54 research outputs found
Direct Cloning of a Xylanase Gene from Pawan-Riau Hot Spring
No full textA functional gene containing an Open Reading Frame (ORF) encoding a -1, 4-endoxylanase glycosyl hydrolase family 11 was cloned directly using metagenomic PCR-cloning method from Pawan Hot Spring sample in Riau. The gene consisted of 642 nucleotides, encoded for 213 amino acids. The amino acid sequence analysis using BLAST showed that the gene has high homology (93%) with xylanase gene from Bacillus subtilis. The gene showed its function when it was subcloned into an expression vector and overexpressed in E. coli. The crude extract of the recombinant enzyme had activity for 170 U/ml at 50 oC. The result of this work showed that metagenomic approach was a powerful short cut method to obtain recombinant biocatalyst that was useful for industrial application
The development of papain‐like protease from SARS‐CoV‐2, a potential drug target for antiviral screening: A review
No full textThe SARS‐CoV‐2 outbreak caused a global pandemic, claiming numerous lives and becoming this century’s most widespread life‐threatening disease. The virus relies on two specific enzymes to facilitate replication, 3‐chymotrypsin‐like protease (3CLPro) and papain‐like protease (PLpro). These enzymes are crucial in breaking down nonstructural polypeptides into functional proteins. PLpro with LXGG↓X recognition and cleavage sites also play a role in deubiquitylase (DUB) and delSGylase by cleaving after the double glycine residue of ubiquitin (Ub) and ISG15 as a mechanism to suppress the host’s innate immune response. Despite its important role in the viral infection cycle and the potential for drug discovery, no antivirals have been approved as PLpro inhibitors. Therefore, this review focuses on PLpro protein, its recombinant product development and purification, and its application as a protein target in drug discovery for COVID‐19 screening to develop effective COVID‐19 drugs
APPLICATION OF RECOMBINANT TRIACYLGLYCEROL LIPASE AND CARBOXYLESTERASE ENZYMES FROM Bacillus velezensis STRAIN S3 FOR POLYESTER SURFACE MODIFICATION
Get PDFEnzymatic polyester surface modification can be performed with lipase and esterase enzymes. In this study, the polyester fabric modification utilized triacylglycerol lipase (TGA) and carboxylesterase (CES) recombinant enzymes. The effect of these treatments was observed by determining the hydrophilicity level, dye absorption level, hydroxyl group measurement, and fiber surface morphology. The results revealed an elevated hydrophilicity level in polyester fabric, followed by dye absorption improvement and carboxyl group increase. The water absorption times required by the fabric based on the results of TGA, CES, comparative lipase, and negative control treatments were 3±0.05 seconds, 3.5±0.07 seconds, 5±0.05 seconds, and 80±11.54 minutes, respectively. Dye absorption test in polyester fabric based on these groups mentioned above were 52±0.5, 58±0.5, 178±0.5, and 2968±290 seconds. The total hydroxyl group measurement in polyester fabric was observed at 30.9±0.09, 30.5±0.05, 28.6±0.09, and 3 meq/100 g. The SEM observation showed that the enzymatic hydrolysis could alter the porous structure and surface of the fibers
INCREASING RECOMBINANT PENICILLIN G ACYLASE PRODUCTION: GENETIC, PROTEIN ENGINEERING, AND PRODUCTIVITY IMPROVEMENT
Get PDFB-lactam derived antibiotics are the most used globally for treatment against different infections caused by pathogenic bacteria and comprises 65% of the world antibiotics. Recently, penicillin G acylase (PGA) is used as biocatalyst for those B-lactam antibiotics production by which 6-aminopenicillanic acid (6-APA) or 7-aminodeacetoxycephalosporanic acid (7-ADCA) as the building blocks is produced. Commercialized PGA from native microbial resources are still limited to E. coli. Therefore, genetic engineering approach such as cloning and expression in other microbial hosts were assessed to enhance bacterial strains that produce PGA. However, such improvement could increase immature precursors accumulation and lowering the enzyme yield, activity, or stability. This review focus on the review of PGA recombinant produced by several microbial host, their expression levels, and improvement achieved by some modification such as replacement of signal peptide and promoter continued to protein engineering to utilize the enzymes in synthetizing amoxicillin rather than to hydrolyses Penicillin G
Direct Cloning of a Xylanase Gene from Pawan-Riau Hot Spring
Get PDFA functional gene containing an Open Reading Frame (ORF) encoding a ?-1, 4-endoxylanase glycosyl hydrolase family 11 was cloned directly using metagenomic PCR-cloning method from Pawan Hot Spring sample in Riau. The gene consisted of 642 nucleotides, encoded for 213 amino acids. The amino acid sequence analysis using BLAST showed that the gene has high homology (93%) with xylanase gene from Bacillus subtilis. The gene showed its function when it was subcloned into an expression vector and overexpressed in E. coli. The crude extract of the recombinant enzyme had activity for 170 U/ml at 50 oC. The result of this work showed that metagenomic approach was a powerful short cut method to obtain recombinant biocatalyst that was useful for industrial application. Key words: ?-1, 4-endoxylanase, metagenomic DNA, Pawan-Riau hot-sprin
