一株产纤维素酶的甲醇利用细菌的鉴定及其纤维素降解条件优化

采用刚果红染色法,从废弃矿山周边土壤中筛选出一株产纤维素酶的甲醇利用细菌,命名为xt-04。形态特征、生理试验及16SrDNA序列和gyrB序列分析表明,该菌株属于Bacillusmethylotrophicus。为提高该菌所产纤维素酶的降解能力,首先通过单因子实验考察了底物CMC—Na浓度、反应温度及缓冲液pH值对纤维素酶活力的影响;然后采用响应面分析法对影响纤维素酶活力的3个单因子进行了优化。结果表明,单因素实验得出的适宜反应温度、缓冲液pH和底物浓度分别为70℃、5．0和2％（20mg／mL）;响应面法得出的最高酶活力条件：反应温度、pH和底物浓度分别为66．1℃、4．81和19．01mg／mL。在最优条件下,酶活力达到17．85U／mL,比优化前的酶活力12．84U／mL提高了39．01％。因此,鉴于这种纤维素酶能耐受较高温度和酸性条件,该菌株所产纤维素酶可能在工业中具有良好的应用前景。     

一株纤维素降解细菌的筛选、鉴定及产酶条件分析

目的筛选高活性的纤维素降解细菌,并进行初步鉴定和产纤维素酶条件分析。方法采集吉首旗帜山松树林的土壤样品,通过富集培养和刚果红平板染色法筛选分离纤维素降解细菌;通过形态观察、生理生化特性检测和基于16S rRNA基因序列的系统发育分析对分离的菌株进行初步鉴定。利用单因素实验对产纤维素酶条件进行优化。结果分离获得1株高活性纤维素降解细菌JDM11,初步鉴定其为Bacillus velezensis;菌株JMD11产纤维素酶最佳培养温度、最适初始pH和培养时间分别为28℃、7.0～7.5和32h,在该条件下其滤纸酶(FPase)和羧甲基纤维素酶(CMCase)活力分别为260.32U/ml和651.75U/ml。结论菌株JDM11是1株高活性纤维素降解的Bacillus velezensis。     

降解棉秸秆耐热菌株的鉴定及降解条件优化

【目的】鉴定从新疆棉花秸秆高温堆肥中分离出的两株耐热真菌Z1、Z2的属种,并通过优化影响菌株产生纤维素酶的因素来提高菌株对秸秆的降解率。【方法】经形态学和菌株的ITS区克隆与序列分析确定属种,以液体摇瓶发酵产滤纸酶活性(FPA)变化为衡量指标,对Z1、Z2以及二者混合菌(MS)的纤维素酶产生条件进行优化。【结果】菌株Z1为曲霉属烟曲霉(Aspergillus fumigatus Fresen),Z2为蚀丝霉属(Myceliophthora Cost.)。确定Z1以棉秸秆为碳源、以NaNO3为氮源、起始pH 9.5、接种量11%、50°C摇床培养10 d,对棉秸秆降解率为10.19%;Z2以麦秸秆为碳源、以NaNO3为氮源、起始pH 5.5、接种量9%、50°C摇床培养10 d,对麦秆降解率为27.50%;MS以棉花秸秆为碳源、以蛋白胨为氮源、起始pH 5.5、接种量11%、50°C摇床培养10 d,对棉秸秆的降解率为53.45%。【结论】实验表明,MS(Z1、Z2混合)对秸秆的降解效果优于单株菌,降解率达到一半以上,本研究中的两株耐热真菌在降解棉花秸秆、小麦秸秆等农作物废弃秸秆中具有较高的应用价值。     

一株高效菲降解菌的筛选及降解条件研究

从南京某石化厂排污口附近采集土样,以菲为碳源的选择性培养基分离筛选到一株菲高效降解菌F10a,根据形态和生理生化特性初步鉴定为芽孢杆菌属,并对其降解菲的特性及各种影响因素进行了研究.结果表明,F10a在50 mg·L^-1的条件下,28 ℃振荡培养27 h,菲的降解率达到98.12%;静置培养8 h,菲的降解率达到98.7%.pH值分别为、6、8时,F10a对菲具有良好的降解效能;pH值为10时F10a不生长.Zn²⁺与Pb²⁺的存在不影响F10a的降解效能,Cu²⁺可以延缓菲的降解,Cr²⁺对F10a有毒性.F10a在菲浓度为200 mg·L^-1时,28 ℃振荡培养8 h,降解率为99.6%.菲的降解程度与细菌数量的增长呈正相关关系.     

