Nucleotide sequence analysis and strain improvement for high expression of penicillin G acylase gene from Bacillus megaterium UN-1
2
Issued Date
2024
Copyright Date
1993
Resource Type
Language
eng
File Type
application/pdf
No. of Pages/File Size
xv, 130 leaves : ill.
Access Rights
open access
Rights
ผลงานนี้เป็นลิขสิทธิ์ของมหาวิทยาลัยมหิดล ขอสงวนไว้สำหรับเพื่อการศึกษาเท่านั้น ต้องอ้างอิงแหล่งที่มา ห้ามดัดแปลงเนื้อหา และห้ามนำไปใช้เพื่อการค้า
Rights Holder(s)
Mahidol University
Bibliographic Citation
Thesis (M.Sc. (Microbiology))--Mahidol University, 1993
Suggested Citation
Kanchana Weeradechapon Nucleotide sequence analysis and strain improvement for high expression of penicillin G acylase gene from Bacillus megaterium UN-1. Thesis (M.Sc. (Microbiology))--Mahidol University, 1993. Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/100002
Title
Nucleotide sequence analysis and strain improvement for high expression of penicillin G acylase gene from Bacillus megaterium UN-1
Alternative Title(s)
การหาลำดับ nucleotide และการปรับปรุงสายพันธุ์ของ Bacillus megaterium UN-1 ที่สร้างเอนไซม์ penicillin G acylase
Author(s)
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.
Description
Microbiology (Mahidol University 1993)
Degree Name
Master of Science
Degree Level
Master's degree
Degree Department
Faculty of Science
Degree Discipline
Microbiology
Degree Grantor(s)
Mahidol University