title: Identification and Characterization of a New 7-Aminocephalosporanic Acid Deacetylase from Thermophilic Bacterium Alicyclobacillus tengchongensis.
abstract: Deacetylation of 7-aminocephalosporanic acid (7-ACA) at position C-3 provides valuable starting material for producing semisynthetic β-lactam antibiotics. However, few enzymes have been characterized in this process before now. Comparative analysis of the genome of the thermophilic bacterium Alicyclobacillus tengchongensis revealed a hypothetical protein (EstD1) with typical esterase features. The EstD1 protein was functionally cloned, expressed, and purified from Escherichia coli BL21(DE3). It indeed displayed esterase activity, with optimal activity at around 65°C and pH 8.5, with a preference for esters with short-chain acyl esters (C2 to C4). Sequence alignment revealed that EstD1 is an SGNH hydrolase with the putative catalytic triad Ser15, Asp191, and His194, which belongs to carbohydrate esterase family 12. EstD1 can hydrolyze acetate at the C-3 position of 7-aminocephalosporanic acid (7-ACA) to form deacetyl-7-ACA, which is an important starting material for producing semisynthetic β-lactam antibiotics. EstD1 retained more than 50% of its initial activity when incubated at pH values ranging from 4 to 11 at 65°C for 1 h. To the best of our knowledge, this enzyme is a new SGNH hydrolase identified from thermophiles that is able to hydrolyze 7-ACA.
  
  
  OBJECTIVE
  Deacetyl cephalosporins are highly valuable building blocks for the industrial production of various kinds of semisynthetic β-lactam antibiotics. These compounds are derived mainly from 7-ACA, which is obtained by chemical or enzymatic processes from cephalosporin C. Enzymatic transformation of 7-ACA is the main method because of the adverse effects chemical deacylation brought to the environment. SGNH hydrolases are widely distributed in plants. However, the tools for identifying and characterizing SGNH hydrolases from bacteria, especially from thermophiles, are rather limited. Here, our work demonstrates that EstD1 belongs to the SGNH family and can hydrolyze acetate at the C-3 position of 7-ACA. Moreover, this study can enrich our understanding of the functions of these enzymes from this family.
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, School of Life Sciences, Yunnan Normal University, Kunming, China.
authors.forename: Jun-Mei
authors.initials: JM
authors.lastname: Ding
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, School of Life Sciences, Yunnan Normal University, Kunming, China.
authors.forename: Ting-Ting
authors.initials: TT
authors.lastname: Yu
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, School of Life Sciences, Yunnan Normal University, Kunming, China.
authors.forename: Nan-Yu
authors.initials: NY
authors.lastname: Han
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, School of Life Sciences, Yunnan Normal University, Kunming, China.
authors.forename: Jia-Lin
authors.initials: JL
authors.lastname: Yu
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, School of Life Sciences, Yunnan Normal University, Kunming, China.
authors.forename: Jun-Jun
authors.initials: JJ
authors.lastname: Li
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, School of Life Sciences, Yunnan Normal University, Kunming, China.
authors.forename: Yun-Juan
authors.initials: YJ
authors.lastname: Yang
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, School of Life Sciences, Yunnan Normal University, Kunming, China.
authors.forename: Xiang-Hua
authors.initials: XH
authors.lastname: Tang
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, School of Life Sciences, Yunnan Normal University, Kunming, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Xu
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, School of Life Sciences, Yunnan Normal University, Kunming, China.
authors.forename: Jun-Pei
authors.initials: JP
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China tanghongzhi@sjtu.edu.cn huangzunxi@163.com.
authors.forename: Hong-Zhi
authors.initials: HZ
authors.lastname: Tang
authors.affiliation: Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, School of Life Sciences, Yunnan Normal University, Kunming, China tanghongzhi@sjtu.edu.cn huangzunxi@163.com.
authors.forename: Zun-Xi
authors.initials: ZX
authors.lastname: Huang
mesh_terms: D056450:Alicyclobacillus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D002511:Cephalosporins / Q000378:metabolism*; D003001:Cloning, Molecular; D004950:Esterases / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D008969:Molecular Sequence Data; D010802:Phylogeny
chemical_list: D002511:Cephalosporins; C030735:7-aminocephalosporanic acid; D004950:Esterases