一株敌草胺降解菌的分离鉴定及降解性能

为探讨酰胺类除草剂敌草胺的微生物降解机理和土壤中敌草胺降解的生物强化性能,从长期使用敌草胺的烟田土壤中分离到一株敌草胺降解细菌菌株,命名为LGY06.经形态特征、生理生化特性及16S rDNA序列分析将其鉴定为蜡状芽孢杆菌.在纯培养条件下,菌株LGY06对敌草胺的降解符合一级动力学特征,7 d内对50 mg·L^-1敌草胺降解率达到75.7%;LGY06降解敌草胺的最适温度和最适pH值分别为35 ℃和8.0.通过气相色谱-质谱鉴定了菌株LGY06降解敌草胺的降解产物并分析了其降解途径,α-萘酚和丙酰胺是主要的降解产物,LGY06对敌草胺的作用方式主要有脱烷基、氧化(或水解),为矿化过程.室内模拟条件下,LGY06能有效促进土壤中敌草胺残留的降解,与未接种灭菌土、非根际土和根际土相比,敌草胺在接种LGY06土壤中的降解半衰期分别缩短了79.5%、36.6%和41.1%.     

玉米赤霉烯酮降解菌的鉴定及其降解特性

【背景】玉米赤霉烯酮(Zearalenone,ZEN)是一种具有类雌激素作用的霉菌毒素,常会污染谷物和饲料,严重威胁动物和人类的健康。生物脱毒作为理想的去除ZEN的方法,广受关注,然而相关菌株较少,仍有待进一步筛选。【目的】明确一株玉米赤霉烯酮降解菌的生物学分类地位,并优化其赤霉烯酮降解菌降解条件。【方法】通过菌株的16S rRNA基因序列比对,构建系统发育进化树,并开展了相关培养条件的单因素优化和玉米赤霉烯酮降解动力曲线的绘制。【结果】实验菌株WLB-29经鉴定为斯塔普氏菌属(Stappia),其16S rRNA基因序列在GenBank上登录号为MT196321,该序列与模式菌株Stappia indica B106T相似性最高为97.47%,初步确定为斯塔普氏菌属潜在新种。单因素优化表明,菌株降解玉米赤霉烯酮的最佳条件为LB培养基、37℃培养、pH 8.0、2%接种量和玉米赤霉烯酮初始浓度为10mg/L,在此条件下培养144h后,玉米赤霉烯酮的降解率最高可达92.56%。【结论】菌株WLB-29具有较好的ZEN降解作用,为进一步解析菌株降解ZEN作用机理提供了研究基础,也为进一步开发利用菌株开展ZEN的生物脱毒提供了新的菌株资源。     

Vitamin B 12 formation by a gram-variable methanol-utilizing bacterium

Summary A methanol-utilizing bacterium isolated from soil was studied for ability to produce vitamin B 12. The isolated strain XF is gram-variable, pink-pigmented and rod-shaped. Effects of growth conditions on vitamin B 12 productivity were examined. The maximal yield of vitamin B 12 was 280 g/l in shaked flasks and 236 g/l in fermentation jars.     

Construction of a promoter-probe vector for the methanol-utilizing bacterium acetobacter methanolicus MB 58

We report here the construction of a promoter-probe vector, pRS2, which can be utilized in either Acetobacter methanolicus MB 58 or Escherichia coli due to the presence of broad-host-range replicon RSF 1010. The vector provides several unique restriction sites for promoter cloning as well as resistance markers for the selection of transformants. The promoter-probe vector was constructed by inserting an EcoRI-SalI-polylinker fragment of pUC 19 into EcoRI/SalI digested pMK 16. The resulting plasmid, pRS1, was cloned into the unique EcoRI site of the broad-host-range plasmid RSF 1010. The vector was used to clone promoter-containing sequences derived from the A. methanolicus MB 58 chromosome as well as the E. coli lac-promoter.     

