Characterization of penicillin G acylase from bacillus subtilis (pBA 401)
3
Issued Date
2024
Copyright Date
1994
Resource Type
Language
eng
File Type
application/pdf
No. of Pages/File Size
xii, 112 leaves : ill. (some col.)
Access Rights
open access
Rights
ผลงานนี้เป็นลิขสิทธิ์ของมหาวิทยาลัยมหิดล ขอสงวนไว้สำหรับเพื่อการศึกษาเท่านั้น ต้องอ้างอิงแหล่งที่มา ห้ามดัดแปลงเนื้อหา และห้ามนำไปใช้เพื่อการค้า
Rights Holder(s)
Mahidol University
Bibliographic Citation
Thesis (M.Sc. (Biochemistry))--Mahidol University, 1994
Suggested Citation
Arunsri Sritanaitipol Characterization of penicillin G acylase from bacillus subtilis (pBA 401). Thesis (M.Sc. (Biochemistry))--Mahidol University, 1994. Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/99046
Title
Characterization of penicillin G acylase from bacillus subtilis (pBA 401)
Alternative Title(s)
การศึกษาคุณสมบัติของเอนไซม์เพนนิซิลลินจี เอซีเลส จากเชื้อจุลินทรีย์ Bacillus subtilis (pBA 401)
Author(s)
Advisor(s)
Abstract
Penicillin G acylase (PA) is the enzyme used commercially for the production of semisynthetic penicillins. Penicillin G acylase gene from Bacillus megaterium has been cloned into Bacillus subtilis which can release the enzyme extracellularly. The enzyme was extracted from the medium by using celite adsorption and eluted with 24% ammonium sulfate. The enzyme was then further purified on FPLC using Mono Q column (HR10/10) and Superose-12 column (S16/50). Two peaks of penicillin G acylase, i.e., peak I (PAI) and peak II (PAII), were obtained from Mono Q column. Molecular weight of the native enzyme of both PAI and PAII were found to be 79 KDa on Superose-12 column. SDS-polyacrylamide gel electrophoresis revealed that both PAI and PAII consisted of two nonidentical subunits with a molecular weight of 59 and 22 KDa. The isoelectric point of PAI and PAII were 6.78 and 6.52, respectively. The pH optimum of PAI and PAII were found to be in the range of 7-9. PAI was stable at pH range from 6.5-11.5 whereas PAII was stable at pH range from 6.5-10. In studying heat stability, PAI and PAII were stable upto 45 degree C, both peaks rapidly lost activities at temperature above 55 degree C. The Michaelis-Menten constant (Km) of PAI and PAII for penicillin G were found to be 7.232 and 0.69 mM, respectively. The Km value for 6-nitro-3-phenylacetamido benzoic acid (NIPAB) of PAI and PAII were 0.268 and 0.308 mM, respectively. When the effect of ammonium sulfate on enzyme activity was studied, it was found that 0.2-5 mM ammonium sulfate had no significant effect on activities of PAI and PAII, both with and without preincubation of the enzyme with ammonium sulfate prior to the assay. However, activities of PAI and PAII increased about 1.6 time when They were preincubated with 260 mM ammonium sulfate prior to the assay. About 1.2 fold increase in activities of PAI and PAII were observed when 260 mM ammonium sulfate were added to the assay mixture without preincubation. It was found that 0.2-5 mM potassium bromide had no significant effect on enzyme activity, both with and without preincubation of potassium bromide with the enzyme prior to the assay. The results from time course study showed that only one peak of penicillin G acylase was obtained when B.subtilis was cultured for 24 h and 36 h while two peaks of enzyme were obtained from 48 h culture. However, enzyme at 72 h-culture showed only peak corresponded to PAI.
Description
Biochemistry (Mahidol University 1994)
Degree Name
Master of Science
Degree Level
Master's degree
Degree Department
Faculty of Science
Degree Discipline
Biochemistry
Degree Grantor(s)
Mahidol University