Lytic polysaccharide monooxygenase of soil actinomycete with potential use for lignocellulose biodegradation

Aims@#Lytic polysaccharide monooxygenase (LPMO) is an enzyme capable of cleaving glycoside bonds of recalcitrant polysaccharides through an oxidative mechanism. LPMO activity, in synergy with hydrolytic enzymes, increases the production of monomer sugars from the biodegradation of lignocellulose. This study was aimed at evaluating actinomycete S2 strain LPMO activity based on the release of xylose as one of reducing sugar and hydrogen peroxide (H2O2) in the course of lignocellulosic biodegradation. @*Methodology and results@#The oxidation activity of LPMO from actinomycete S2 strain was measured by using the substrate of Avicel supplemented with ascorbic acid and copper ions (Cu2+) to identify its effect on the release of xylose as one of reducing sugar. The optimum incubation time for the LPMO production was also conducted. Further, H2O2 quantitative analysis was performed as by-product of LPMO activity and 16S rRNA gene sequence of actinomycete S2 strain were subsequently determined. We found that supplementation of 1 mM ascorbic acid and 0.2 mM Cu2+ increased xylose as one of reducing sugar production by up to 5-fold from 255.03 to 1290 μg/mL after an optimal incubation period of 6 days. Based on H2O2 production, the LPMO activity of actinomycete S2 strain was 0.019 ± 0.001 U/mL. There is likelihood that LPMO activity derived from actinomycete S2 strain has a synergistic effect with the activity of other lignocellulose-degrading enzymes. This actinomycete showed 99% similarity to the 16S rRNA gene sequence of Streptomyces avermitilis strain EAAG80. @*Conclusion, significance, and impact of study@#LPMO enzyme activity from actinomycete S2 strain as determined by the production of reducing sugar and H2O2 was greatly increased by supplementation with ascorbic acid as an electron donor and Cu2+ ions. To the best of our knowledge, this is the first elucidation of LPMO activity from an indigenous Indonesian actinomycete.

Suppression of Feed Intake in Response to Rice Bran Oil Supplementation in Normal Rat

@#Introduction: Rice bran oil (RBO) is used in Asian countries as a daily dietary supplement. RBO is known in particular for its hypolipidemic effect. There has been increasing interest recently in the use of RBO as a means to maintain body weight and prevent obesity, though the mechanism of how this happens is still not well understood. We have investigated the effect of RBO on expression of genes that might influence energy homeostasis and feed intake. Methods: This study assessed Sprague-Dawley male rats at 12-weeks that were split into three groups over a 28-day period. A control group was fed a diet of standard rat chow, a standard group was fed standard rat chow with Orlistat (10.8 mg/kg bw/day), and a treatment group was fed standard rat chow with RBO (57.6 mg oryzanol/day). All supplementation was given by oral gavage. Possible adiposity was investigated through a histological analysis of adipocytes size measurement of intra-abdominal white adipose tissue in the rats. Changes in gene expression in the liver were determined by microarray. Results: The data suggest that RBO supplementation of a regular diet did not result in excess body weight and adiposity. A microarray analysis of the rats’ livers found that RBO altered the expression of genes related to energy homeostasis and feeding behavior, by upregulating genes such as Olr522, RGD1561231 and Rgs16. Conclusion: It is suggested that RBO supplementation can be used to maintain body weight by lowering appetite.

Potential effect of striatin (DLBS0333), a bioactive protein fraction isolated from Channa striata for wound treatment

Objective To characterize proteins and other nutrients in striatin (DLBS0333), a bioactive protein fraction isolated from snakehead fish (Channa striata) and to investigate its wound healing activity. Methods Proteins and other constituents in striatin were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two dimension electrophoresis, immunoblotting assay, spectroscopy and high performance liquid chromatography. The wound healing activity of striatin was studied in vitro using 3T3 fibroblast cells and in vivo using wound-induced animal model. Various parameters related to wound healing process were evaluated. Results Striatin contained four major bioactive proteins with approximate molecular weight of 8.3, 10.9, 15.4 and 16.7 kDa. In addition to proteins, striatin also contained amino acids (10 essential and 7 non essential amino acids), fatty acids (palmitic acid, oleic acid, stearic acid, linoleic acid, arachidonic acid), vitamins (vitamin A, vitamin B

Characterization and identification of mannanolytic actinomycete Nonomuraea sp. ID06-379

