Site-Directed Mutagenesis of Recombinant Penicillin-G Acylase from Escherichia coli and Effect on the Hydrolytic Activity
Penulis/Author
Sunni Sofiah Aniqah (1); Prof. Dr. apt. Sismindari, S.U. (2); Purwanto, M.Sc., Ph.D., Apt. (3)
Tanggal/Date
1 2024
Kata Kunci/Keyword
Abstrak/Abstract
Penicillin-derived antibiotics such as amoxicillin are one of the antibiotics that are widely used. Amoxicillin is synthesized through the availability of raw materials, 6-aminopenicillanic acid (6-APA), with penicillin-G acylase (PGA) as the catalyst. Previous studies revealed a recombinant PGA through the utilization of expression hosts. However, the low activity of the expressed enzyme is still an obstacle in the optimum utilization of its enzyme. In this study, we applied site-directed mutagenesis computationally for initial prediction by creating 36 PGA mutants through homology modeling and predicted their binding affinity as well as bounding interaction mode to penicillin G through molecular docking. Our research found that 4 mutants had better potential activity than wild-type PGA. As the first experiment, only βThr68Tyr PGA mutant was selected to be explored further. The mutant PGA gene inserted into pET-22b plasmid was transformed to E. coli BL21(DE3) by heat shock method. For enzyme expression, IPTG and arabinose were used as inducers. As the result, the specific enzyme activity of mutant PGA showed insignificantly lower values in comparison to wild-type PGA. This activity appeared to be influenced by formation of inclusion bodies during post-translational protein maturation, as described more in this paper.
Rumpun Ilmu
Farmasi Umum dan Apoteker
Bahasa Asli/Original Language
English
Level
Internasional
Status
Dokumen Karya
No
Judul
Tipe Dokumen
Aksi
1
Purwanto_JAPS journal_Submission and Under review.pdf
