【作者】 吴石金；

【导师】 陈建孟；

【作者基本信息】 浙江工业大学 ， 生物化工， 2009， 博士

【摘要】 本文通过选择性富集培养,从长期被卤代脂肪烃污染的废水和活性污泥中分离纯化二氯甲烷高效降解菌,并对所分离菌株进行生化鉴定以及16S rDNA系统发育研究,进一步开展了降解特性、关键酶基因克隆与表达方面的研究,以期为探索微生物对异型生物质（Xenobiotics）的转化与降解机理、典型有机污染物的生物处理、污染环境的生物修复提供创新的技术支持与方法论指导。本研究获得的主要结论如下:1.从废水和活性污泥中分离筛选得到2株二氯甲烷降解菌株,分别命名为WZ-12和wh22菌株。通过细菌形态学观察、生理生化测试、抗生素抗性试验、碳源利用试验（Biolog）、全细胞脂肪酸分析、G+C mol%含量分析以及16S rDNA序列同源性分析,证明WZ-12和wh22菌株应分别属于芽孢杆菌属及梭杆菌属。其中WZ-12菌株被鉴定为环状芽孢杆菌（Bacillus circulans）的又一菌株;推测菌株wh22应当是梭形杆菌（Lysinibacillus sphaericus）的又一菌株,首次发现了环状芽孢杆菌菌株和梭形杆菌菌株具有二氯甲烷降解活性。2.WZ-12和wh22两株菌都有较宽的温度、pH及二氯甲烷底物浓度适应范围。在菌体生长最适的pH环境中,菌株对二氯甲烷的降解效率也最高;在30℃、初始pH6.0的MM培养基中,WZ-12菌株72 h对二氯甲烷的降解效率达85.23%。而wh22菌株在37℃、初始pH6.0的MM培养基中48 h的降解效率约80.09%。有机物的加入可使降解菌WZ-12对二氯甲烷的降解加速,其中酵母膏及葡萄糖效果优于蛋白胨。有机物的加入对wh22菌株的降解能力影响不大;WZ-12和wh22菌株会因二氯甲烷浓度增加而产生抑制,降解率下降,符合非线性的高姆比兹模型。菌株WZ-12能适应高盐浓度生长,NaCl浓度在10-60 g L^-1范围生长良好,对CH₂Cl₂、CH₂BrCl、C₂H₂Cl₂和C₂H₄Cl₂均有降解能力,72 h降解效率分别为78.28%、58.19%、60.32%和45.08%。3.菌株wh22含有1个降解性质粒,分子量为48.8 kb,命名为pRC11。初步建立了质粒pRC11的物理图谱。该质粒还是一个汞盐抗性质粒,可在不同种属菌株之间转移,以E.coli DH5（dcm^-）作为受体菌做质粒的接合转移试验,阳性转化子的转化频率为1.65×10⁵/μg of plasmid DNA。4.通过设计基于二次多项式数学模型,对试验结果进行多元回归分析,得到了如下适用于DCM降解率预测的经验关联式回归模型:采用响应面分析法,在摇瓶水平上,得到了菌株WZ-12生物降解工艺的最优条件为:DCM初始浓度380 mg/L（X₁）、葡萄糖添加浓度13.72 mg/L（X₂）、H₂O₂添加浓度115 mg/L（X₃）。在此条件下,预测得到的最大降解率为93.18%。对以上获得的最佳工艺参数进行摇床水平上的降解率试验验证,得到好氧降解率平均值为92.88±0.27%,与模型预测值（93.18%）吻合较好,该降解率回归模型为菌株WZ-12在生物法处理含DCM废气的应用有一定的指导意义。5.对来自菌株WZ-12的脱卤素酶进行了分离纯化和酶学性质研究,获得二氯甲烷脱卤素酶,纯酶分子量为31 kDa,比酶活提高了8.27倍,得率为34.83%,纯化倍数为8.27。该酶为诱导酶,最佳产酶温度为30℃,粗酶液在50℃以下具备一定的耐热性能和稳定性,最佳产酶pH为6.5;该酶在pH5-7之间稳定较好,但当pH大于7或小于5时,稳定性急剧下降。Ca²⁺、Mg²⁺对酶活有增强作用,而Cu²⁺、Zn²⁺和Ba²⁺则抑制酶活性,Hg²⁺对酶活性抑制影响最大。该脱卤素酶表观Km值在30℃（pH7.0）为5.25×10^-3mol/L,Vmax为3.67×10^-4mol/L·min,K_cat为6.97×10⁴S^-1。该酶的底物特异性不高,可以催化CH₂Cl₂、CH₂Br₂、CH₂I₂和CH₂BrCl脱卤。6.从菌株WZ-12中克隆得到二氯甲烷脱卤素酶基因dcmR,并对克隆产物进行了Southern杂交鉴定。dcmR编码基因序列为864 bp,编码脱卤素酶蛋白大小为288个氨基酸残基,预测分子量32±1 kDa。BLAST比对结果显示,克隆的基因片段与Methylobacterium sp.DM4的二氯甲烷脱卤酶基因序列同源性达98.6%。蛋白的氨基末端约20-80 aa与GST有类似的结构域,中间约第50位-80位aa间具有多个跟GSH的结合位点有关的超二级结构模体结构域。将所得到的菌株相关基因序列用SEQUIN软件上传至GenBank,得到序列登录号FJ405230。7.通过构建具T7强启动子的pET高效表达载体（pET21a和pET15b）,转化E coli（DE3）RP,构建了二氯甲烷脱卤酶的原核表达系统。构建了3种表达载体,pET-21a-dcmR（no-tag）不含任何标签,其转化子的二氯甲烷脱卤素酶酶活(21.95U·mL^-1)高于原始菌株WZ-12(14.26U·mL^-1),可直接用于二氯甲烷生物降解的工程应用研究;pET-21a- dcmR with his-tag引入了6个组氨酸残基“标签”（His-tag）,为后续酶蛋白分离纯化获得纯化酶的研究非常有意义;pET-15b-dcmRwith his-tag and LVPRGS thrombin在引入6个组氨酸残基“标签"（His-tag）的同时,增加了凝血酶酶切位点,方便了His-tag的切除。将3种原核表达载体转化E.coli（DE3）RP并诱导表达,得到了具有酶活性的融合蛋白。8.探讨重组酶的表达特点和部分酶学性质。重组菌经IPTG诱导后,细菌总蛋白表达量为0.76g/L,其中融合蛋白占总蛋白的32.00%,酶活最高达25.78U/mL,酶的比活为88.86 U/mg蛋白。重组菌周质中酶活2.92 U/mL,胞内酶活22.86 U/mL。重组菌产生的酶活力与比活较原降解菌株高1-2倍。利用融合蛋白N末端的His-tag,经金属螯合亲和层析纯化后,得到了纯度较高的融合酶蛋白,酶蛋白的得率为72%,比活为144.73 U/mg。凝血酶切除His-tag后,经SDS-PAGE测定,重组蛋白的分子量为33±1 kDa,与理论计算值34 kDa相符。重组的二氯甲烷脱卤素酶在pH 6.5,温度30℃有最大相对酶活。但重组的二氯甲烷脱卤素酶对温度和pH要敏感。最后,对重组菌的生长特性和降解特性的研究表明,重组菌在LB培养基中的生长特性与原始菌株没有差别,生长至对数期A_600nm值都可达到2.4左右。重组菌株dcmR-1在25 h的降解率达90%以上,降解效率比原降解菌株有明显提高。更多还原

【Abstract】 In this thesis, two dichloromethane-degrading bacterial were:isolated by using traditional incubation method. Their identification were based on standard morphological and physiological properties, cellular fatty acid composition, mol% G+C and nucleotide sequence analysis of enzymatically amplified 16S ribosomal deoxyribonucleic acid. The factors influencing growth of dichloromethane-degrading bacteria and degradation of dichloromethane, the cloning and expression of dichloromethane degrading gene of two strains, were studied in this thesis. The results were expected to supply useful