高浓度氯苯优势降解菌的筛选及其降解酶的纯化

[目的]分离纯化出一株高浓度氯苯优势降解菌株,对其所产氯苯降解酶进行分离与纯化,为该菌株及其氯苯降解酶的研究提供理论参考.[方法]利用梯度富集培养技术和无菌滤纸片平板法分离菌株,通过形态特征及16S rRNA基因序列分析初步鉴定菌株,用气相色谱法测定培养液中氯苯浓度,以单位细胞氯苯降解率评价菌株对氯苯的降解能力,以氯苯降解率表示氯苯降解酶的活性.取纯化菌株的发酵酶液制备粗酶液,经硫酸铵梯度盐析、透析脱盐、DE-52离子交换层析、G-100凝胶层析和透析浓缩后,进行SDS-PAGE凝胶电泳检验酶的纯度并测定酶的分子量.[结果]从氯苯长期驯化的成熟期活性污泥中筛选到一株以氯苯为唯一碳源和能源的氯苯优势降解细菌LW13,该菌株在以2000 mg/L氯苯为唯一碳源的无机盐培养基中仍能正常生长,其单位细胞氯苯降解率可达1.37 ×10-10.扫描电镜观察到该菌株细胞大小约为2.3 ×0.8μm,长有数根端生鞭毛.16S rRNA基因序列相似性比较表明该菌株与Lysinibacillus fusiformis(溶藻菌)的相似性达95.5％.所纯化的氯苯降解酶为胞外酶,带正电荷,其分子大小约为57 kDa.整个纯化过程中酶纯化倍数化达8.0倍,酶活回收率达52.51％,酶量回收率达6.57％.纯化后的氯苯降解酶在30℃-55℃和pH在6.0-8.0之间都保持较高的酶活性,其最适反应温度和pH分别在40℃和pH8.0左右.[结论]所分离的氯苯优势降解菌属于Lysinibacillus属菌株,该菌株能有效降解高浓度(500-2000 mg/L)氯苯废水,通过逐级分离纯化,可获得氯苯降解酶纯酶,纯化指标符合分离纯化基本规律,纯化效果较为理想.     

The growth dynamics of a methanol-utilizing bacterium L3 in a batch bioreactor

The growth dynamics of a methanol utilizing bacterium, L3, in batch bioreactors were experimentally investigated. Formaldehyde, a key intermediate of methanol oxidation, is indicated to have a significant role in the complex batch growth behavior of L3. The intricate batch growth dynamics of many microorganisms can be elegantly characterized by examining the specific rates of exchange of nutrients and products between the cells and the cellular environment. Following such an analysis, the batch growth of L3 on methanol was characterized by the presence of unbalanced and balanced growth phases. The nature and significance of nutrient and product concentration profiles or semilog-arithmic profiles of nutrient and product exchange rates during balanced and unbalanced growth phases are also outlined.     

一株氯嘧磺隆降解菌分离鉴定及降解条件优化

为解决氯嘧磺隆残留对土壤、水体污染及后茬敏感作物药害问题,为污染土壤微生物修复提供降解菌种资源,文中采用富集培养、逐级驯化等方法,从氯嘧磺隆污染土壤中分离到1株高效氯嘧磺隆降解菌T9DB-01,经形态特征、生理生化及16S rDNA序列分析,鉴定为假单胞菌Pseudomonas sp.。采用单因素实验探究温度、pH值、底物浓度、装液量和接种量对菌株T9DB-01降解氯嘧磺隆的影响,采用正交试验及验证,优化菌株T9DB-01对氯嘧磺隆降解条件。结果表明,在30℃,pH 8.0,底物浓度200 mg/L,装液量100 mL/250 mL,接种量4%的条件下,5 d后降解率达到93.7%。该降解菌株对氯嘧磺隆污染土壤原位生物修复具有一定的应用潜力。     

新的纤溶酶产生菌培养条件及其酶性质研究

采用定向筛选方法,分离到一株能产生强烈分解纤维蛋白的枯草芽孢杆菌（Bacillus subtilis)EM29-5。对其生长,产酶条件,酶性质进行系统研究,结果表明：生长和产酶温度25－37℃,37℃时最好,生长最适pH6.4-7.0,产酶为pH7.0-8.0;12种碳源氮源均能利用,但对生长和产酶均有利的是大豆浸出淮EM29－5。纤溶酶为外分泌蛋白酶,发酵上清液经硫酸铵沉淀,透析脱盐,上离子交换柱和凝胶过滤柱后得纯酶。经SDS－PAGE电泳得单一条带,分子量为32500左右;等电聚焦电泳分析PI9．0左右;酶蛋白在pH5.0-10.0范围内稳定,作用最适pH9.0,为碱性蛋白酶;5mmPMSF能完全抑制酶活性,为氨酶蛋白酶;不能分解人工合成的氨基酸的酯,而能分解纤维蛋白（原）,特别对交联纤维蛋白具更高亲和性。     

