应用现状及展望

地衣芽孢杆菌是一种革兰氏染色呈阳性的益生菌,俗称整肠生,其细胞呈杆状排列、单生,能产生近中生的芽孢,形态为椭圆状,孢囊稍膨大。它在生活中主要应用于饲料添加、医药、工业等方面,本文综述了地衣芽孢杆菌的主要应用。     

地衣芽孢杆菌20386株特性及其生态制剂的研究

作者在1986年从1例正常待产妇女的阴道中,分离出1株革兰氏阳性需氧地衣芽孢杆菌(Bacillus licheniformis)BL_(20386)株。该株体外试验(In Vitro Test)对金葡萄和白念珠菌具有明显的拮抗作用;体内试验(In vivo Test)表明具有调整肠道菌群和治疗作用。对BL_(20386)株的生物特性、生物毒性、生物拮抗、生物耗氧研究显示了安全、有效的指征,符合生态制剂生产用菌株。用该菌株制备的生态制剂—整肠生, 治疗婴幼儿感染性腹泻和非感染性腹泻治愈率分别为70％和85％;成年人细菌感染性腹泻3天治愈率为50％,5天为81％,7天为96.2％。整肠生具有治疗和预防肠道疾病的前景。     

地衣芽孢杆菌TS-01固态发酵培养基的优化

分离到一株饲用地衣芽孢杆菌TS-01,初步研究了该菌的固态发酵培养基,得到的最优发酵培养基为(g/kg干固体物料)含水量600、红糖7、米糠420、麸皮580.当采用此种配比的培养基,接种量为10%(v/w),发酵2 d后,菌体浓度可达9.15×109CFU/g鲜曲,芽孢浓度可达7.50×109CFU/g鲜曲,芽孢形成率在80%以上.     

地衣芽孢杆菌对家兔体液免疫功能的影响研究

本研究采用地衣芽孢杆菌制成微生态制剂,饲喂断奶后的家兔４０天,再用兔病毒性出血病（ＲＨＤ）组织灭活疫苗免疫,检测家兔产生的血清抗体对ＲＨＤ病毒的特异性血凝抑制价和血清免疫球蛋白的含量。结果,试验组家兔的血清抗体对ＲＨＤ病毒的特异性血凝抗体和血清免疫球蛋白含量均明显高于对照组。表明,地衣芽孢杆菌对家兔的体液免疫功能有促进作用,ＲＨＤ组织灭活疫苗与地衣芽孢杆菌制剂相结合较单独使用疫苗的效果更佳     

地衣芽孢杆菌H-1的鉴定及其产木聚糖酶性质的研究

本实验室分离到一株木聚糖酶高产菌,经形态、生理以及生化鉴定,确认为地衣穿孢杆菌,定名为H-1。对H-1的胞外木聚糖酶进行初步的研究。从碳源利用方面,认为 H-1的木聚糖酶为组成型合成。木聚糖和纤维二糖对它有诱导作用,而木糖和葡萄糖对酶的产生有阻抑作用。对酶解产物分析表明,有小分子糖产生、但并非都是单糖。H-1胞外木聚糖酶经60％硫酸铵沉淀,过DEAE-50柱,以及超过滤,得到了部分纯化酶。该酶作用的最适pH为7.6,在pH4～5范围较为稳定;最适温度为50℃; 70℃25min则完全失活。米氏常数为10.42×10~(-3)g/ml,最大反应速度为0.345μmol/min。2mM Ag~+使酶活增加了1.5倍,2mM EDTA抑制了 22％的酶活性,而2mM Cu~(++)则使酶完全失活。     

一株地衣芽孢杆菌产凝乳酶酶学性质研究

目的：对地衣芽孢杆菌所产凝乳酶的酶学特性进行研究。方法：在不同的温度、pH值、底物浓度、不同金属离子等条件下测定地衣芽孢杆菌产凝乳酶相对酶活力。结果：地衣芽孢杆菌所产凝乳酶最适凝乳温度为70℃;40℃以上热处理后凝乳活性有不同程度的损失,75℃热处理10min后凝乳酶活性丧失;pH值为5～8时凝乳活性随乳pH值的降低而增强,pH值为7时凝乳酶最稳定;Ca2+ 、Fe2+、Fe3+、Mn2+、Mg2+、Al3+对酶活性有一定的促进作用;Li2+、Na+、Cu2+、Zn2+对酶活性有抑制作用;底物浓度最适为250 g/L、Ca2+的最适浓度为0.014 g/L。     

地衣芽胞杆菌活菌制剂的临床应用进展

整肠生是应用二十多年的地衣芽胞杆菌活菌制剂,已有大量的研究报道其临床有效性和安全性。地衣芽胞杆菌大量分泌种类丰富的消化酶,通过抑制肠道有害菌生长和促进有益菌增殖调节肠道菌群,并产生杆菌肽、地衣素和乙酸等生物活性物质发挥益生作用。本文总结了益生菌的益生特点,并重点分析了芽胞杆菌的益生特点,归纳了整肠生的作用机制与临床研究现状,揭示了其在肝脏疾病、溃疡性结肠炎、肠易激综合征、幽门螺旋杆菌感染以及病毒感染等疾病中的治疗作用,并对整肠生未来的临床应用方向进行了展望。     

