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1.
从Meth ylomonas sp.GYJ3菌株中经DNEAE-SepharoseCl-6B阴离子交换层析和SephacrylS300凝胶层析分离出纯化出甲烷加氧酶羟基酶组分,经HPLC分析,纯度大于90%,分子量为240kD,纯化们数为3.9,比活为225nmol环氧丙烷每分钟毫克蛋白,SDS-PAGE表明,羟基化酶由三个亚基组成,亚基分子量为56、43、27kD.ICPAES测定羟基化酶的Fe  相似文献   

2.
Metylomonassp.GYJ3菌的甲烷单加氧酶(MMO)粗酶提取液经DEAE-SepharoseCL-6B阴离子交换层析、SephadexG-100凝胶过滤层析和DEAE-TSKgelHPLC分离纯化出MMO还原酶组分.经HPLC分析,纯度大于95%,纯化倍数为4.4,加入至MMO羟基化酶和调节蛋白B的体系中表现比活为228nmol环氧丙烷每分钟毫克蛋白.SDS-PAGE电泳表明还原酶由一种亚基组成,分子量42kD.ICP-AES测定还原酶的Fe含量为1.83molFe每mol蛋白.UV-Vis光谱表明还原酶除280nm蛋白质特征峰外在460nm有最大吸收峰,且A280nm/A460nm为2.50,与其它黄素一铁硫蛋白相似,推测还原酶可能含一个FAD辅基和Fe2S2中心.在厌氧条件下,还原酶能够和NADH作用,UV-Vis光谱分析表明还原酶460nm处特征吸收峰消失,说明在MMO催化过程中还原酶接受NADH的电子.DEAE-SepharoseCL-6B阴离子交换层析分离出调节蛋白B,部分纯化的调节蛋白B的分子量大约在20kD,它能够提高MMO比活性40倍,MMO还原酶和调节蛋白B单独存在时不具有MMO  相似文献   

3.
Meylomonas sp.GYJ3菌的甲烷单加氧酶(MMO)粗酶提取液经DEAE-Sepharose CL-6B阴离子交换层析,Sephadex G-100凝胶过滤层析和DEAE-TSKgel HPLC分离纯化出MMO还原酶组分,经HPLC分析,纯度大于95%,纯化倍数为4.4,加入至MMO羟基化酶和调节蛋白B的体系中表现比活为228nmol环氧丙烷每分钟毫克蛋白,SDS-PAGE电泳表明的酶由  相似文献   

4.
甲烷氧化细菌能够催化甲烷和一系列小分子烃类化合物的羟基化反应,对控制全球变暖起着重要作用,在工业催化和生物除污中具有非凡的潜能。应用层析方法纯化了Ⅱ型甲烷氧化细菌MethylosinustrichosporiumIMV3011中甲烷单加氧酶的羟基化酶,并对其进行了表征。凝胶过滤法测定了该酶分子量为201.3kD;SDS-PAGE表明羟基化酶含有三个亚基(αβγ),分子量分别为58kD、36kD和23kD,比较两种方法证明该羟基化酶是一个同型二聚体构型(αβγ)2,总分子量为234kD。薄层等电聚焦测定该酶的等电点为5.2。酶的比活力为603.6nmol/(min.mg),活力回收为34.3%。HPLC法测定该酶的纯度在95%以上。原子吸收光谱显示每分子羟基化酶中含有3.02个Fe原子。羟基化酶的稳定性pH值为6.2~7.5,稳定性温度为低于35℃。菌株IMV3011的细胞表观构型呈现了长型、稍微弯曲的杆状形态。  相似文献   