Cloning of formate dehydrogenase gene from a methanol-utilizing bacterium Mycobacterium vaccae N10

The gene of NAD⁺-dependent formate dehydrogenase (FDH) from Mycobacterium vaccae N10 was cloned into Escherichia coli by hybridization with digoxigenin-labeled DNA probes, which were prepared by amplification of the chromosomal DNA from the bacterium by the polymerase chain reaction with degenerate primers. The primers were designed on the basis of the most conserved parts of known sequences of FDH from different organisms. An open-reading frame of 1200 bp exhibited extremely high sequence similarity to the FDH gene of Pseudomonas sp. 101. The deduced amino acid sequence of FDH from Mycobacterium vaccae N10 (McFDH) was identical to that of Pseudomonas sp. 101 (PsFDH) except for two amino acid residues: isoleucine-35 (threonine in PsFDH) and glutamate-61 (lysine in PsFDH). The physicochemical properties of both enzymes appeared to be closely similar to each other, but the thermostability of McFDH was a little lower than that of PsFDH. To examine the role of the two amino acid residues in the thermostability of the enzymes, glutamate-61 of McFDH was replaced by glutaminyl, prolyl and lysyl residues by site-directed mutagenesis. All the mutant enzymes showed higher thermostability than the wild-type McFDH. The negative charge of glutamate-61 contributes to the stability of the wild-type enzyme being lower than that of PsFDH.     

Isolation of a homocysteine gamma-lyase-producing bacterium and study of its enzyme production conditions

Aim: To investigate the possibility of finding a new homocysteine (Hcy) γ‐lyase with the desired properties for Hcy measurement in bacteria. Methods and Results: Through a process of enrichment, the Hcy γ‐lyase‐producing bacterium strain N2‐1 was isolated from soil. Based upon its morphological, physiological, and biochemical characteristics, as well as its 16S rDNA sequence and phylogenetic tree analysis, this isolate belongs to the genus Serratia. The effects of pH, aeration, inducers, carbon (C) and nitrogen (N) sources on enzyme production were studied. Methionine, yeast extract, and glucose were selected as the optimal inducer, C and N sources, respectively. Maximum production of Hcy γ‐lyase was obtained when the isolate was cultured at 30°C at pH 6·5 for about 36 h in the optimum medium. Results also showed that this Hcy γ‐lyase has relatively high specificity towards Hcy. Conclusions: Because of its high specificity for Hcy, this bacterial Hcy γ‐lyase has the potential application in Hcy determination. Significance and Impact of the Study: In addition to isolating a bacterium that produces Hcy γ‐lyase suitable for Hcy determination, this study also indicates that the bacterium could be a source for production of Hcy γ‐lyase for clinical applications.     

Isolation of a thermostable uricase-producing bacterium and study on its enzyme production conditions

A uricase-producing bacterium was isolated from soil with a medium containing uric acid as the only carbon source. Based on its morphological and physiological characteristics, as well as 16S rDNA sequence and phylogenetic tree analysis, this new isolate belong to the genus Microbacterium. After heat treatment at 70 °C for 30 min, the uricase retained about 100% of the initial activity. The enzyme activity remained largely unchanged when it was stored in borate buffer at pH 8.5 at 37 °C for 40 days. The effects of different factors on the enzyme production were studied. Maize milk was the best C and N resources, and the uric acid showed to be an inducer for uricase production. When the strain was cultured at 30 °C at pH 7.5 for 30–36 h, the uricase activity peaked at 1.0 U/ml.     

