角蛋白酶kerC基因的克隆及序列分析

为克隆分析角蛋白酶ker C基因,本研究以羽毛为底物从11株实验室保存的芽孢杆菌中筛选角蛋白酶产生菌。得到了一株具有高效降解羽毛能力的枯草芽孢杆菌BS10,该菌株3 d即可降解一根完整的羽毛,以羽毛粉、天青角蛋白为底物测定其酶活力,分别达(1.88±0.10)U/mL、(1.79±0.49)U/mL。以同源克隆的方法克隆ker C基因,获得了一条全长1 149 bp的kerC基因(Gen Bank登录号:KX108888),编码383个氨基酸。利用BLAST、ProtParm、SOPMA和MEGA等生物信息学工具对其理化性质、二级结构及系统进化树等进行分析,发现其与NCBI中的角蛋白酶基因相似性达到85%;相对分子质量为39.095 kD,等电点为9.23;该基因蛋白为亲水性蛋白;系统进化树分析结果与菌株信息相吻合。羽毛降解菌的获得可促进废弃羽毛资源化利用;角蛋白酶基因的获得及其分子特征的分析,为进一步通过基因工程手段提高角蛋白酶活性提供了一定的理论依据。     

一株羽毛降解菌产酶特性的初步研究

通过富集培养从土壤中分离到一株能降解羽毛角蛋白的芽孢八叠球菌（编号为GIMN1.015）。以天然羽毛为底物,初步研究了温度、起始pH、辅助碳源以及羽毛底物含量对该菌株的蛋白酶水解活性的影响。结果表明,在羽毛发酵培养基中,菌株GIMN1.015在初始pH 11.0、温度30℃时,蛋白酶活力最强;与培养基中只含有羽毛的发酵过程相比,添加葡萄糖有利于提高蛋白酶的活性;底物浓度为1.5%时蛋白酶活性最高。本试验结果为进一步利用角蛋白降解微生物实现羽毛角蛋白的资源化利用奠定了基础。     

一株羽毛降解菌的分离与鉴定

【目的】从土壤中分离并鉴定羽毛降解菌,测定其生长最适温度及起始pH,并观察酶活动态。【方法】采用系列稀释法和选择培养基法筛选目的菌株,基于16S rRNA基因序列及Biolog方法鉴定其分类地位,利用全自动生长曲线分析仪监测菌株的最适生长条件,并通过测定蛋白水解活性观察其酶活动态。【结果】从混合羽毛的土壤样品中筛选到一株羽毛降解菌,命名为菌株GIMN1.015,初步判定该菌株属于芽孢八叠球菌属(Sporosarcina)。最适生长pH为9.0,温度为30°C。蛋白水解活性最高值出现在培养后96 h。【结论】菌株GIMN1.015在利用羽毛角蛋白资源中具有潜在的应用价值。这是芽孢八叠球菌在羽毛降解方面的首次报道。     

高效羽毛降解菌株的鉴定及发酵条件的优化

通过前期研究筛选获得一株高效降解羽毛的菌株BS8,该菌株在48 h内可将完整羽毛全部降解。研究其对不同动物毛发的降解能力发现,该菌株对羽毛类硬蛋白降解效果最明显。通过形态学、生理生化特性及16S r DNA分析,结果表明该菌株为枯草芽孢杆菌(Bacillus subtilis)。对菌株的最适培养条件进行了单因素试验优化,同时将显著因素进行正交试验。试验结果表明,枯草芽孢杆菌BS8液体发酵的最适培养条件为:起始p H 8.0,发酵温度37℃,接种量2%,羽毛含量1%,转速180 r/min,摇床培养48 h。在上述条件下测得其角蛋白酶活力为23.6 U/m L,相对于优化前18.2 U/m L提高了29.7%。废弃羽毛经枯草芽孢杆菌BS8发酵酶解不仅有利于环境保护,其发酵产物可广泛用于饲料蛋白源及有机肥料。     

酸解羽毛粉代替蛋白胨研制新型细菌培养基

【目的】从工厂下脚料——酸解羽毛粉氨基酸含量高且种类丰富出发,开发出低成本新型细菌培养基,以期资源化该类废弃物。【方法】将酸解羽毛粉代替常用细菌培养基(LB培养基)中的蛋白胨,进行细菌液体发酵试验,比浊法在波长600 nm处比色测定菌液的吸光值,可培养计数法监测细菌数量。【结果】以LB培养基为对照,酸解羽毛粉完全代替蛋白胨液体发酵供试菌株时,菌株的生物量与对照无显著性差异或显著高于对照。培养模式菌株大肠杆菌和枯草芽孢杆菌24 h后,与对照相比,生物量分别增加了21.59%和27.83%。菌株生长曲线表明,生长初期,菌株在新型培养基中生长稍延迟,但含酸解羽毛粉培养基能延长枯草芽孢杆菌的对数生长期,并且两菌株到达稳定期时的生物量均高于对照。可培养计数法结果同样表明,含酸解羽毛粉培养基所培养活菌数量与对照(LB培养基)相比,差异不显著。【结论】用酸解羽毛粉代替LB培养基中蛋白胨进行细菌培养是可行的,可以大大降低生产成本。     