reference for building up alert index systems in transformation and biodegradation mechanism of xenobiotics, and for environmental quality evaluation and for bioremediation of halogenated hydrocarbon pollution.Here are presented the main results of this study:1. Two strains which could use dichloromethane as sole carbon source were isolated from halogenated hydrocarbon contaminated sample. Their identification were based on standard morphological and physiological properties, G+C content and nucleotide sequence analysis of enzymatically amplified 16S ribosomal deoxyribonucleic acid. Strain named WZ-12 （GenBank accession no.EF100968） was isolated and identified as Bacillus circulans and strain named wh22 （GenBank accession no. FJ418643） as Lysinibacillus sphaericus which were the first representative of Bacillus circulans and Lysinibacillus sphaericus able to degrade dichloromethane very fast at high experimental concentration.2. Both WZ-12 and wh22 has wide temperature, pH range for growth and degradation, and could tolerate high concentration of dichloromethane. The optimal growth conditions of strain WZ-12 （pH6.0, 37℃,degradation rate 85%） and wh22 （pH7.0, 30℃,degradation rate 80%）, respectively. Addition of yeast extraction, peptone or glucose could promote the growth and dichloromethane degradation ability of both WZ-12 and wh22 to different degree. Biodegradation of DCM followed the modified Gompertz model. WZ-12 degrade CH₂C1₂,CH₂BrCl,C₂H₄C1₂ and C₂H₂C1₂ efficiently in the medium containing NaCl at concentrations of 15 g L^-1 in 72 h. Kinetic analysis revealed that there was an inverse relationship between the velocity of the degradation reaction and salt concentration over the range between 5 and 60 g NaCl L^-1 and a linear reciprocal relationship （R²=from 0.85 to 0.94） was observed.3. The strain wh22 harbored a novel degradative plasmid, pRC11 （48.8 kilobases）. The genes coding for the metabolism of dichloromethane were found to be plasmid-borne, and a physical map of the plasmid has been established. The purified plasmid was transformed to dcm^- Escherichia coli DH5 at a rate of 1.65×10⁵.The transformed cells were able to grow on dichloromethane at concentration of 5-16 mM, and can be further used as a excellent source for genetic manipulations leading to construction of genetically modified microbial strains or genetically engineered microorganisms.4. Response surface methodology （RSM） was employed to evaluate the optimum aerobic biodegradation of dichloromethane （DCM） in batch culture. The parameters investigated include the initial DCM concentration, glucose as an inducer and hydrogen peroxide as terminal electron acceptor （TEA）. Maximum aerobic biodegradation efficiency was predicted to occur when the initial DCM concentration was 380 mg/Lwith the glucose of 13.72 mg/L and the H₂O₂ of 115 mg/L.Under these conditions the aerobic biodegradation rate reached up to 93.2%, which was significantly higher than that obtained under original conditions. Without additives, the degradation efficiencies≤80% were obtained with the DCM concentrations < 326 mg/L. In order to achieve an 80% or higher biodegradation efficiency, DCM concentrations should be lower than 350 mg/L and the addition of glucose is necessary. When concentrations of DCM were more than 480 mg/L, the addition of H₂O₂ did not significantly contribute to increase DCM degradation efficiency. When DCM concentrations was increased from 240 mg/L to 480 mg/L, the overall DCM degradation efficiency decreased from 91% to 60% as the presence of H₂O₂ for 120mg/L.5. Dichloromethane dehalogenase from B. circulans WZ-12 was purified to 8.27-fold with a yield of 34.83%. The electrophoretically homogeneous-purified enzyme exhibited a specific activity of 118.82 U/mg. