Nucleotide sequence analysis and strain improvement for high expression of penicillin G acylase gene from Bacillus megaterium UN-1

Abstract

Penicillin G acylase (PAC) catalyzes the hydrolysis of benzylpenicillin to 6-aminoper,icillanic acid (6-APA), a key intermediate, in the synthesis of semisynthetic peniciliins. The penicillin G acylase gene (pac) gene was isolated from Bacillus megaterium UN-1, a high penicillin G acylase producer mutant of Bacillus megaterium ATCC 14945. Complete nucleotide sequence of pac gene was determined and found that the gene contained an open reading frame encoding for 802 amino acids with MW of about 92,000 Da. This polypeptide consisted of a 26 amino acid signal peptide. The putative -35 and -10 sequences were TTGAAT and TATAAG, respectively. Comparison of pac nucleotide sequence to that from Arthrobacter viscosus revealed 97% homology. Results from SDS-PAGE suggested that the penicillin G acylase of Bacillus megaterium UN-1 comprised of two subunits MW of about 23,000 Da and 57,000 Da. Therefore, this enzyme seem to be synthesized first as a single polypeptide and later being processed during maturation to two subunits as reported in the other pac gene of various microorganisms. In order to increase the yield of enzyme production, four plasmids containing pac gene were constructed and transfomed to Bacillus megaterium UN-CAT1, a penicillin G acylase negative mutant of Bacillus megaterium UN-1, and Bacillus subtilis MI113. The expression of the plasmids containing pac gene in Bacillus megaterium UN-CAT1, the mutant host, was higher than that in Bacillus subtilis MI113. The penicillin G acylase activity in Bacillus megaferium UN-CAT1 and Bacillus subtilis MI113, both carrying pBA402, a plasmid containing pac gene with its self-promoter ) were approximate 26 times and 13 times higher than that in Bacillus megaterium UN-1, a donor penicillin G acylase producer which contained no plasmid. Hence, Bacillus megaterium UN-CAT1 carrying pBA402, is a good potential strain to be used for penicillin G acylase production.