5.
甲烷氧化细菌中的关键酶系甲烷单加氧酶是一个含双核铁的多组份氧化酶,常温、常压下能够催化甲烷转化为甲醇。对甲烷氧化细菌Methylomonas sp.GYJ3中溶解性甲烷单加氧酶基因和16SrDNA进行了测序与分析。利用已知相关基因数据库信息,设计了PCR引物和测序引物,获得了满意的测序结果。全长的溶解性甲烷单加氧酶基因为5690bp,部分16S rDNA的序列长度为1280bp。与已发表的甲烷氧化细菌中甲烷单加氧酶进行了比较,结果表明MMOX组份中氨基酸序列的同一性为78%到99%,基因序列的同一性为71%到97%,6个组份中orfY片段的同一性相对较低。MMOX氨基酸序列的多序列联配表明,MMOX序列具有高度保守性,特别是在双核铁中心区域。16S rDNA进化分析显示Methylomonas sp.GYJ3与γ蛋白细菌是相关联的,基于MMOX氨基酸序列的进化分析证明,与Methylomonas sp.GYJ3最近似的菌株是Ⅰ型甲烷氧化细菌Methylomonas sp.KSWⅢ。综合分析表明,菌株GYJ3属于Ⅰ型甲烷氧化细菌Methylomonas sp.属。这个结果为Ⅰ型甲烷氧化细菌也能表达溶解性甲烷单加氧酶提供了新的证据。羟基化酶的理论等电点是6.28,理论分子量为248874.41Da。  相似文献   

6.
甲烷甲基单胞菌的一个新变种   总被引:1,自引:0,他引:1  
赵树杰 《微生物学报》1991,31(3):183-186
对甲烷氧化细菌761M菌株做了进一步鉴定。结果表明,该菌株为甲烷甲基单胞菌的一个新变种,命名为甲烷甲基单胞菌成都变种(Methylomonas methanica vas.chengduensis)。  相似文献   

7.
甲基单胞菌甲烷单加氧酶缺陷突变株的研究   总被引:1,自引:0,他引:1  
用高剂量紫外线诱变I型专性甲烷营养菌(Methylomonas sp.)761 AR菌株,获得了9个甲烷单加氧酶缺陷突变株。对其中76lAR-55突变株的进一步研究表明,此菌株完全丧失了氧化甲烷及氧化丙烯为环氧丙烷的能力,证明其甲烷单加氧酶活性存在缺陷。而其它特性如DNA内切酶谱,DNA中G+c克分子含量,可溶性蛋白电泳谱带,以及细胞内膜结构均与亲本菌株一致。  相似文献   

8.
甲烷单加氧酶的催化性能和活性中心结构   总被引:3,自引:0,他引:3  
甲烷单加氧酶是甲烷利用细菌代谢甲烷过程中的重要酶系,它能够催化烷烃羟基化和烯烃环氧化反应;还能催化降解氯代烃类,可用于环境中氯代烃类化合物污染的治理,是具有广泛应用前景的生物催化剂.甲烷单加氧酶是含有μ-氧桥双核铁催化活性中心的蛋白,它的研究对分子氧的活化、化学催化剂的设计具有重要意义.文章介绍了甲烷单加氧酶催化性能和机理的最新研究进展.  相似文献   

9.
甲烷利用细菌降解三氯乙烯的研究   总被引:5,自引:0,他引:5  
GYJ3菌株细胞微细结构的电镜观察结果表明:它具有Ⅱ型甲烷利用细菌的特征,应归属于Ⅱ型菌。考察了Cu2+浓度、培养气相中甲烷浓度对菌株细胞中甲烷单加氧酶(EC1.14.13.25,简称MMO)活性的影响。结果表明,培养液中Cu2+浓度为1.5μmol/L,培养气相中甲烷:空气比为2∶1时,可溶性甲烷单加氧酶占细胞中MMO总量的95%。研究了GYJ3菌株细胞悬浮液降解三氯乙烯过程。实验结果表明,GYJ3菌株能够降解不同浓度的三氯乙烯,较高浓度的三氯乙烯对降解反应没有明最的抑制作用。加入甲酸盐作为电子给体能够提高三氯乙烯降解反应速率。实验中观察到GYJ3菌株降解三氯乙烯过程中反应速率随着反应的进行而下降,在三氯乙烯降解过程中三氯乙烯氧化产物是导致细胞失活的主要原因。实验室中测定了GYJ3菌株单位重量细胞降解三氯乙烯极限量,它可作为评价细菌降解三氯乙烯能力的重要指标。  相似文献   

