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Constitutive Expression of Candida antarctica Lipase B (CALB) in Pichia pastoris Using pGAPZα Vector

The CalBsyn gene was previously constructed synthetically to encode Candida antarctica lipase B (CALB). Lipase from CalBsyn gene is slightly different from that of wild type CALB (CALB-wt) where it has three amino acids substitutions at different positions, i.e. V210I, A281E, and V221D, to improve enzyme’s thermostability and catalytic efficiency. The CalBsyn gene was isolated from pJ912-CalBsyn vector by digestion using XhoI restriction enzyme. The CalBsyn gene then was ligated to pGAPZα expression vector and transformed into E. coli TOP10F’ in order to obtain recombinant vector pGAPZα-CalBsyn. The result showed that pGAPZα-CalBsyn recombinant vector was successfully transformed into E. coli TOP10F’ with transformation efficiency of 4.11 x 103 cfu/µg plasmid DNA. The pGAPZα-CalBsyn recombinant plasmid was successfully introduced into Pichia pastoris SMD1168H using electroporation method with transformation efficiency of 1.01 x 102 cfu/µg DNA. Qualitative lipase activity assays showed that transformed P. pastoris secreted recombinant lipase (CALB) and has lipolytic activity; while quantitative lipase activity assays showed that the lipase activity was 63.5 Units/ml in 48 hours. Analysis using SDS-PAGE showed that CALB protein was expressed successfully and the recombinant protein’s molecular size was approximately 45 kDa.
 

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Bioactivities Screening of Indonesian Marine Bacteria Isolated from Sponges

The marine bacteria were cultured in liquid medium under shaking condition were extracted with ethyl acetate. Antidiabetes was measured using inhibition of α-glucosidase inhibitory activity method; antioxidant was measured using DPPH free radical scavenging activity method; antibacterial was tested using disc diffusion method.S creening results showed that at sample concentration of 200 µg/ml, there was significant α-glucosidase inhibitory activity was detected in the extracts of strain sp 7.9 (84% inhibition) and 8.10 (75% inhibition),however the antioxidant activity of these two strains were low only around 30% inhibition, antioxidant activities of other strains were very low.Screening for antibacterial activities using 10µl samples showed that extract of strain Sp 8.5was best for Staphylococcusaureus (14 mm inhibition); Sp 7.9 and Sp 8.5 for Bacillus subtilis (18 mm inhibition); Sp 8.10 for Escherichia coli (10 mm inhibition); Sp 8.9 and Sp 8.10 for Pseudomonas aeuriginosa. Based on these results marine bacteria strain Sp 7.9 and Sp 8.10 were selected to be used for further studies in the isolation of bioactive that has potential as antidiabetes and antibacterial.Results of molecular identification conducted by INACC showed that identity of both strain based on BLAST Homology using NCBI database were Bacillus thuringiensis strain Ou2.
 

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Identification of a New Compound as α-Glucosidase Inhibitor from Aspergillus aculeatus

Terestrial fungi are of great importance as potential sources of pharmaceutical agent. Aspergillus aculeatus,a fungus isolated from soil sample collected in Indonesia, was cultured in liquid media to investigate a novel compound as inhibitor α-glucosidase. The mycelium extract of A. aculeatus shows potential activity against Saccharomyces cereviseae α-glucosidase and mild activity against mammalian α-glucosidase with IC50 values of 9.57 µg/mL and 470.76 mg/mL, respectively. Enzyme assay-guided fractionation of this extract afforded rubrofusarin (1). Rubrofusarin, a linear naphtho-γ-pyrone, is a natural pigment from Aspergillus sp. Interestingly, compound 1 shows potential inhibitory activity against mammalian α-glucosidase (IC50 of 92.7 µg/mL), but no to S.cereviseae α-glucosidase. The results suggest that A. aculeatus is a promising natural source as a lead compound in the discovery of antidiabetic drug.

 

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Isolation and Characterization of Compounds from the Leaves of Pterocarpus indicus Willd and Their Antioxidant Activity

The flavone glycoside was isolated from ethyl acetate fraction of ethanol extract of leaves Pterocarpus indicus Willd. The isolation was conducted by gravitation column chromatography and eluted successively with hexane, ethyl acetate and methanol by gradient, and purified by sephadex-LH20. The structure was elucidated base on spectroscopy data of NMR (1D and 2D), UV, LC-MS and FT-IR. Antioxidant was evaluated using 2,2-diphenyl-1-picrylhidrazyl (DPPH) radical scavenging. The isolation and identification led a stigmasterol as Compound 1 and a new flavonol-glycoside [(2R)-7-hydroxy-3-(3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yloxy)-2-(3,4,5-trihydroxy phe nyl)chroman-4-one] or ptevon-3-D- glycoside as Compound 2. Antioxidant activity of Compound 2 showed IC50 for 18.53 mmol and blank of quercetin was 7.94 mmol and Vitamin C was 40.25 mmol. These compounds and antioxidant activities are the first time reported from this plant.

 

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Massive In Vitro Propagation Of Sandalwood Through Friable Embryogenic Callus

Sandalwood (Santalum album), which belongs to Santalaceae family, is a commercially important tree in Indonesia due to its many application. However,its population has significantly depleted since the planting materials using conventional methods are difficult to be provided. This study was conducted to mass propagate sandalwood using in vitro methods through friable embryogenic callus (FEC). The somatic embryos were formed using leaves cultured in MS +0.5 mg/l +1 mg/l indole acetic acid (IAA), and MS +1 mg/l IAA + 0.2 mg/l kinetin as well as 0.5 MS+1 mg/l Gibberellic acid (GA3). Primary somatic embryos (PSE) and secondary somatic embryos (SSE) then formed friable embryogenic callus when they were repetitively transferred to MS +1.5 mg/l BAP + 1.2 mg/l kinetin every 3 weeks. The maturation and regeneration of FEC was best done in the MS +1.5 mg/l BAP + 1.2 mg/l kinetin for 4-8 weeks. The acclimatization of sandalwood plantlets can be best conducted in the medium containing soil, sand and compos in ratio of 1:1:1 with the companion plant Murraya paniculata, which gave the best percentage of survival rate and the lowest percentage of falling leaves.

 

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