崔中利
环境微生物学与环境微生物结构生物学;微生物分子生态学。
个性化签名
- 姓名:崔中利
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学术头衔:
博士生导师, 教育部“新世纪优秀人才支持计划”入选者
- 职称:-
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学科领域:
微生物学
- 研究兴趣:环境微生物学与环境微生物结构生物学;微生物分子生态学。
崔中利,教授,南京农业大学微生物学系系主任,农业部农业环境微生物工程重点开放实验室副主任,中国微生物学会环境微生物专业委员会委员、基础微生物专业委员会委员和中国土壤学会土壤生物与生物化学专业委员会委员,土壤学报编委,入选教育部“跨世纪优秀人才”和江苏省“青蓝工程”中青年骨干人才计划。
长期从事环境有毒污染物微生物降解和环境生物技术的研究。主持完成国家“863”计划项目“高效安全的农药残留降解菌剂的研制及其在绿色农产品生产中的应用”,通过项目实施建立了国内最大的农药残留降解微生物资源库,在小区试验的基础上提出了农药残留微生物降解田间应用标准,并进行了大规模的试验示范,项目获得国家科技进步二等奖。目前正在主持国家自然科学基金项目“有机磷水解酶mpd结构与功能的初步研究”,进行了有机磷水解酶基因的高效表达和MPH结构与功能的初步研究。先后参加了本课题组承担的农业部和江苏省的科技攻关课题“多功能农药残留降解菌的构建”, 863预演项目“农药残留降解基因工程菌的构建”(项目编号SZ0308)及农业部、财政部农业科技跨越计划项目“农药残留降解技术在蔬菜和茶叶上的应用示范”。在这些项目中担负着农业残留降解菌的基因工程、分子生物学等方面的研究,将现代生物技术成功地应用到环境微生物的研究中,并取得了一定的成果。在日本京都大学进行了蛋白质晶体结构解析方面的研究工作,熟悉蛋白质晶体培养、X-衍射数据分析,解析了海藻糖降解途径中的鼠李糖苷酶的结构。发表论文30余篇,其中SCI论文8篇,均与污染物降解及其分子生物学相关,为国家科技进步二等奖的主要完成人之一(第二完成人)。
获奖:
农药残留微生物降解技术的研究与应用,李顺鹏、崔中利、沈标、刘智、何健、杨新民、王新华、张瑞福、蒋建东和洪青,国家科技进步二等奖,2005
研究方向:
环境微生物学与环境微生物结构生物学
微生物分子生态学
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成果数
11
崔中利, Zhong-Li Cui∗, Xiao-Zhou Zhang, Zhong-Hui Zhang & Shun-Peng Li
Biotechnology Letters 26: 1115-1118, 2004.,-0001,():
-1年11月30日
A facilitative and efficient promoter-trapping vector, pUC-mpd, was constructed with the promoterless methyl parathion hydrolase gene as the reporter. This reporter gene is easily used to clone promoters with different promoting strength on selective plates. Promoter regions of the ytkA and ywoF genes with strong promoting and signal peptide functions were cloned from the Bacillus subtilis 168 genomic promoter library with this vector.
Bacillus subtilis,, gene expression,, genomic promoter library,, methyl parathion hydrolase,, promotertrapping vector
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【期刊论文】High-Level Expression and Secretion of Methyl Parathion Hydrolase in Bacillus subtilis WB800
崔中利, Xiao-Zhou Zhang, Zhong-Li Cui, Qing Hong, and Shun-Peng Li*
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2005, p. 4101-4103,-0001,():
-1年11月30日
The methyl parathion hydrolase (MPH)-encoding gene mpd was placed under the control of the P43 promoter and Bacillus subtilis nprB signal peptide-encoding sequence. High-level expression and secretion of mature, authentic, and stable MPH were achieved using the protease-deficient strain B. subtilis WB800 as the host.