10.
以丙烯氧化反应为指标研究了不同外源电子给体对甲烷细菌(Methylomonas sp.GYJ30)休止细胞催化活性的影响。结果表明甲烷、甲醇、甲醛和甲酸盐作为电子给体加入反应中,将甲烷单加氧酶催化丙烯环氧化反应活性分别提高5.3,12.7,10和12.4倍。以甲烷和甲醛作为外源电子给体时提高初始浓度对甲烷单加氧酶具有抑制作用;而以甲醇和甲酸盐作为电子给体时提高初始浓度对甲烷单加氧酶催化活性无明显抑制作用。研究了甲醇作为电子给体时它的代谢、环氧丙烷的积累以及催化反应活性与反应时间的关系  相似文献   

11.
A soluble methane monooxygenase (sMMO: EC 1.14.13.25) was purified from a type II obligate methanotroph, Methylocystis sp. M. Ion exchange chromatography elution separated the sMMO into three components, I, II, and III. Components II and III were purified to homogeneity and were essential for the sMMO activity. Components II and III had molecular masses of approximately 233,000 and 39,000, respectively. Component II consisted of three subunits with molecular masses of 55,000, 44,000, and 21,000, which appeared to be present in stoichiometric amounts, suggesting a (αβγ)2 configuration in the native protein. Component II contained 1–4 mol of iron and was considered to be a hydroxylase. Component III was a flavoprotein, which contained 1 mol of FAD as well as 1–2mol of iron. It catalyzed the reduction of K3Fe(CN)6 and 2,6-dichloroindophenol by NADH. Component I, which was partially purified and not essential for sMMO activity, stimulated the activity by about 11-fold. Its stimulation could be replaced by addition of Fe2+. The molecular mass of the partially purified component I was estimated to be from 35,000 to 40,000 based on gel filtration, which suggested the presence of a new type of regulatory protein of sMMO.  相似文献   

12.
Soluble methane monooxygenase (sMMO) of Methylosinus trichosporium OB3b is a three-component oxygenase that catalyses the O2- and NAD(P)H-dependent oxygenation of methane and numerous other substrates. Despite substantial interest in the use of genetic techniques to study the mechanism of sMMO and manipulate its substrate specificity, directed mutagenesis of active-site residues was previously impossible because no suitable heterologous expression system had been found for expression in a highly active form of the hydroxylase component, which is an (αβγ)2 complex containing the binuclear iron active site. A homologous expression system that enabled the expression of recombinant wild-type sMMO in a derivative of M. trichosporium OB3b from which the chromosomal copy of the sMMO-encoding operon had been partially deleted was previously reported. Here we report substantial development of this method to produce a system for the facile construction and expression of mutants of the hydroxylase component of sMMO. This new system has been used to investigate the functions of Cys 151 and Thr 213 of the α subunit, which are the only nonligating protonated side chains in the hydrophobic active site. Both residues were found to be critical for the stability and/or activity of sMMO, but neither was essential for oxygenation reactions. The T213S mutant was purified to >98% homogeneity. It had the same iron content as the wild type and had 72% wild-type activity toward toluene but only 17% wild-type activity toward propene; thus, its substrate profile was significantly altered. With these results, we have demonstrated proof of the principle for protein engineering of this uniquely versatile enzyme.  相似文献   

13.
The soluble MMO (sMMO) gene clusters from group I methanotrophs were characterized. An 8.1-kb KpnI fragment from Methylomonas sp. strain KSWIII and a 7.5-kb SalI fragment from Methylomonas sp. strain KSPIII which contained the sMMO gene clusters were cloned and sequenced. The sequences of these two fragments were almost identical. The sMMO gene clusters in the fragment consisted of six open reading frames which were 52 to 79% similar to the corresponding genes of previously described sMMO gene clusters of the group II and group X methanotrophs. The phylogenetic analysis of the predicted amino acid sequences of sMMO demonstrated that the sMMOs from these strains were closer to that from M. capsulatus Bath in the group X methanotrophs than to those from Methylosinus trichosporium OB3b and Methylocystis sp. strain M in the group II methanotrophs. Based on the sequence data of sMMO genes of our strains and other methanotrophs, we designed a new PCR primer to amplify sMMO gene fragments of all the known methanotrophs harboring the mmoX gene. The primer set was successfully used for detecting methanotrophs in the groundwater of trichloroethylene-contaminated sites during in situ-biostimulation treatments.  相似文献   

