野生Bc菌株产黑色素的研究*

Bc58是一株野生蜡状芽孢杆菌菌株,经L-酪氨酸诱导后可产生红棕色色素。通过红外光谱及各种化学测定证明该色素与S igm a公司标准黑色素(M elan in)的性质相似。生测结果显示添加Bc58黑色素的B t制剂经紫外照射5 h后的LC₅₀为16.1μg/mL,与未经紫外照射的B t制剂的LC5015.2μg/mL基本相同,而比未添加黑色素的B t制剂经紫外照射后的杀虫毒力高出近1倍。经SDS-PAGE检测表明该黑色素可保护苏云金杆菌晶体蛋白在紫外光下基本不降解,表明Bc58黑色素     

一株产微生物絮凝剂菌株的分离鉴定及特性*

从活性污泥中分离筛选到一株产絮凝剂的细菌 A2 5,鉴定为巨大芽孢杆菌 Bacillusmegaterium。该菌株产絮凝剂的最适碳源为麦芽糖 ,最适氮源为酵母提取物 ,最适 p H为 7.0～ 10 .0。絮凝剂的形成与菌体生长同步 ,均在 10 h达到最高值。该絮凝剂主要分布在发酵液中 ,另外还有一部分存在于菌体上。所产絮凝剂对供试的各种悬浮液和菌悬液都具有良好的絮凝效果。     

细菌耐热植酸酶基因的克隆及表达*

设计筛选培养基,从203株细菌菌株中筛选到四株可以分解植酸的菌株：SD01N,SD01X,SD01B和SD01D,设计植酸酶基因特异性引物P1和P2,分别以这四株菌的基因组DNA为模板进行扩增,其中菌株SD01N出现一条明显的扩增条带,大小约1．2kb。对PCR产物进行序列分析表明,该片段含有一个编码383个氨基酸的开放阅读框架。将该片段与载体pQE-30连接后转化大肠杆菌M15,得到重组菌株SDLiuTP01,对该菌株进行培养,经IPIG诱导基因表达,与携带空载体菌株相比较,在菌株SDLiuTP01中可检测到植酸酶活力。对重组菌株的植酸酶活力考察表明,该酶在25℃～95℃温度范围均具生物活性,最适反应温度为75℃,属于耐热性植酸酶。在各pH缓冲系统中反应结果,酶活力出现两个较高值,分别为pH4．6和pH7．5。     

向日葵籽壳黑色素的分离提取及生物活性研究*

目的:天然黑色素安全性高,兼具多种生物活性,深受消费者青睐,发展前景十分广阔。研究向日葵籽壳黑色素制备条件,以及抗氧化和吸附重金属生物活性,为其进一步应用提供理论基础。方法: 采用热碱提取、酸水解、有机溶剂洗涤和反复沉淀法,结合单因素与响应面分析法实现了黑色素的高效制备,利用氯化硝基四氮唑蓝还原法、分光光度法和静态吸附法进行抗氧化活性和吸附活性测定。结果: 成功制备了向日葵籽壳黑色素。提取结果表明最优工艺为氢氧化钠浓度0.11 mol/L、提取温度75.19℃、提取时间181.16 min、料液比为15.77∶1,向日葵籽壳黑色素得率为2.95%,与预测值相近,说明模型拟合良好,该优化工艺准确可行。抗氧化活性测定结果表明,向日葵籽壳黑色素清除超氧阴离子自由基、DPPH自由基的能力和还原力都高于合成型黑色素。吸附活性测定结果表明,向日葵籽壳黑色素对Pb²⁺、Cu²⁺和Cr³⁺的吸附效率高于合成型黑色素。结论: 与其合成型黑色素相比,天然黑色素既可作为着色剂,又兼抗氧化剂和吸附剂,且不亚于合成型黑色素的效果,可用于代替合成型黑色素开发的重要候选。     

产胞外黑色素菌株的筛选*

对不同来源获得的47株菌株在酪素培养基上生长、产色素情况进行了对比研究。从中选取了T4和Neurospora crassa AS3．1602,比较了二利用5种不同培养基产黑色素的能力。对T4菌株产生的黑色素做了初步研究,并初步鉴定T4菌株为奇异变形杆菌(Proteus mirabilis）。     