耐高温厨余垃圾降解菌的分离、驯化与应用

为提高厨余垃圾减量效率,从不同来源的垃圾样品中分离筛选高效耐高温降解菌株应用于厨余垃圾降解。经平板涂布和划线分离法筛选共获得18株耐高温菌株,进一步利用平板透明圈法考察菌株对淀粉、蛋白、脂肪和纤维素的降解能力,最终获得4株耐高温强降解能力的菌株;经形态学和分子生物学鉴定,菌株分别为枯草芽孢杆菌(Bacillus subtilis)、短小芽孢杆菌(Bacillus pumilus)、特基拉芽孢杆菌(Bacillus tequilensis)和嗜温鞘氨醇杆菌(Sphingobacterium thalpophilum);为进一步提高厨余垃圾减量性能,以厨余垃圾浸出液作为培养基,对菌株进行定向驯化并制备复合菌剂,应用于厨余垃圾处理器,48 h后厨余垃圾质量减少了73.8%,与对照组相比,质量减少率分别提高约10%和35.1%。本研究利用厨余垃圾浸出液对筛选获得的菌株进行驯化和制备培养,不仅提高了菌剂降解活性,而且实现了废弃物的资源化利用。     

短小芽孢杆菌肉碱转运系统opuC基因的克隆与序列分析

以前期获得的ω-1-羟基脂肪酸高产突变菌株短小芽孢杆菌(Bacillus pumilus)M-F641的总DNA为模板,利用Primer Premier 5.0软件设计4对引物,对决定长链脂肪酸无效降解途径中肉碱转运的OpuC转运系统的基因进行克隆,成功获得了opuCA、opuCB、opuCC和opuCD的基因序列,并利用MEGA 3.1、DNAStar等软件进行序列分析.研究内容将为进一步利用短小芽孢杆菌长链脂肪酸高效转化生产ω-1-羟基脂肪酸菌株奠定基础.     

利用淀粉的碱性蛋白酶工程菌的培养条件

自70年代开始,国内曾筛选出短小芽孢杆菌(Bacillus pumilus) 289、209两株产碱性蛋白酶的菌株。由于这两个菌株以葡萄糖为速效碳源,菌株产碱性蛋白酶的能力受培养基中总糖的制约,且两株菌易受短小芽孢杆菌烈性噬菌体PP_1—PP_10。的感染,因而不利于作为碱性蛋白酶制剂生产用菌。作者已从制革厂毛泥池中分离得到一株碱性蛋白酶产生菌——短小芽孢杆菌cl72,该菌株对PP_1—pp_10。噬菌体不敏感,对地衣芽孢杆菌pL_1噬菌体亦无交叉感染,是野生型的噬菌体抗性菌株。C172菌无淀粉酶基因,仍     

角蛋白单体获得及诱导S.maltophilia DHHJ产角蛋白酶研究

可降解羽毛微生物能以羽毛粉为唯一营养源生长,而羽毛粉是一种大颗粒的不溶性底物,不能直接进入细胞内作为营养源同时作为一级信使来诱导酶基因表达.本文通过化学还原法水解羽毛得到可溶性羽毛角蛋白溶液,利用电泳和质谱验证其分子量约10 kD,为角蛋白单体.分别以该可溶性羽毛角蛋白及角蛋白单体酶解片段为诱导源,在无诱导源、羽毛粉为对照的情况下,测定72 h内嗜麦芽窄食单胞菌(S.maltophilia)DHHJ产生的角蛋白酶的酶活.在无诱导源时,角蛋白酶基因表现本底表达(0.5 U/mL);培养基中添加羽毛粉及角蛋白单体时,角蛋白酶表达量高,分别可达15 U/mL和20 U/mL.并且角蛋白单体酶解片段诱导酶表达较低(2.3 U/mL).该结果初步表明,水溶性角蛋白单体作为信号源与细菌细胞接触或进入细胞内,控制角蛋白酶基因表达.该结果为细菌降解角蛋白分子机制研究奠定基础.     

