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All Journal HAYATI Journal of Biosciences Makara Journal of Science Jurnal Bioteknologi & Biosains Indonesia
Achnafani, Dini
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Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Earth & Planetary Sciences

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Optimizing the Expression of Polyethylene Terephtalate Hydrolase-Encoding Synthetic Gene in Escherichia coli Arctic Express (DE3) Nataniel, Jocelyn; Ulfah, Maria; Achnafani, Dini; Nurhayati, Niknik; Sabbathini, Gabriela Christy; Wulandari, Sri Rezeki; Abinawanto, Abinawanto; Helianti, Is
Makara Journal of Science Vol. 28, No. 2
Publisher : UI Scholars Hub

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Abstract

The waste of polyethylene terephthalate (PET) plastic waste in Indonesia is a pressing concern due to its slow degradation and potential environmental damage. One promising solution is to utilize polyethylene terephthalate hydrolase from Ideonella sakaiensis (IsPETase), an enzyme that specifically degrades PET. However, inducing the expression of IsPETase synthetic gene in Escherichia coli BL21 (DE3) has been challenging because much of it remains insoluble. This study aimed to express IsPETase in E. coli Arctic Express (DE3) and optimize the conditions to enhance its production. First, pET22b(+)pelB-IsPETase was inserted into E. coli Arctic Express (DE3). The recombinant E. coli Arctic Express (DE3) was induced with isopropyl-β-D-1-thiogalactopyranoside (IPTG) and incubated at 10 °C. The fraction expressing soluble IsPETase was determined in different culture media, IPTG concentrations, induction times, and soni-cation durations. Parameters were optimized using a one-factor-at-a-time approach and then evaluated based on esterase specific activity and SDS-PAGE analysis. Results showed that IsPETase can be expressed in extracellular, periplasmic, and cytoplasmic soluble fractions. However, the extracellular fraction should be concentrated. Subsequent optimization focused only on the cytoplasmic fraction under optimal conditions, achieving a threefold increase in PETase specific activity compared with that under uninduced IPTG conditions. The reaction of PETase enzyme with PET and PCL was proven by weight loss, Scanning electron microscope (SEM), and Fourier transform infrared spectroscopy (FTIR). Although successful IsPETase expression and production optimization have been achieved, the specific activity remains low, prompting the need for ongoing expression optimization.
Screening for Penicillin G Acylase (PGA)-Producing Bacteria and Gene Cloning Using Degenerate Oligonucleotide Primed-PCR Masdalifah, Masdalifah; Wulandari, Sri Rezeki; Sabbathini, Gabriela Christy; Ulfah, Maria; Achnafani, Dini; Wibisana, Ahmad; Sriherfyna, Feronika Heppy; Helianti, Is; Nurhayati, Niknik
Makara Journal of Science Vol. 29, No. 1
Publisher : UI Scholars Hub

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The growing concern over antibiotic resistance has driven global efforts to explore innovative solutions, including the use of Penicillin G acylase (PGA) to produce semisynthetic β-lactam antibiotics. This study screened four potential in-tracellular PGA-producing bacteria: Alcaligenes faecalis InaCC B444 (AfPGA), Kluyvera cryocrescens InaCC B850 (KcPGA), Providencia rettgeri InaCC B25 (Pr25PGA), and P. rettgeri InaCC B466 (Pr466PGA). Penicillin G Acylase encoding genes (pgas) were isolated from them using a Degenerate Oligonucleotide Primed-PCR (DOP-PCR) approach and sequenced. Microbiological assays confirmed all tested crude extracts to exhibit inhibitory effects. Penicillin G was used for evaluating hydrolytic activity and 6-Amino Penicillanic Acid (6-APA) coupled with D-p-Hydroxyl-phenylglycine methyl ester hydrochloride (DHPGME) for the synthetic activity. Pr466PGA and Pr25PGA showed the highest synthetic and hydrolytic activities, respectively. DOP-PCR successfully amplified a 2,517 bp pga-encoding Pr25PGA. The deduced amino acid sequence shared 95.1% identity with the known PGA from P. rettgeri PX04. Sec-ondary structure analysis of Pr25PGA revealed 35% α-helices, 16% β-sheets, and 49% coils, suggesting that the enzyme may be flexible and dynamic, with structural stability primarily provided by the α-helices and β-sheets. These findings offer valuable insights for the future design and application of Pr25PGA, particularly in the production of semisynthetic β-lactam antibiotics.
MICROBIAL L-ASPARAGINASES AND STRATEGIES TO IMPROVE THEM Achnafani, Dini; Sari, Aniska Novita
Jurnal Bioteknologi & Biosains Indonesia (JBBI) Vol. 10 No. 2 (2023)
Publisher : BRIN - Badan Riset dan Inovasi Nasional

