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All Journal HAYATI Journal of Biosciences Makara Journal of Science
Sabbathini, Gabriela Christy
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Agriculture, Biological Sciences & Forestry Earth & Planetary Sciences

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Sequence-Structure Based Comparison of Structurally Homologous Thermophilic and Mesophilic Polyethylene Terephthalate (PET) Hydrolases Hasan, Khomaini; Ulfah, Maria; Nurhayati, Niknik; Sabbathini, Gabriela Christy; Wulandari, Sri Rezeki; Putra, I Gede Eka Perdana; Helianti, Is
HAYATI Journal of Biosciences Vol. 31 No. 2 (2024): March 2024
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.31.2.348-356

Abstract

Protein structure has a direct impact on thermostability. Deviations in the primary sequence can affect structural changes, leading to alterations in thermostability properties. However, the molecular basis of protein thermostability is unspecified; thus, elucidation of key factors that role particular protein thermostability is required when engineering proteins to be thermostable. To address this challenge, the amino acid composition, hydrophobicity/hydrophilicity ratio, cysteine bridges, and intrinsic features of two structurally homologous but different thermostability, poly(ethylene terephthalate) hydrolase (PETase) were compared. According to the findings, thermostable and thermolabile PETases have similar folds, compactness, and disulfide bridges. Interestingly, an abundance gap of aromaticity, hydrophobic cluster area, polar amino acid and hydrogen bond network compositions demonstrated dominant trends of variations for both PET hydrolases, indicating a pivotal role of these features in the thermostability of PET hydrolase. Furthermore, increased hydrophobic amino acid frequency in the inner surface of thermostable proteins contributed significantly to thermostability by forming more internal hydrophobic interactions and a less hydrophobic patch. There are no consistent trends in insertions and deletions between both PETases. Taken together, these observations demonstrate that hydrophobicity and hydrogen bond networks are essential factors in thermostability of thermostable PETase.
The Potential of Bacillus altitudinis B538 and Alcaligenes faecalis B947 in PET and PCL Plastic Degradation Dini, Muthia Rahmah; Nurcholis, Mochamad; Ulfah, Maria; Sabbathini, Gabriela Christy; Wulandari, Sri Rezeki; Helianti, Is
HAYATI Journal of Biosciences Vol. 31 No. 4 (2024): July 2024
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.31.4.621-629

Abstract

Polyethylene terephthalate (PET) plastic is the most widely used type of plastic that produces waste and causes various environmental and health problems. The treatment of PET plastic waste with chemically and mechanically recycling approaches still has shortcomings, so biological processing using microorganisms or enzymes has new potential. Two bacterial isolates from the Indonesian Culture Collection of National Research and Innovation Agency (InaCC, BRIN), namely isolate InaCC B538 and InaCC B947, were further observed for their potential in PET plastic degradation. Firstly, both isolates were determined by the molecular marker 16S rDNA. The potential of both isolates was measured with following method: 10 days of degradation using PET and PCL substrates, esterase enzyme activity assay, and observation of the PET plastic surface using Scanning Electron Microscope (SEM). Species identification was performed using DNA sequencing of 16S rDNA. InaCC B538 and InaCC B947 were closely related to Bacillus altitudinis TBMAX41 and Alcaligenes faecalis AN-13, respectively. InaCC B947 isolate has a better potential in degrading PET plastic and PCL with a degradation percentage of 0.32% for PET plastic and 3.22% for PCL film for 10 days, respectively, and esterase activity of 0.06 U/ml; while InaCC B538 did not cause weight loss of PET and 2.49% for PCL, respectively, with esterase activity of 0.04 U/ml. The degradation of PET plastic by the isolates InaCC B947 was able to cause damage to the plastic surface leading to the degradation of PET plastic.
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
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
Publisher : UI Scholars Hub

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Abstract

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.
Co-Authors Abinawanto Abinawanto Achnafani, Dini Ahmad Wibisana, Ahmad Dini, Muthia Rahmah Feronika Heppy Sriherfyna I Gede Eka Perdana Putra, I Gede Eka Perdana IS HELIANTI KHOMAINI HASAN Maria Ulfah Masdalifah, Masdalifah Mochamad Nurcholis Nataniel, Jocelyn NIKNIK NURHAYATI Wulandari, Sri Rezeki