角蛋白降解菌的筛选及其UV诱变选育

从长年堆积废羽毛的土壤,屠鸡场下水道、池塘底污泥中分离出一株角蛋白降解菌C-1,摇瓶发酵测得其角蛋白酶活达35.6U,经形态观察和生理生化鉴定该菌株属于芽孢杆菌属.对菌株进行Uv诱变,在照射时间为100 s下得到一株产角蛋白酶活较高突变株C-1-4,其酶活比原始出发菌株提高了122%,且遗传性状稳定.     

短小芽孢杆菌(Bacillus pumilus) WHK4以羽毛粉为底物产蛋白酶条件的优化

探索Bacillus pumilusWHK4以羽毛粉为底物产酶的最佳条件和最佳培养基组成。以羽毛粉发酵培养基为基础,首先采用单因子试验考察底物浓度、初始pH、接种量、外加碳源、外加氮源对WHK4产酶活力的影响。在单因子试验的基础上采用正交试验设计对底物浓度、温度、初始pH、接种量、外加(NH4)2SO4、外加麦芽糖进行优化。结果显示:Bacillus pumilusWHK4最佳的产酶条件为初始pH7.38,菌龄16 h,接种量5%,37℃。最佳的培养基组成为:1 L基础发酵培养基,40.0 g羽毛粉,10.0 g(NH4)2SO4和10.0 g麦芽糖。在优化的条件下Bacillus pumilusWHK4 24 h产蛋白酶活力为每毫升90 U。对培养条件和培养基的优化为Bacillus pumilusWHK4产蛋白酶的分离纯化奠定了基础。     

Keratinases and sulfide from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> SLC to recycle feather waste

The aim of this study is to investigate the culture conditions of chicken feather degradation and keratinolytic enzyme production by the recently isolated Bacillus subtilis SLC and to evaluate the potential of the SLC strain to recycle feather waste discarded by the poultry industry. The SLC strain was isolated from the agroindustrial waste of a poultry farm in Brazil and was confirmed to belong to Bacillus subtilis by rDNA gene analysis. There was high keratinase production when the medium was at pH 8 (280 U ml⁻¹). Activity was higher using the inoculum propagated for 72 h on 1% whole feathers supplemented with 0.1% yeast extract. In the enzymatic extract, the keratinases were active in the pH range from 2.0 to 12.0 with a maximum activity at pH 10.0 and temperature 60°C. For gelatinase the best pH was 5.0 and the best temperature was 37°C. All keratinases are serine peptidases. The crude enzymatic extract degraded keratin, gelatin, casein, and hemoglobin. Scanning electron microscopy showed Bacillus cells adhered onto feather surfaces after 98 h of culture and degraded feather filaments were observed. MALDI-TOF mass spectrometric analysis showed multiple peaks from 522 to 892 m/z indicating feather degradation. The presence of sulfide was detected on extracellular medium probably participating in the breakdown of sulfide bridges of the feather keratin. External addition of sulfide increased feather degradation.     

Degradation of raw feather by a novel high molecular weight extracellular protease from newly isolated Bacillus cereus DCUW

Biotreatment of feather wastes and utilization of the degraded products in feed and foodstuffs has been a challenge. In the present study, we have demonstrated the degradation of feather waste by Bacillus cereus DCUW strain isolated during a functional screening based microbial diversity study on East Calcutta Wetland Area. A high molecular weight keratinolytic protease from feather degrading DCUW strain was purified and characterized. Moreover, utilization of degraded products during feather hydrolysis was developed and demonstrated. The purified keratinolytic protease was found to show pH and temperature optima of 8.5 and 50 degrees C, respectively. PMSF was found to inhibit the enzyme completely. The purified enzyme showed molecular weight of 80 kDa (from SDS-PAGE). The protease was found to have broad range substrate specificities that include keratin, casein, collagen, fibrin, BAPNA and gelatin. The protease was identified as minor extracellular protease (Vpr) by RT-PCR and northern blotting techniques. This is the first report describing the characterization of minor extracellular protease (Vpr) and its involvement in feather degradation in B. cereus group of organisms.     