Aims: The study focused on screening, identification and characterization of mannanolytic actinomycetes isolated from soil and leaf litter samples obtained from several sites in Indonesia. Methodology and results: A total of 337 isolates of actinomycetes isolated from soil and leaf litter samples collected from various areas in Indonesia were screened for their mannanolytic activity. Mannanase activity was analysed using locus bean gum (LBG) as the substrate. The strain ID06-0379 displayed significant mannanase activity. The strain ID06- 0379 was analysed for its mannanase activity by determining the rate of enzyme production when cultured in the presence of palm kernel cake (PKC) as a substrate. The highest mannanase activity from ID06-0379 was 4.40 U/mL at 5% PKC concentration at 5 days incubation. Chemotaxonomic and phenotypic characterisation of mannanolytic actinomycete was done and the strain ID06-0379 contained meso-diaminopimelic acid, and madurose was the diagnostic sugar in whole cell sugar. The polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, and hydroxy-phosphatidylethanolamine. The predominant menaquinone of strain ID06-0379 was MK-9(H4). The major cellular fatty acids were C16:0 (31.47%), cis9-C16:1 (15.23%) and iso-C16:0 (10.84%), and the G+C content of the DNA was 71.7 mol%. Phylogenetic analysis based on the 16S rDNA sequences revealed that strain ID06- 379 was closely related to species of Nonomuraea jabiensis A4036T with 99% nucleotide similarity. Conclusion, significance and impact study: The results from this study revealed that the mannanolytic actinomycete strain ID06-379 belongs to the genus Nonomuraea that closely related to N. jabiensis A4036T . Mannanase production using agricultural waste such as palm kernel cake may contribute to the development and utilisation of biomass bioconversion processes. Keywords: Indonesian actinomycetes, mannanase enzyme, locus bean gum, palm kernel cake, Nonomuraea sp. ID06- 0379.

Quantity and quality of bromelain in some Indonesian pineapple fruits.

This research aimed to extract bromelain from all parts of some variant of Indonesian pineapple fruits in order to evaluate their amount and characteristics as well. Each part of pineapple (crown, flesh, core, and peel) was separately blended with sodium phosphate buffer 0.01 M pH 7.0 at ratio of 1:1 (w/v) then filtered using cheese cloth. The filtrate was centrifuged at 1250 g for 10 minutes. The supernatant was collected and then centrifuged at 10000 g for 15 minutes. All extracts from each stage (blended mixture, pulp, filtrate, supernatant 1, pellet 1, supernatant 2, and pellet 2) were collected and characterized. The concentration of protein was measured by using BSA as standard and the proteolytic activity was determined using casein 1% (w/v) as substrate under the standard conditions (37 oC, pH 8.00, 10 min). The optimum temperature (ranged 27-80°C) and pH (ranged 3-10) was also evaluated. The result showed that three stepwise extraction process (blending with buffer – filtration – centrifugation) could be applied to obtain bromelain from all parts of pineapple, but some amount of bromelain was still left in the residue. Due to its price and weight, Subang pineapple was the most potential Indonesian pineapple as the source of bromelain. The maximum protein content and bromelain activity in one of Subang pineapple were 236.29 mg and 787.92 unit, respectively, while those in Bogor and Palembang pineapples were 168.27-190.10 mg and 464.97-510.37 unit, respectively.

Purification of methanol dehydrogenase from mouth methylotrophic bacteria of tropical region

Aims: Purification of methanol dehydrogenase (MDH) from methylotrophic bacteria was conducted to obtain pure enzyme for further research and industrial applications due to the enzyme’s unique activity that catalyzes oxidation of methanol as an important carbon source in methylotrophic bacteria. Methodology and Results: The enzyme was screened from methylotrophic bacteria isolated from human mouth. Purification of this enzyme was conducted using ammonium sulphate precipitation followed by cation exchange chromatography. Two types of media were used to produce the enzymes: luria broth and standard mineral salts media (MSM). MSM produced MDH with higher specific activity than LB. Specific activity was also increased along with the purification steps. Application of ammonium sulphate increased the purity of enzyme and was more effective for the enzyme produced in LB. Using sepharose increased the enzyme activity 10 -57 folds. Conclusion, significant and impact of this study: With this, ammonium sulphate precipitation coupled with single cation exchange chromatographic system has been proved to provide sufficient purified of methanol dehydrogenase from methylotrophic bacteria origin of human mouth with high specific activity for further application.