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All Journal Makara Journal of Science Jurnal Bioteknologi & Biosains Indonesia
Achnafani, Dini
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Biochemistry, Genetics & Molecular Biology

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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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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.
Co-Authors Abinawanto Abinawanto Ahmad Wibisana, Ahmad Feronika Heppy Sriherfyna IS HELIANTI Maria Ulfah Masdalifah, Masdalifah Nataniel, Jocelyn NIKNIK NURHAYATI Sabbathini, Gabriela Christy sari, aniska novita Wulandari, Sri Rezeki