一株代谢煤油兼性厌氧菌的筛选鉴定和特性*

从大港油田的油层水样中分离得到一株能以煤油为碳源代谢生成表面活性剂、产酸和产气的菌株DG7,该菌为革兰氏阴性杆菌 ,兼性厌氧 ,运动 ,能在NaCl浓度为 18.5%的培养基中生长 ,经鉴定 ,可能为气单胞杆菌属 (Aeromonassp.)中的一个新种 ,但还需进一步确定。     

用快中子法选育细菌脂肪酶高产菌株*

研究了从土壤中筛选产脂肪酶的菌株,利用紫外线、快中子、快中子和磁场复合、γ射线、γ射线和磁场复合诱变,以酶活为筛子进行诱变育种。结果,出发菌酶活较低的一株得到了一株酶活为３９６．２２Ｕ／ｍＬ的诱变株,此酶活比出发菌株高９２倍,并发现此菌对紫外线和快中子比较敏感;而出发菌酶活较高的一株得到了酶活为４２４．６０Ｕ／ｍＬ发酵液的诱变株,此酶活为出发菌株３．０倍。在此基础上,初步探讨了快中子、γ射线及磁场复合处理在产脂肪酸菌种诱变中的作用,并认为,在产脂肪酸菌株的诱变中快中子诱变更为有效。     

脂肽的分离纯化与结构研究*

采用酸沉淀分离、有机溶剂提取、分级沉淀、脱色吸附及制备薄板的方法,从枯草芽孢杆菌HSO121的培养液中分离得到一种脂肽类化合物。茚三酮试验、红外光谱分析表明该脂肽具有环状结构,电喷雾离子化质谱结果显示,该脂肽是分子量为1022D和1036D的两个同系物,氨基酸分析及串联质谱分析表明,脂肽的肽链结构为Leu-Leu-AspValLeu-Leu-Glu,说明它是Surfactin的结构类似物。     

一株海绵放线菌的分离鉴定及活性研究*

从大连潮间带繁茂膜海绵中分离到一株具有很强抑制革兰氏阳性菌活性和较强抗肿瘤活性的链霉菌。通过其形态观察,生理生化测定和16SrDNA序列的测定,初步判定属于假浅灰链霉菌(Streptomyces pseudogriseolus)。并对其活性物质的生产条件进行了初步探索和优化,确定了用天然海水配制的TSB培养基为最佳发酵培养基,3d为最适发酵时间。     

Diverse genes of alkaliphilic Bacillus firmus OF4 that complement K+-uptake-deficient Escherichia coli include an ftsH homologue

Seven clones isolated from libraries of DNA from alkaliphilic Bacillus firmus OF4 restored the growth of a K⁺-uptake-deficient Escherichia coli mutant on only 10mM K⁺. None of the clones contained genes with apparent homology to known K⁺ transport systems in other organisms. Based on sequence homologies, the newly isolated alkaliphile loci included: ftsH; a dipeptide transport system; a gerC locus with hydrophobic open reading frames not found in the comparable locus of Bacillus subtilis; a sugar phosphotransferase enzyme; and a capBC homologue. The ftsH gene provided a new and striking example of a recognized property of extracellular and external regions of polytopic alkaliphile proteins: a significant paucity of basic amino acid residues relative to neutrophile counterparts. The alkaliphile ftsH gene was able to complement a mutant of E. coli with a temperature-sensitive ftsH gene product. Received: 5 August 1996 / Accepted: 14 October 1996     

坚强芽孢杆菌三个淀粉酶基因的克隆和表达

以pUC18为载体,用鸟枪法从产淀粉水解酶的坚强芽孢杆菌725菌株中得到三个产淀粉水解酶的重组质粒,在大肠杆菌中表达。用高压液相色谱分析了三个表达的酶的淀粉水解产物,其中pBA135和pBA150表达的酶的淀粉水解产物主要是麦芽糖,具β-淀粉酶的性质。pBA140表达的酶的淀粉水解主要产物除麦芽糖外还有一糖,三糖和四糖。pBA135编码的酶有较好的热稳定性,60℃保温30min,活性保留70％以上,最适反应温度55-60℃。而在同样条件下pBA150编码的酶仅保留37％的酶活,最适反应温度50℃。     