14.
15.
Methylomonas sp. GYJ3 is a methanotrophic bacterium containing methane monooxygenase (MMO), which catalyses the epoxidation of propene to epoxypropane. In this study, the cell suspension of Methylomonas sp. GYJ3 has been used for epoxypropane biosynthesis from propene. When propene is epoxidized, the product epoxypropane is not further metabolized and accumulates extracellularly. Unfortunately, continuous production of epoxypropane is usually difficult due to exhaustion of reductant and the accumulation of toxic products. Hence, in order to address these problems, batch experiments were performed to explore the possibility of producing epoxypropane by a co-oxidation process. Methane was chosen as the most suitable electron-donating co-substrate since it did not result in molecular toxicity and provided abundant reductant for epoxidation. It was found that the maximum production of epoxypropane occurred in an atmosphere of 30% methane. Batch experiments also indicated that continuous removal of product was necessary to overcome the inhibition of epoxypropane. In continuous experiments, optimum mixed gaseous substrates were continuously circulated through the stirred tank bioreactor to remove product from the cell suspension. Initial epoxypropane productivity was 268 mol/day. The bioreactor has been allowed to operate continuously for 12 days without obvious loss of epoxypropane productivity, and more than 96% of initial MMO activity was retained.  相似文献   

16.
17.
Methane monooxygenase (MMO) catalyzes the oxidation of methane to methanol as the first step of methane degradation. A soluble NAD(P)H-dependent methane monooxygenase (sMMO) from the type II methanotrophic bacterium WI 14 was purified to homogeneity. Sequencing of the 16S rDNA and comparison with that of other known methanotrophic bacteria confirmed that strain WI 14 is very close to the genus Methylocystis. The sMMO is expressed only during growth under copper limitation (<0.1 μM) and with ammonium or nitrate ions as the nitrogen source. The enzyme exhibits a low substrate specificity and is able to oxidize several alkanes and alkenes, cyclic hydrocarbons, aromatics, and halogenic aromatics. It has three components, hydroxylase, reductase and protein B, which is involved in enzyme regulation and increases sMMO activity about 10-fold. The relative molecular masses of the native components were estimated to be 229, 41, and 18 kDa, respectively. The hydroxylase contains three subunits with relative molecular masses of 57, 43, and 23 kDa, which are present in stoichiometric amounts, suggesting that the native protein has an α2β2γ2 structure. We detected 3.6 mol of iron per mol of hydroxylase by atomic absorption spectrometry. sMMO is strongly inhibited by Hg2+ ions (with a total loss of enzyme activity at 0.01 mM Hg2+) and Cu2+, Zn2+, and Ni2+ ions (95, 80, and 40% loss of activity at 1 mM ions). The complete sMMO gene sequence has been determined. sMMO genes from strain WI 14 are clustered on the chromosome and show a high degree of homology (at both the nucleotide and amino acid levels) to the corresponding genes from Methylosinus trichosporium OB3b, Methylocystis sp. strain M, and Methylococcus capsulatus (Bath).  相似文献   

18.
Methylobacterium sp. strain CRL-26 grown in a fermentor contained methane monooxygenase activity in soluble fractions. Soluble methane monooxygenase catalyzed the epoxidation/hydroxylation of a variety of hydrocarbons, including terminal alkenes, internal alkenes, substituted alkenes, branched-chain alkenes, alkanes (C1 to C8), substituted alkanes, branched-chain alkanes, carbon monoxide, ethers, and cyclic and aromatic compounds. The optimum pH and temperature for the epoxidation of propylene by soluble methane monooxygenase were found to be 7.0 and 40°C, respectively. Among various compounds tested, only NADH2 or NADPH2 could act as an electron donor. Formate and NAD+ (in the presence of formate dehydrogenase contained in the soluble fraction) or 2-butanol in the presence of NAD+ and secondary alcohol dehydrogenase generated the NADH2 required for the methane monooxygenase. Epoxidation of propylene catalyzed by methane monooxygenase was not inhibited by a range of potential inhibitors, including metal-chelating compounds and potassium cyanide. Sulfhydryl agents and acriflavin inhibited monooxygenase activity. Soluble methane monooxygenase was resolved into three components by ion-exchange chromatography. All three compounds are required for the epoxidation and hydroxylation reactions.  相似文献   

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