Conjugational Transformation of Wild Type Bacillus halodurans CM1 by Methylated Recombinant Plasmid Harbouring a Gene Encoding for Alkaline Protease and the Protease Activity Assay of the Transformant
Get PDFProtease contributes significantly to various industrial sectors, as shown by its increasing demand. An indigenous previously isolated bacteria, Bacillus halodurans CM1, is a wild-type bacterium whose ability to produce alkalotermophilic protease. In this study, the transformation of methylated pBBRE194 plasmid containing alkaline protease gene from this strain (PBBRE194-prot-CM1) into itself using a conjugation approach was conducted, and the measurement of the alkaline protease activity of the recombinant bacterium was carried out. The recombinant B. halodurans CM1 has been verified to carry the methylated pBBRE194 prot-CM1 by examining its ability to degrade protein in media containing skim milk and tetracycline, but also by amplifying tetracycline resistance gene sequence with 1,024 bp length of plasmid pBBRE194 prot-CM1 by PCR method from the recombinant bacterium. The results confirmed that recombinant B. halodurans CM1 was positively harboring plasmid pBBRE194 prot-CM1. Alkaline protease produced by recombinant CM1 reached higher activity than wild type between 18-36 h of cultivation. The alkalotermophilic wild type B. halodurans could accept the recombinant plasmid into their cells via conjugational transformation is firstly reported to our further knowledge
In Silico and In Vitro Inhibitory Activity of Indonesian Herbal Compound Extracts against SARS-COV-2 Recombinant Papain-Like Protease
Get PDFThe SARS-CoV-2 papain-like protease (PLpro) is essential for viral replication and a promising target for drug discovery. This study explored the inhibitory potential of compounds from Indonesia herbals Butterfly pea flower (Clitoria ternatea L), Star fruit leaves (Averrhoa carambola L.), and Java plum leaves (Syzygium cumini (L.) Skeels) against PL pro through molecular docking and in vitro assays. The molecular docking method utilized the target protein PLpro (PDB ID: 7CMD), with the native ligand obtained from compounds identified in these plant extracts. The compounds were identified using the KNApSAcK database and analyzed for drug-likeness based on Lipinski\u27s Rule of Five. The physicochemical characteristics affecting absorption, distribution, metabolism, excretion, and toxicity (ADMET) were determined using the pkCSM descriptor algorithm protocol. Validation was performed using the redocking method, achieving an RMSD score of 0.728 Å, which indicated validity (RMSD <2.0 Å). The results identified four ligands with the lowest binding affinities from these extracts: (-)-Epicatechin 3-O-gallate, folic acid, petunidin 3-glucoside, and ellagic acid, with binding scores of -8.6, -8.3, -7.1, and -7.1 kcal/mol, respectively. Prior to conducting the PLpro in vitro inhibition assay, a fluorescence-based inhibition assay was performed using Z-RLRGG-AMC as the substrate and GRL0617as the control inhibitor. All extracts were subjected to 70% ethanol maceration. The IC50 value of GRL0617 was 3.38 μM, while fluorescence tests showed that Java plum leaf extract exhibited the highest inhibition percentage at 66.10±3.22%. These findings indicate that all three plant extracts contain compounds capable of inhibiting PLpro activity
Kloning dan Sekuensing Gen Xilanase dengan Produk Gen Berukuran 30 kDa dari Bacillus halodurans CM1 pada Escherichia coli DH5α
No full textThe paper industry contributed the environment pollution due to chlor substances. Utilization of alkalothermophilic xylanase enzyme as a biocatalyst in the production of paper may become an environmentally friendly biobleaching alternative. Bacillus halodurans CM1 produces xilanase enzyme that had optimal activity at pH 9 and temperature 70°C. Previous study showed that this CM1 strains has several xilanase genes. The cloning of one of these alkalothermophiic xylanase (alkxyn) gene has been already conducted. This study aimed to clone alkxyn gene that encode alkalothermophilic xylanase enzyme from B. halodurans CM1 into Escherichia coli DH5α. Amplification of alkxyn has been carried out using primers for amplification xylanase 30 kDa. The alkxyn gene fragment was inserted into pGEM-T Easy vector and then transformed into E. coli DH5α. The results showed that the recombinant of E. coli DH5α harboring alkxyn gene from B. halodurans CM1 has been obtained. The sequences analysist based on BLAST showed that alkxyn fragment has homology (99%) with the alkaliphilic xylanase gene from Bacillus sp. 31 which encodes alkaliphilic xilanase (Genebank assession number: JF912895.1). Keywords: cloning, Bacillus halodurans CM1, xylanase, alkalothermophilic.</jats:p
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