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified DCM dehalogenase gave a distinct band with an estimated molecular mass of 31 kDa. Enzyme activity was optimized at 30℃to 35℃and pH from 6.5 to 7.0. The enzyme was stable in the pH range of 5.0-8.0. The reaction was apparently accelerated by addition of Mg²⁺and Ca²⁺,causing an 22 to 27% stimulation, whereas it was inhibited by the addition of Hg²⁺,Cu²⁺,Zn²⁺ and Ba²⁺.DCM dehalogenase exhibited extremely broad substrate and the apparent Km value at 30℃（pH7.0） for DCM was 5.25×10^-3 mol·L^-1. Vmax was 3.67×10^-4mol·L^-1/min and K_cat was 6.97×10⁴ S^-1,respectively.6. The gene dcmR encoding a novel dichloromethane dehalogenases（DehalA）, has been cloned from strain WZ-12. Its accession number in Genbank was FJ418643. The open reading frame of dcmR, spanning 864 bp, encoded a 288-amino-acid protein. A homology search with the BLAST program revealed that the nucleotide sequence of the dcmR gene was almost identical （98.6%） to Methylobacterium sp DM4.7. The gene dcmR of strain WZ-12 was recombined and expressed in E.coli BL21（DE3） successfully. A high level of soluble dehalogenases （DehalA） was expressed in E.coli BL21（DE3） from a pET expression system（pET21a and pET15b） and the activity of recombined enzyme protein expressed by recombinant obtained was much more than that by the original WZ-12 strain in primary detection. The gene dcmR was subcloned into pET21a vector at Nde I and Xho I sites with no any tag, the gene dcmR was subcloned into pET21a vector at Nde I and Xho I sites with his-tag and the gene dcmR was subcloned into pET15b vector at Nde I and BamH I sites with his-tag and LVPRGS thrombin. The recombined vectors were confirmed by DNA sequencing and transformed into the E. coli strain ArcticExpress^TM （DE3） RP for expression optimization. The expressions were analyzed by SDS-PAGE followed by Coomassie blue staining. There were obvious expression band and there were soluble fusion protein when induced at low temperature.8. Then plasmid pET-15b-dcmR with his-tag and LVPRGS thrombin was introduced into Escherichia. coli BL21（DE3）. Expression was induced by IPTG, and the enzyme activity reached 25.78 U/mL, the specific enzyme activity reached 88.86 U/mg protein. The periplasmic and cytoplasmic enzyme activity reached 2.92 U/mL and 22.86 U/mL respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified recombinant DCM dehalogenase gave a distinct band with an estimated molecular mass of 33±1kDa. All results analysis demonstrated that the E.coli. strain carrying the dcmR gene could produce dichloromethane dehalogenase efficiently. The growth characteristics of dcmR-l was compared with the original strain, and the result showed that there was no difference,A₆₀₀ nm of dcmR-1 in LB medium could reach about 2.4 in logarithmic period, which was the same as that of the original strain. The recombinant strain dcmR-1 showed the higher degrading ability than B. circulans WZ-12 and with more than 90% removal efficiency of 120 mM CH₂C1₂ in 25 h.All these results indicated that recombinant strain dcmR-1 was a promising strain in bioremediation of CH₂Cl₂ contaminated environment.更多还原