一株苯酚降解菌的鉴定及其降解特性

【目的】鉴定从某化工厂附近土样中分离到的一株耐高浓度苯酚的菌株T10,通过优化菌株的培养条件提高菌株对苯酚的降解率。【方法】根据菌株的形态、生理生化鉴定及16S rDNA测序分析确定其种属,以液体摇瓶培养菌株T10对苯酚的降解率为指标,对菌株的生长条件进行优化。【结果】菌株T10属恶臭假单胞菌(Pseudomonas putida)。添加葡萄糖、蛋白胨能有效缩短T10菌的生长周期,并使苯酚的降解率提高1.7倍。在菌体初始接种浓度为10%、温度为30°C、转速为180 r/min条件下,对初始苯酚浓度、pH和装液量的响应面优化结果如下:初始苯酚浓度3 000 mg/L、pH 7.5和装液量80 mL/250 mL,苯酚去除率最高可达到87.56%。【结论】T10菌能够耐受较高浓度的含酚废水,并且对苯酚有较强的降解能力,为下一步利用生物法处理含酚废水提供科学依据。     

一株产表面活性剂细菌鉴定及发酵条件优化

本文利用16S rDNA序列并结合形态学特征,鉴定实验室前期从油污土壤中分离的产表面活性剂菌株1098-3为大肠埃希氏菌(Escherichia coli).通过单因子实验初步确定了其产生表面活性剂的最适条件:37℃,初始pH7.0,转速200 r/min,培养基配比为可溶性淀粉2.5％,胰蛋白胨1.5％,氯化钠0.3％,磷酸二氢钾0.5％,氯化钙0.004％,硫酸铵0.6％,硫酸镁0.07％,酵母粉0.06％,500 mL三角瓶装液量为200 mL.在此条件下,发酵液表面张力由69.3 mN/m降至34.3mN/m,此时发酵液中表面活性剂相对浓度(RBC)为500.     

Methylobacterium soli sp. nov. a methanol-utilizing bacterium isolated from the forest soil

A Gram-negative, pink-pigmented, non-spore-forming rod shaped, methanol-utilizing bacterium, strain YIM 48816(T), was isolated from forest soil collected from Sichuan province, China. Strain YIM 48816(T) can grow at 4-37 °C, pH 5.0-7.0 and 0% NaCl (w/v). Based on 16S rRNA gene sequence similarity studies, it belonged to the genus Methylobacterium, and formed a phyletic line. The 16S rRNA gene sequence similarities were 96.2% to Methylobacterium mesophilicum DSM 1708(T) and 96.0% to Methylobacterium brachiatum DSM 19569(T), and the phylogenetic similarities to all other Methylobacterium species with validly published names were less than 96.0%. The major menaquinones detected were Q-10 (97.14%) and Q-9 (2.86%). The major fatty acids were C18:1 ω7c (80.84%). The DNA G + C content was 66.2 mol%. It is apparent from the genotypic and phenotypic data that strain YIM 48816(T) belongs to a novel species of the genus Methylobacterium, for which the name Methylobacterium soli sp. nov. is proposed. The type strain is YIM 48816(T) (CCTCC AA 208027(T) = KCTC 22810(T)).     

一株异养型细菌对无机硫化物的降解特性和培养条件优化

畜禽屠宰加工、鱼粉饲料加工等一些食品工业生产过程中会释放出大量的硫化氢恶臭气体,导致周边环境的严重污染。为实现以培养异养型细菌脱除硫化氢气体的目的,取分离到的异养脱硫细菌XJ-2,通过诱变筛选得到一株高效脱硫菌株ZJNB-B3,其脱硫率达97%。基于形态学研究、API 50 CHB生理生化鉴定及16S r RNA基因测序,鉴定该菌为蜡状芽胞杆菌Bacillus cereus ZJNB-B3。该菌Gen Bank登录号为MF679650。降解特性研究表明,ZJNB-B3菌株对有毒的硫化物有较高耐受性,耐受上限高达300 mg/L。采用响应面法优化环境因素对菌株降解硫化物效率的影响,得到在最适培养温度30℃下,初始S~2–浓度为211.8 mg/L、初始p H值6.72、接种量为5.04%时,菌株氧化脱硫效果最显著,经过实测在48 h产生的硫酸盐浓度为63.8 mg/L,脱硫率达97.3%。菌株在氧化硫化物时不会产生硫酸抑制菌株的生长,可以在p H值温和的环境条件下脱硫,因此,该菌有较高的工业应用价值。本研究为异养型细菌应用于工业反应器脱除硫化氢恶臭气体提供了小试研究基础。