Characterization of a recombinant (+)-γ-lactamase from Microbacterium hydrocarbonoxydans which provides evidence that two enantiocomplementary γ-lactamases are in the strain

• Published in: Applied microbiology and biotechnology Vol. 99; no. 7; pp. 3069 - 3080
• Main Authors: Wang, Jianjun, Zhu, Yaxin, Zhao, Guogang, Zhu, Junge, Wu, Sheng
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.04.2015; Springer Berlin Heidelberg
• Subjects: Actinomycetales - chemistry; Actinomycetales - enzymology; Amidohydrolases - chemistry; Amidohydrolases - genetics; Amidohydrolases - metabolism; Amino Acid Sequence; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; clones; Cloning, Molecular; Cosmids; Escherichia coli - genetics; Gene Library; genome; high performance liquid chromatography; Hydrogen-Ion Concentration; Life Sciences; Microbacterium; Microbial Genetics and Genomics; Microbiology; Molecular Sequence Data; Mutagenesis, Site-Directed; open reading frames; plasmids; polymerase chain reaction; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; screening; Sequence Homology, Amino Acid; Substrate Specificity; Temperature; γ-Lactamase; Cosmid library; Enantiocomplementary; Isochorismatase
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-014-6114-8

A two-step method, i.e., the transfer acyl analysis and then the chiral HPLC analysis, was employed in the screening of the cosmid library of Microbacterium hydrocarbonoxydans genome. Two enantiocomplementary γ-lactamase clones were found. A 40-kb cosmid showed (−)-γ-lactamase activity, and the activity was from Mhg which was reported previously according to the results of PCR identifying experiment. The 37-kb (+)-γ-lactamase cosmid was further constructed into a pUC18 plasmid library and screened by the same two-step method. A plasmid clone harboring a 1.6-kb fragment showed (+)-γ-lactamase activity. A 555-bp ORF in the 1.6-kb fragment showed high (+)-γ-lactamase activity when it was expressed under the control of T₇promoter. The coding protein showed significant homology with bacterial isochorismatase. The (+)-γ-lactamase was characterized and compared with the (−)-γ-lactamase Mhg. This is another report that two enantiocomplementary γ-lactamases are present in the same strain.