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.55981/jbbi.2023.2020

Abstract

L-asparaginase is a type of hydrolase enzyme that has been used in anticancer treatment, mainly Acute Lymphoblastic Leukemia (ALL). L-asparaginase reduces the blood supply of L-asparagine needed by cancer cells to survive. The commercially approved L-asparaginase by the FDA originated from E. coli and E. chrysantemi. However, reports of immunogenic effects in more than 50% of cases due to the use of these enzymes have become the driving force for the need to explore other sources of L-asparaginase. In this review, various alternative sources of L-asparaginase other than these two microbes will be explained. Microbes from the group of Gram-positive bacteria, actinomycetes, and fungi produce L-asparaginase with a higher affinity for L-asparagine than L-glutamine. Protein engineering is an alternative strategy to produce L-asparaginase that is not recognized by antibodies to reduce the immune reaction. Besides, the fermentation process also needs to be considered to determine the appropriate substrate and bioprocess system to obtain the enzyme.
A Comparative Study of Penicillin G Acylase Expression in Two Escherichia coli Strains: BL21 (DE3) and Arctic Express Cendana, Kartika Sari; Wulandari, Sri Rezeki; Sabbathini, Gabriela Christy; Ulfah, Maria; Achnafani, Dini; Abinawanto; Aniqah, Sunni Sofiah; Helianti, Is; Nurhayati, Niknik
HAYATI Journal of Biosciences Vol. 33 No. 3 (2026): May 2026
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.33.3.596-604

Abstract

The growing demand for semisynthetic beta-lactams has directed attention towards enzymes, specifically Penicillin G Acylases (PGAs), for their potential in synthesizing these antibiotics. This study delves into the expression of Achromobacter xylosoxidans PGA (AxPGA) in Escherichia coli, with a focus on enhancing the yield of active PGA, often constrained by a complex maturation process. The optimization of PGA expression included variations in IPTG concentration and the addition of CaCl2. Furthermore, the study compared PGA expression in E. coli BL21 (DE3) with that in E. coli Arctic Express (DE3), capable of co-expressing chaperones (chaperonin Cpn60 and Cpn10). Induction with 0.5 mM IPTG resulted in the highest hydrolytic activity in both strains, with Arctic Express (AE) exhibiting significantly higher activity due to improved folding facilitated by cold-adapted chaperonins. Alongside optimal IPTG induction, the addition of 10 mM CaCl2 in the culture media significantly increased PGA activity in both strains, highlighting that Ca2+ supplementation is an effective strategy to enhance the yield of functional PGA. Subcellular fractionation demonstrates that the periplasmic fraction yielded higher volumetric and specific activities compared to the cytoplasmic fractions in both E. coli strains, highlighting the importance of periplasmic processing for PGA maturation. This suggests that extracting the periplasmic fraction is an effective strategy for recovering active PGA while avoiding or reducing contamination either from co-expressed cytoplasmic chaperones or other intracellular proteins. These findings emphasize that induction strategy, ionic stabilization, and host strain selection play synergistic roles in increasing active recombinant PGA expression.
Co-Authors Abinawanto Abinawanto Abinawanto Ahmad Wibisana, Ahmad Aniqah, Sunni Sofiah Cendana, Kartika Sari Feronika Heppy Sriherfyna IS HELIANTI Maria Ulfah Masdalifah, Masdalifah Nataniel, Jocelyn NIKNIK NURHAYATI Sabbathini, Gabriela Christy sari, aniska novita Wulandari, Sri Rezeki