野生Bc菌株产黑色素的研究

Bc58是一株野生蜡状芽抱杆菌菌株,经L-酪氨酸诱导后可产生红棕色色素。通过红外光谱及各种化学测定证明该色素与Sigma公司标准黑色素（Melanin）的性质相似。生测结果显示添加Bc58黑色素的Bt制剂经紫外照射5h后的LC50为16．1μg／mL,与未经紫外照射的Bt制剂的LC50 15．2μg／mL基本相同,而比未添加黑色素的Bt制剂经紫外照射后的杀虫毒力高出近1倍。经SDS—PAGE检测表明该黑色素可保护苏云金杆菌晶体蛋白在紫外光下基本不降解,表明Bc58黑色素是一种优良的紫外保护剂。     

苏云金芽孢杆菌酪氨酸酶基因的克隆表达及应用初探

酪氨酸酶基因编码的酪氨酸酶是生物体合成黑色素的关键酶。采用比较酪氨酸酶的同源保守结构域氨基酸序列的方法设计引物 ,从苏云金芽胞杆菌 (Bacillusthuringiensis) 4D11中通过PCR扩增得到了包含酪氨酸酶基因的DNA片段。将该片段亚克隆到载体pGEM_7zf上并转入大肠杆菌DH5α ,所得到的转化子在添加了L_酪氨酸的LB培养基中能合成可溶性的黑色素。测定该菌株黑色素的产量和在紫外光照射后的菌体活力 ,结果表明该基因产生的黑色素能在一定程度上保护菌体免受紫外辐射     

An Extracellullar Nuclease from Bacillus Firmus VKPACU-1: Specificity and Mode of Action

An extracellular nuclease from Bacillus firmus VKPACU-1 was multifunctional enzyme, this nuclease hydrolyzed poly U rapidly and more preferentially than the other homopolyribonucleotides. Hydrolysis of RNA this enzyme released mononucleotides in the order 5′UMP > 5′AMP > 5′GMP where as in hydrolysis of DNA the mononucleotides in the order of 5′dAMP > 5′dGMP > 5′dTMP and oligonucleotides. Uridylic linkages in RNA and adenylic linkages in DNA were preferentially cleaved by the nuclease. Nuclease produced oligonucleotides having only 3’ hydroxyl and 5’ phosphate termini. Present nuclease hydrolyzed RNA and DNA released oligonucleotides as major end products and mononucleotides, suggesting an endo mode of action.     

Production of cyclodextrin from starch by cyclodextrin glycosyltransferase from Bacillus firmus and characterization of purified enzyme

During screening for cyclodextrin-forming microorganisms, an alkalophilic Bacillus sp, which produced high activity of cyclodextrin glycosyltransferase, was isolated and identified as Bacillus firmus. The crude enzyme transformed starch to mainly β-and γ-cyclodextrin. The purified enzyme had an optimum pH of 7.5–8.5 and its optimum temperature was 65°C, which is the highest optimum temperature as compared to other cyclodextrin glycosyltransferases except that produced by Bacillus amyloliquefaciens. Received 06 January 1997/ Accepted in revised form 20 March 1997     

Sequence analysis and functional studies of a chromosomal region of alkaliphilic Bacillus firmus OF4 encoding an ABC-type transporter with similarity of sequence and Na+ exclusion capacity to the Bacillus subtilis NatAB transporter

