PERAN MUTASI GEN ACY II TERHADAP PRODUKSI ANTIBIOTIK SEFALOSPORIN

The Roles of AcyII Gene Mutations for Production of Antibiotics Derived From CephalosporinSemisynthetic antibiotics cephalosporins are widely used to treat infectious diseases, especially those caused by gram-negative bacteria. Various types of semisynthetic antibiotics could be synthesized using 7-aminocephalosporanic acid (7-ACA) as the main raw material. 7-ACA is obtained by conversion of cephalosporin C, either chemically or enzymatically. Converting cephalosporin C to 7-ACA enzymatically in one step involves the cephalosporin acylase enzyme. Currently, all of cefalosporin acylase enzymes produced by wild-type microbes have only high activity on glutaryl-7-ACA as the main substrate. Genetic engineering of the encoding gene of cefalosporin acylase is required to obtain recombinant enzyme having high activity on cephalosporin C. In this paper, the engineering attempts made on acyII gene from Pseudomonas SE83 using directed mutagenesis, error prone PCR, and structural modeling are described. Keywords: AcyII gene, cephalosporin, cephalosporin C acylase, enzyme activity, mutation ABSTRAKAntibiotik sefalosporin semisintetik banyak digunakan untuk mengatasi penyakit infeksi, khususnya yang ditimbulkan oleh bakteri gram negatif. Berbagai jenis antibiotik semisintetk dapat disintesis menggunakan senyawa asam 7-aminosefalosporanat (7-ACA) sebagai bahan baku utamanya. Senyawa 7-ACA diperoleh melalui konversi sefalosporin C, baik yang dilakukan secara kimiawi maupun enzimatis. Konversi sefalosporin C menjadi 7-ACA secara enzimatis dalam satu langkah melibatkan enzim sefalosporin asilase. Hingga saat ini, seluruh enzim sefalosporin asilase yang dihasilkan oleh mikroba wild type hanya mempunyai aktifitas yang tinggi terhadap glutaryl-7-ACA. Rekayasa genetik terhadap gen pengkode enzim sefalosporin asilase diperlukan untuk memperoleh enzim rekombinan yang mempunyai aktifitas tinggi terhadap substrat sefalosporin C. Dalam ulasan ini diuraikan upaya-upaya rekayasa yang telah dilakukan terhadap gen acyII dari Pseudomonas SE83 menggunakan teknik mutasi terarah, error prone PCR, dan pemodelan struktur.Kata kunci: Aktivitas enzim, gen acyII, mutasi, sefalosporin, sefalosporin C asilase Received: 14September 2017 Accepted: 19 December 2017 Published: 30 December 2017 Received: 14September 2017 Accepted: 19 December 2017 Published: 30 December 2017

PRODUKSI REKOMBINAN SEFALOSPORIN ASILASE SEBAGAI BIOKATALIS UNTUK PRODUKSI ASAM 7-AMINOSEFALOSPORANAT

Production of Cephalosporin Acylase Recombinant as Biocatalyst for 7-Aminocephalosporanic Acid Production7-aminocephalosporanic acid (7-ACA) is a precursor for the production of semisynthetic cephalosporin derivatives. The enzymatic 7-ACA production can use two-stage and one-step enzymatic methods. Two-stage enzymatic method uses D-amino acid oxidase (DAAO) enzyme to produce glutaryl-7-aminocephalosporanic acid (GL-7-ACA) in the first stage and glutaryl-7-aminocephalosporanic acid acylase to produce 7-ACA in the second stage. The one-stage enzymatic method using cephalosporin acylase (CPC acylase) converts the CPC to 7-ACA directly. The aim of this research was to produce recombinant CPC acylase in Escherichia coli BL21(DE3). Transformantion culture E. coli BL21(DE3) was induced with concentrations of IPTG 0; 0.25; 0.5; 0.75; 1; 2 mM for 5 hours. The induction time of IPTG was determined at 0, 1, 2, 3, 4, and 5 hours. The results showed that CPC acylase produced by E. coli BL21(DE3) with optimum condition of CPC acylase production was 0.5 mM IPTG and optimal induction time of IPTG was 5 hours.Keywords: Cephalosporin, cephalosporin acylase, 7-ACA, protein expression, Escherichia coli BL21(DE3) ABSTRAKAsam 7-aminosefalosporanat (7-ACA) merupakan prekursor untuk produksi turunan sefalosporin semisintetik. Produksi 7-ACA secara enzimatik dapat menggunakan metode dua tahap dan satu tahap enzimatik. Metode enzimatik secara dua tahap menggunakan enzim asam D-amino oksidase (DAAO) untuk menghasilkan asam glutaril-7-aminosefalosporinat (GL-7-ACA) pada tahap pertama dan menggunakan asam glutaril-7-aminosefalosporinat asilase untuk menghasilkan 7-ACA pada tahap kedua. Metode enzimatik satu tahap dengan sefalosporin asilase (CPC asilase) mengubah CPC menjadi 7-ACA secara langsung. Tujuan penelitian adalah memproduksi rekombinan CPC asilase di dalam sel Escherichia coli BL21(DE3). Kultur Transforman E. coli BL21(DE3) diinduksi dengan konsentrasi IPTG 0; 0,25; 0,5; 0,75; 1; 2 mM selama 5 jam. Waktu induksi IPTG ditentukan pada 0, 1, 2, 3, 4 dan 5 jam. Hasil penelitian menunjukan bahwa CPC asilase diproduksi oleh E. coli BL21(DE3) dengan kondisi optimal produksi CPC asilase adalah konsentrasi IPTG 0,5 mM dan waktu induksi IPTG optimal adalah 5 jam.

The N-terminal Nucleophile Serine of Cephalosporin Acylase Executes the Second Autoproteolytic Cleavage and Acylpeptide Hydrolysis

Cephalosporin acylase (CA) precursor is translated as a single polypeptide chain and folds into a self-activating pre-protein. Activation requires two peptide bond cleavages that excise an internal spacer to form the mature αβ heterodimer. Using Q-TOF LC-MS, we located the second cleavage site between Glu159 and Gly160, and detected the corresponding 10-aa spacer 160GDPPDLADQG169 of CA mutants. The site of the second cleavage depended on Glu159: moving Glu into the spacer or removing 5–10 residues from the spacer sequence resulted in shorter spacers with the cleavage at the carboxylic side of Glu. The mutant E159D was cleaved more slowly than the wild-type, as were mutants G160A and G160L. This allowed kinetic measurements showing that the second cleavage reaction was a first-order, intra-molecular process. Glutaryl-7-aminocephalosporanic acid is the classic substrate of CA, in which the N-terminal Ser170 of the β-subunit, is the nucleophile. Glu and Asp resemble glutaryl, suggesting that CA might also remove N-terminal Glu or Asp from peptides. This was indeed the case, suggesting that the N-terminal nucleophile also performed the second proteolytic cleavage. We also found that CA is an acylpeptide hydrolase rather than a previously expected acylamino acid acylase. It only exhibited exopeptidase activity for the hydrolysis of an externally added peptide, supporting the intra-molecular interaction. We propose that the final CA activation is an intra-molecular process performed by an N-terminal nucleophile, during which large conformational changes in the α-subunit C-terminal region are required to bridge the gap between Glu159 and Ser170.