title: A novel cephalosporin deacetylating acetyl xylan esterase from Bacillus subtilis with high activity toward cephalosporin C and 7-aminocephalosporanic acid.
abstract: A cephalosporin deacetylating acetyl xylan esterase was cloned from the genomic DNA of Bacillus subtilis CICC 20034 and functionally expressed in Escherichia coli. Its gene contained an open reading frame of 957 bp encoding 318 amino acids with a calculated mass of 35,607 Da, and it displayed significant identity to acetyl xylan esterases from Bacillus sp. 916, B. subtilis 168, and Bacillus pumilus Cect5072. The enzyme was a native homohexamer but a trimer under the condition of 1% sodium dodecyl sulfate (SDS); both forms were active and could transit to each other by incubating in or removing SDS. The enzyme belongs to carbohydrate esterase family 7 and had a double specificity on both the acetylated oligosaccharide and cephalosporin C (CPC) and 7-aminocephalosporanic acid (7-ACA). The activity of this purified enzyme toward CPC and 7-ACA was highest among all the acetyl xylan esterase from CE family 7, which were 484 and 888 U/mg, respectively, and endowed itself with great industrial interest on semi-synthetic β-lactam antibiotics. The optimum pH of the purified enzyme was 8.0, and the optimum temperature was 50 °C, and the enzyme had high thermal stability, broad range of pH tolerance, and extremely organic solvent tolerance.
authors.affiliation: College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing, Jiangsu, China.
authors.forename: Qianqian
authors.initials: Q
authors.lastname: Tian
authors.forename: Ping
authors.initials: P
authors.lastname: Song
authors.forename: Ling
authors.initials: L
authors.lastname: Jiang
authors.forename: Shuang
authors.initials: S
authors.lastname: Li
authors.forename: He
authors.initials: H
authors.lastname: Huang
mesh_terms: D000115:Acetylesterase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology*; D002511:Cephalosporins / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002511:Cephalosporins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; C025163:cephalosporin C; C030735:7-aminocephalosporanic acid; D000115:Acetylesterase; C081851:acetylxylan esterase