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【期刊论文】Expression, puriWcation, and characterization of a novel methyl parathion hydrolase
崔中利, Guoping Fu, a, b Zhongli Cui, a Tingting Huang, a and Shunpeng Lia, *
Protein Expression and PuriWcation 36(2004)170-176,-0001,():
-1年11月30日
The mpd gene coding for a novel methyl parathion hydrolase (MPH) was previously reported and its putative open reading frame was also identiWed. To further conWrm its coding region, the intact region encoding MPH was obtained by PCR and expressed in Escherichia coli as a hexa-His C-terminal fusion protein. The fusion protein was puriWed to homogeneity by metal-aYnity chromatography. The enzyme activity and zymogram assay showed that the fusion protein was functional in degrading methyl parathion. The amino terminal sequencing of the puriWed recombinant MPH indicated that a signal peptide of the Wrst 35 amino acids was cleaved from its precursor to form active MPH. A rat polyclonal antiserum was raised against the puriWed mature fusion protein. The results of Western blot and zymogram demonstrated that mature MPH in native Plesiomonas sp. strain M6 was also processed from its precursor by cleavage of a putative signal peptide at the amino terminus. The production of active MPH in E. coli was greatly improved after the coding region for the signal peptide was deleted. HPLC gel Wltration of the puriWed mature recombinant MPH revealed that the MPH was a monomer.
Methyl parathion hydrolase, Signal peptide
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崔中利, CUI ZHONGLI, LI SHUNPENG, * AND FU GUOPING
APPLIED AND ENVIRONMENTAL MICROBILOGY, Oct. 2001, p.4922-4925,-0001,():
-1年11月30日
A degradative bacterium, M6, was isolated and presumptively identified as Plesiomonas sp. strain M6 was able to hydrolyze methyl parathion to p-nitrophenol. A novel organophosphate hydrolase gene designated mpd was selected from its genomic library prepared by shotgun cloning. The nucleotide sequence of the mpd gene was determined. The gene could be effectively expressed in Esherichia coli.
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崔中利, Ruifu Zhang, Zhongli Cui, Xiaozhou Zhang, Jiandong Jiang, Ji-Dong Gu & Shunpeng Li, *
Biodegradation (2006)17: 465-472,-0001,():
-1年11月30日
Seven organophosphorus pesticide-degrading bacteria harboring the methyl parathion degrading (mpd) gene were isolated from a methyl parathion contaminated site. In this study, the 4.7 kb mpd gene cluster, conserved in all seven bacteria capable of degrading methyl parathion, was cloned and further analysis revealed that this cluster contained five ORFs and the mpd gene was associated with a mobile element, IS6100. In addition to mpd gene ORF and tnpA ORF, three other ORFs showed high homology to the permease component of ABC-type transport system, the general secretion pathway protein B, and the RNA polymerase sigma 70 factor, respectively. The mpd genes of these 7 strains were subcloned and expressed in E. coli, SDS-PAGE and zymogram analysis showed that two expression products of mpd genes in E. coli were found, but the one without signal peptide showed the hydrolytic activities. Our evidences collectively suggest that mpd gene cluster may be disseminated through horizontal gene transfer based on phylogenetic analysis of the cluster and their host bacterial strains, and comparisons of GC content of the cluster and respective host’s chromosome.
horizontal gene transfer,, organophosphorus hydrolase gene cluster,, signal peptide,, degradation
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崔中利, Zhongli Cui, a, b Yukie Maruyama, a Bunzo Mikami, c Wataru Hashimotoa and Kousaku Murataa*
Acta Cryst. (2006). F62, 646-648,-0001,():
-1年11月30日
α-L-Rhamnosidases play important roles in the metabolism of plant cell walls, glycosides and bacterial biofilms. This enzyme is also used industrially for debittering citrus fruits by releasing rhamnose from the plant flavonoid naringin. Bacillus sp. GL1 α-l-rhamnosidase (RhaB) is a member of glycoside hydrolase (GH) family 78. Native and selenomethionine-derivative enzymes were crystallized at 293 K by hanging-drop vapour diffusion with polyethylene glycol 8000 as a precipitant. This is the first report of the crystallization of a family GH78 enzyme.