耐碱性木聚糖酶基因在短小芽孢杆菌中高效分泌表达的研究

从木聚糖酶高产短小芽孢杆菌 (Bacilluspumilus)BP5 1中克隆得到木聚糖酶基因xynA ,将其构建在芽孢杆菌表达载体pWH1 5 2 0中得到重组质粒pWSX1 1。xynA由木糖诱导xylA启动子调控xynA表达。采用同源高效表达策略 ,以原生质体转化方法将pWSX1 1转回原始菌株BP5 1中 ,获得重组菌株BPX1 1。通过木糖诱导重组菌株中的xy nA基因高效分泌表达 ,使木聚糖酶产酶活力比原菌株BP5 1提高了 87% ,同时对重组表达的木聚糖酶的酶学性质进行了初步研究     

Cross-Species Induction of Antimicrobial Compounds, Biosurfactants and Quorum-Sensing Inhibitors in Tropical Marine Epibiotic Bacteria by Pathogens and Biofouling Microorganisms

Enhancement or induction of antimicrobial, biosurfactant, and quorum-sensing inhibition property in marine bacteria due to cross-species and cross-genera interactions was investigated. Four marine epibiotic bacteria (Bacillus sp. S3, B. pumilus S8, B. licheniformis D1, and Serratia marcescens V1) displaying antimicrobial activity against pathogenic or biofouling fungi (Candida albicans CA and Yarrowia lipolytica YL), and bacteria (Pseudomonas aeruginosa PA and Bacillus pumilus BP) were chosen for this study. The marine epibiotic bacteria when co-cultivated with the aforementioned fungi or bacteria showed induction or enhancement in antimicrobial activity, biosurfactant production, and quorum-sensing inhibition. Antifungal activity against Y. lipolytica YL was induced by co-cultivation of the pathogens or biofouling strains with the marine Bacillus sp. S3, B. pumilus S8, or B. licheniformis D1. Antibacterial activity against Ps. aeruginosa PA or B. pumilus BP was enhanced in most of the marine isolates after co-cultivation. Biosurfactant activity was significantly increased when cells of B. pumilus BP were co-cultivated with S. marcescens V1, B. pumilus S8, or B. licheniformis D1. Pigment reduction in the quorum-sensing inhibition indicator strain Chromobacterium violaceum 12472 was evident when the marine strain of Bacillus sp. S3 was grown in the presence of the inducer strain Ps. aeruginosa PA, suggesting quorum-sensing inhibition. The study has important ecological and biotechnological implications in terms of microbial competition in natural environments and enhancement of secondary metabolite production.     

新的产弹性蛋白酶菌株的筛选与鉴定

本研究从秸秆中分离得到一株弹性蛋白酶高产菌, 并对该菌株进行鉴定, 以期为实现其工业化生产提供理论依据。采用酪蛋白(脱脂奶粉)进行初筛后, 以弹性蛋白(牛筋)进行复筛, 并对筛选结果进行检验, 然后对所得菌株结合形态、生理生化特性和16S rRNA基因序列进行分类鉴定, 得到一株弹性蛋白酶高产菌株LSF-97。结果显示, 菌株LSF-97与短小芽孢杆菌同源率达到100%, 形态及生理生化特征与模式菌也显示高度一致性, 故将其确定为短小芽孢杆菌。     

Characterization of Bacillus probiotics available for human use

Bacillus species (Bacillus cereus, Bacillus clausii, Bacillus pumilus) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties. Three B. cereus strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize. Spores of one B. cereus strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids. Spores of all strains were immunogenic when they were given orally to mice, but the B. pumilus strain was found to generate particularly high anti-spore immunoglobulin G titers. Spores of B. pumilus and of a laboratory strain of B. subtilis were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of B. pumilus and B. subtilis induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon. The B. pumilus strain and one B. cereus strain (B. cereus var. vietnami) were found to produce a bacteriocin-like activity against other Bacillus species. The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect. However, the three B. cereus strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use.     

Keratinolytic potential of a novel Bacillus sp. P45 isolated from the Amazon basin fish Piaractus mesopotamicus

Bacillus sp. P45, isolated from the intestine of the Amazon basin fish Piaractus mesopotamicus, showed proteolytic activity when grown on skimmed milk and feather meal agar plates. The keratinolytic potential of this strain was evaluated on whole feather broth and human hair broth. Bacillus sp. P45 degraded almost 90% of chicken feathers after 72 h of submerged cultivation on whole feather broth, and the production of extracellular proteases was observed. The formation of thiol groups was also detected during growth, indicating the contribution of sulphitolysis to the efficient hydrolysis of feather keratin. Nevertheless, Bacillus sp. P45 was unable to degrade hair keratin, possibly due to the conformational diversity of this substrate in comparison to feather keratin. Additionally, preliminary results demonstrated that this strain might be utilized in the degradation of recalcitrant collagen-containing wastes. The keratinolytic character of Bacillus sp. P45 might be utilized in environmental-friendly processes such as bioconversion of waste feathers, representing an alternative way of waste management that could lead to the production of value-added products such as microbial biomass, protein hydrolysates and proteolytic enzymes.