Development of an asporogenic Bacillus licheniformis for the production of keratinase

AIMS: Bacillus licheniformis PWD-1 is a keratin-degrading, spore-forming bacterium isolated from a poultry waste digester. A sporulation-deficient mutant of B. licheniformis PWD-1, named B. licheniformis WBG, was developed and characterized. METHODS AND RESULTS: The mutation was generated using the splicing by overlap extension PCR method (Gene SOEing) to create 256 bp deletion in the spoIIAC gene, which encodes an essential sporulation-specific sigma factor. In vivo gene replacement was accomplished with the use of a temperature-sensitive plasmid that is able to integrate and excise the nucleotide fragment 256 bp from the B. licheniformis chromosome. PCR analysis and DNA sequencing confirmed the spoIIAC gene deletion. Heat-treatment assays and electron microscopy verified the absence of spores. CONCLUSIONS: This asporogenic strain is able to express normal levels of keratinase when compared with its wild-type host. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, a method of constructing a stable sporulation-defective strain was developed. It can be potentially useful as a tool to generate asporogenic strains of Bacillus that retain their industrial capabilities for production of exoproteases and other exozymes.     

Detection and characterization of naturally occurring plasmids in Bacillus licheniformis

Twenty-two Bacillus licheniformis strains, freshly isolated from pasture-land, were studied for the presence of plasmid DNA. Among these strains, 14 were shown to harbor one or more plasmids of different size. Southern-hybridization experiments showed a high homology between all plasmids investigated and a 2.2-kb PvuII/HindIII fragment of pBL1, a B. licheniformis plasmid previously isolated. Three fragments of pBL1, including the 2.2-kb PvuII/HindIII region, were cloned into pJH101 vector. The resulting chimeras were able to transform Bacillus subtilis. The fragment with high homology probably contains the region with the replicative functions of plasmids from B. licheniformis species.     

地衣芽胞杆菌培养条件的研究

应用单因素和正交试验设计试验法对地衣芽胞杆菌在摇瓶水平上进行了培养基配方和培养条件的研究。结果表明：最适培养基配方为山芋淀粉1．0％,豆粕0．6％,玉米芯粉0．8％,K2HPO4 0．1％,MgSO40．1％。最适培养条件为37℃,摇床转速150r／min,250mL三角瓶装液50mL,接种量5．0％,振荡培养48h,pH7．0。在20L自动发酵罐中进行了扩大培养试验,考察溶氧对菌体生长的影响,并根据试验结果进一步扩大至1m^3发酵罐,通过控制搅拌速度和通气量,1m^3发酵罐中地衣芽胞杆菌培养液菌浓为7．2×10^9efu／mL,芽胞率达到90％。     

Co-linear scaffold of the Bacillus licheniformis and Bacillus subtilis genomes and its use to compare their competence genes

We have established the co-linear regions of Bacillus licheniformis, an industrially important bacterium, and Bacillus subtilis, a model bacterium. In the co-linear regions, revealed by PCR, gene content and order are presumed to be conserved. These regions constitute approximately 60% of the compared chromosomes. Sequencing of the competence genes of B. licheniformis allowed us to validate the approach, and to demonstrate how it can be used for the comparative analysis of complex genetic systems. A new insertion sequence, designated IS3Bli1, was discovered in the competence region of the analyzed B. licheniformis strain.     

Heterodimeric Aminopeptidase A from Bacillus licheniformis NS115

An aminopeptidase A (EC 3.4.11.7) was purified to homogeneity from Bacillus licheniformis NS115 and its enzymatic properties were characterized. The enzyme had an apparent molecular mass of 64 kDa, consisting of heterodimeric 42 kDa and 22 kDa subunits, and is a new enzyme from N-terminal analysis of heavy and light subunits. The light suhunit had no catalytic activity against the substrate and apparent K_m values of heavy and whole enzyme were 0.26 and 0.087 mM of γ-glutamyl-p-nitroanilide, respectively.     

Genotypic diversity among Bacillus licheniformis strains from various sources

Bacillus licheniformis is exploited industrially for the production of enzymes and has been shown to exhibit pathogenic properties. Because of these divergent characteristics, questions arise concerning intraspecies diversity. A comparative study by means of combined repetitive polymerase chain reaction, rpoB and gyrA sequencing, 16S rDNA targeted probe analysis, DNA-DNA hybridizations, gelatinase tests and antibiotic susceptibility tests was performed on a set of strains from diverse sources, including strains with pathogenic potential. B. licheniformis was found to consist of two lineages that are distinguished genotypically.