A 14.1-kb DNA fragment was cloned from a lambda library containing inserts of DNA from alkaliphilic Bacillus firmus OF4 on the basis of its hybridization to a probe from a previously sequenced alkaliphile homolog of the natA gene from Bacillus subtilis. Sequence analysis of the entire fragment revealed that, as in B. subtilis, the natA gene was part of a putative gene locus encoding an ABC-type transporter. In the alkaliphile, the transporter involved three genes, designated natCAB, that are part of a larger operon of unknown function. This is in contrast to the two-gene natAB operon and to another homolog from B. subtilis, the yhaQP genes. Like natAB, however, the alkaliphile natCAB catalyzes Na⁺ extrusion as assessed in a mutant of Escherichia coli that is deficient in Na⁺ extrusion. The full 14.1-kb fragment of alkaliphile DNA sequenced in this study contained several probable operons as well as likely monocistronic units. Among the 17 predicted ORFs apart from natCAB were acsA, a homolog of a halobacterial gene encoding acetylCoA synthetase; sspA, a homolog of a small acid-soluble spore protein; and malK, an ATP-binding component that was unaccompanied by candidates for other mal transport genes but was able to complement a malK-deficient mutant of E. coli. No strong candidates for genes encoding a secondary Na⁺/H⁺ antiporter were found in the fragment, either from the sequence analysis or from analyses of complementation of E. coli mutants by subclones of the 14.1-kb piece. There were a total of 12 ORFs whose closest and significant homologs were genes from B. subtilis; of these, one-third were in apparently different contexts, as assessed by the sequence of the neighboring genes, than the B. subtilis homologs. Received: August 30, 1998 / Accepted: November 13, 1998     

Potential of indole-3-acetic acid-producing rhizobacteria to resist Pb toxicity in polluted soil

Heavy metal toxicity in industrial polluted area is imparting serious consequences on crops. Two Pb-tolerant bacteria were isolated from maize growing in a dumping site of Attock Oil Refinery, Rawalpindi. The oil-polluted field had a higher geo-accumulation index (I_geo) and pollution load index (PLI) value for Pb than standard. The soil accumulated higher Cd, Zn, and Mn contents too. Maize leaves and roots accumulated higher Pb and Zn and exhibited biological concentration factor (BCF), biological accumulating factor (BAC), and translocation factor (TF) Zn. Two bacterial strains (Exiguobacterium aurantiacum and Bacillus firmus) were isolated from maize rhizosphere growing in an oil-polluted field and applied as bio-inoculants on maize in a greenhouse experiment for 80 days. Both bio-inoculants were tolerant to Pb at 500 ppm and had the potential to produce indole-3-acetic acid (IAA) in the presence or absence of Pb. Results revealed that single inoculation of bio-inoculants decreased Pb contents in the soil, leaves, and roots of maize by 30% over the control. Growth and physiological attributes of maize were also improved by 25% in a single application of bio-inoculants. Application of Pb with bio-inoculants decreased the efficiency of PGPR, and there were only 10–15% increases in growth and physiological attributes over single inoculation. Bio-inoculants exhibited the best results in the presence of IAA and Pb application by intensive root growth (60% better than control), reducing Pb toxicity (38%) and increasing growth and physiological attributes by 10–15% over single inoculation of bio-inoculants. Application of bio-inoculants with IAA may decrease the deleterious effects of Pb toxicity in oil-polluted agriculture fields.     

坚强芽胞杆菌主要分泌蛋白的鉴定及其分泌性序列

[目的]坚强芽胞杆菌是一种在自然界普遍存在的益生菌,在对虾养殖中应用较为广泛.为了研究其分泌性蛋白从而为分泌性载体的构建提供理论依据,本文对坚强芽胞杆菌的主要分泌蛋白进行质谱鉴定及分泌性序列的分析.[方法]从本实验室分离保存的1株来自对虾肠道的坚强芽胞杆菌(Bacillus firmus)培养液中获取了分泌性蛋白,进行SDS-PAGE,并对表达量较高的3条蛋白区带进行MALDI-TOF/TOF质谱鉴定及克隆测序,进行生物信息学分析.[结果]鉴定出的蛋白分别是坚强芽胞杆菌几丁质酶(chitinase)、坚强芽胞杆菌肠毒素A(enterotoxin A)和坚强芽胞杆菌BCG9842蛋白(hypothetical protein BCG9842).经在线软件SignaIP 3.0分析,确定chitinase、enterotoxin和BCG9842均存在不同的分泌性信号肽序列bf-43、bf-37和bf-16,通过在线软件PSORT分析表明,bf-43定位于细胞的外膜上,bf-37和bf-16定位于细胞的胞外.[结论]本研究鉴定出了坚强芽胞杆菌的3个主要分泌性蛋白,分析筛选出了3条分泌性序列,为分泌性载体的构建提供理论依据.