Psychrophilic basidiomycete yeast, Glaciozyma antarctica strain PI12, was shown to be a protease-producer. Isolation of the PI12 protease gene from genomic and mRNA sequences allowed determination of 19 exons and 18 introns. Full-length cDNA of PI12 protease gene was amplified by rapid amplification of cDNA ends (RACE) strategy with an open reading frame (ORF) of 2892 bp, coded for 963 amino acids. PI12 protease showed low homology with the subtilisin-like protease from fungus Rhodosporidium toruloides (42% identity) and no homology to other psychrophilic proteases. The gene encoding mature PI12 protease was cloned into Pichia pastoris expression vector, pPIC9, and positioned under the induction of methanol-alcohol oxidase (AOX) promoter. The recombinant PI12 protease was efficiently secreted into the culture medium driven by the Saccharomyces cerevisiae
α-factor signal sequence. The highest protease production (28.3 U/ml) was obtained from P. pastoris GS115 host (GpPro2) at 20°C after 72 hours of postinduction time with 0.5% (v/v) of methanol inducer. The expressed protein was detected by SDS-PAGE and activity staining with a molecular weight of 99 kDa.
Natural products are a vast source of potential compounds that can be developed as anti-obesity agent. One of the mechanisms of anti-obesity agents is inhibition of pancreatic lipase. Assay of 24 crude extracts for their in vitro activity against porcine pancreatic lipase (PPL) detected four extracts demonstrating high (>70%) inhibition, seven extracts had medium (30-70%) inhibition and the remaining 13 extracts exhibited low (<30%) inhibition when incubated with PPL at a concentration of 500 µg/ml for 10 min at 37°C. Phyllanthus niruri extract displayed the most potent PPL inhibitor, followed by Orthosiphon stamineus, Murraya paniculata and Averrhoa bilimbi with the IC50 value of 27.7<34.7< 41.5<55.2 µg/ml, respectively. P. niruri & O. stamineus (the best two extracts) showed noncompetitive and uncompetitive inhibition, respectively. P. niruri & O. stamineus displayed total phenolic content of 431.0 ± 0.01 and 103.0 ± 0.01 mg GAE/g dry extract, while total flavonoid content of 14.8 ± 0.07 and 21.6 ± 0.03 mg QE/g dry extract, respectively. Both P. niruri & O. stamineus extracts showed high antioxidant activity, with EC50 values of 8.4 and 26.3 µg/ml, respectively. The results suggest that P. niruri & O. stamineus may be beneficial for obesity treatment via pancreatic lipase inhibition action.
Introduction: Antilipase from natural resources are a potential tool for the treatment of obesity while antioxidant-rich plants are essential in combating degenerative diseases. The aim of this study is to determine the antilipase and antioxidant activity of Orthosiphon stamineus methanolic extract. Methods: The inhibitory activity against pancreatic lipase was determined by measuring the hydrolysis of p-nitrophenyl butyrate to p-nitrophenol at 405 nm. Antioxidant activity of O. stamineus extract was measured by 2, 2, diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity assay. Results: The O. stamineus crude extract exhibited strong lipase inhibitory activity with an IC50 value of 34.7 µg/ml. The inhibition mode study disclosed that O. stamineus could act as uncompetitive inhibitor. O. stamineus showed high antioxidant activity with an EC50 value of 26.3 µg/ml. Conclusions: The results suggest that O. stamineus has shown potential as a source of natural antilipase and antioxidant.
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