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【期刊论文】对硝基苯酚降解菌P3的分离、降解特性及基因工程菌的构建*
崔中利, 张瑞福, 何健, 李顺鹏**
策生物学报,2002,42(1):19~26,-0001,():
-1年11月30日
分离到一株假单胞菌(Pseudomonassp.)P3,该菌能够以对硝基苯酚为唯一碳源和氮源进行生长。在有外加氮源的条件下,P3降解对硝基苯酚并在培养液中积累亚硝酸根。P3有比较广泛的底物适应性,对多种芳香族化合物都有降解能力。不同金属离子对P3降解对硝基苯酚有不同的作用。葡萄糖的存在对P3降解对硝基苯酚无明显促进作用,而微量酵母粉可以大大促进P3对硝基苯酚的降解。以P3为受体菌,通过接合转移的手段将甲基对硫磷水解酶基因mpd克隆至P3菌中,获得了表达甲基对硫磷水解酶活性的基因工程菌PM,PM能够以甲基对硫磷为唯一碳源进行生长。工程菌PM具有较高的甲基对硫磷降解活性及稳定性。
对硝基苯酚,, 生物降解,, mpd 基因,, 基因工程菌
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崔中利, 傅国平, , 徐玮, 吴旭平, 李顺鹏
微生物学报,2004,44(3):356~360,-0001,():
-1年11月30日
用PCR方法获得甲基对硫磷水解酶编码基因,构建了重组表达质粒pET29a-mpd,将其转化至EscherichiacoliBL21(DE3)中,经IPTG诱导表达,得到C2末端含有6个寡聚组氨酸的甲基对硫磷水解酶,用Ni2NTA亲和层析纯化得到具有活性的甲基对硫磷水解酶。测定了环境因素对酶活性的影响及酶动力学参数。甲基对硫磷水解酶水解甲基对硫磷时,最适pH8.6~8.8,最佳反应温度15℃;Mn2+、Zn2+、Cu2+可使酶活性增加15%~20%,Ca2+、Mg2+微弱地促进酶的作用,Ni2+对酶活性几乎无影响;1mmolPLEDTA•Na2+几乎不影响酶的活性,而10mmolPLEDTA•Na2+对甲基对硫磷水解酶有较强的抑制作用。甲基对硫磷水解酶水解乙基对硫磷时,最适pH816。25℃时,该酶对甲基对硫磷的米氏常数Km为(68.6±511)μmolPL,kcat为(45±6)S-1;对乙基对硫磷的米氏常数Km为(59.5±6.0)μmolPL,kcat为(8±1)S-1。kcatPKm表明甲基对硫磷水解酶对甲基对硫磷的催化效率更高。
甲基对硫磷水解酶,, 基因表达,, Ni2NTA 亲和层析,, 酶活性
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【期刊论文】利用短短小芽孢杆菌启动子和信号肽编码序列构建穿梭分泌表达载体
崔中利, 张晓舟, 闫新, 李顺鹏*
微生物学报,2006,46(4):526~530,-0001,():
-1年11月30日
以短短小芽孢杆菌B15的总DNA为模板,利用PCR技术克隆到其细胞壁蛋白基因串联启动子和信号肽编码序列,测序分析后提交GenBank,登录号为AY956423。重新设计引物扩增该片段并在PCR产物两侧引入BamHI和PstI酶切位点,将PCR产物双酶切后克隆至穿梭载体pP43NMK的相应位点构建分泌表达载体pP15MK,插入片段置于该载体中mpd基因的上游,并使信号肽编码序列与去除了自身信号肽编码序列的mpd基因阅读框恰好融合。将pP15MK导入枯草杆菌构建表达菌株1A751(pP15MK),在短短小芽孢杆菌启动子和信号肽元件的带动下,mpd基因能够在表达菌株的对数生长期和稳定期持续性高效分泌表达,表达产物结合在细胞膜上;发酵液在48h酶活达到最高值7.79U/mL,是出发菌株邻单胞菌M6表达量的8.1倍。
短短小芽孢杆菌, 启动子, 信号肽, 分泌表达, 枯草芽孢杆菌
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【期刊论文】邻单胞菌M6中甲基对硫磷水解酶(MPH)的纯化及性质的研究①
崔中利, 徐玮②, 林恒③, 刘卫东, 李顺鹏
高技术通讯,2006,16(1):84~87,-0001,():
-1年11月30日
通过热变性、硫酸铵沉淀、DEAE-Sephadex-A50阴离子交换层析和CM Sephamse Fast Flow阳离子交换层析等一系列步骤从有机磷农药降解菌Plesiomonas sp. M6中获得了甲基对硫磷水解酶(Methvl parathion hydrolase, MPH, EC 3.1.8.3)的电泳纯。酶谱显示和SDS-PAGE电泳表明纯化的酶只有一条条带,其分子量约为33kD。该酶热稳定性比较高,55℃、15min处理后酶活保持稳定,最适反应pH为9.0,最适反应温度在10℃左右。动力学分析表明其对甲基对硫磷的米氏常数(Km)为2.14mmol/L,最大反应速度(Vmax)为14.08μmol/L. min,转换数(kcat)为2863s-1。
甲基对硫磷水解酶(MPH), 邻单胞菌M6, 纯化, 性质
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