Bacteriocin release protein mediated secretory expression of recombinant chalcone synthase in Escherichia coli

Flavonoids are secondary metabolites synthesized by plants shown to exhibit health benefits such as anti-inflammatory, antioxidant, and anti-tumor effects. Thus, due to the importance of this compound, several enzymes involved in the flavonoid pathway have been cloned and characterized in Escherichia coli. However, the formation of inclusion bodies has become a major disadvantage of this approach. As an alternative, chalcone synthase from Physcomitrella patens was secreted into the medium using a bacteriocin release protein expression vector. Secretion of P. patens chalcone synthase into the culture media was achieved by co-expression with a psW1 plasmid encoding bacteriocin release protein in E. coli Tuner (DE3) plysS. The optimized conditions, which include the incubation of cells for 20 h with 40 ng/ml mitomycin C at OD600 induction time of 0.5 was found to be the best condition for chalcone synthase secretion

Draft genome sequence of roseovarius sp. PS-C2, isolated from sekinchan beach in Selangor, Malaysia

Roseovarius sp. PS-C2 is a bacterium that was isolated from Sekinchan Beach in Selangor, Malaysia, using an ex situ cultivation technique. Here, we present a high-quality annotated draft genome of strain PS-C2 and suggest potential applications of this bacterium

Draft genome sequence of Cellulomonas sp. PS-H5, isolated from Sekinchan beach in Selangor, Malaysia

Cellulomonas sp. PS-H5 was isolated from Sekinchan Beach in Selangor, Malaysia, using an ex situ cultivation method. The present work reports a high-quality draft annotated genome sequence of this strain and suggests its potential glycoside hydrolase enzymes for cellulose, hemicellulose, and starch degradations

Current status and potential applications of underexplored prokaryotes

Thousands of prokaryotic genera have been published, but methodological bias in the study of prokaryotes is noted. Prokaryotes that are relatively easy to isolate have been well-studied from multiple aspects. Massive quantities of experimental findings and knowledge generated from the well-known prokaryotic strains are inundating scientific publications. However, researchers may neglect or pay little attention to the uncommon prokaryotes and hard-to-cultivate microorganisms. In this review, we provide a systematic update on the discovery of underexplored culturable and unculturable prokaryotes and discuss the insights accumulated from various research efforts. Examining these neglected prokaryotes may elucidate their novelties and functions and pave the way for their industrial applications. In addition, we hope that this review will prompt the scientific community to reconsider these untapped pragmatic resources

Polycyclic aromatic hydrocarbons: characteristics and its degradation by biocatalysis remediation

An excessive released of polycyclic aromatic hydrocarbons (PAHs) to surroundings is one of the major factors that cause environmental pollution to increase globally. This issue had gained scientist’s attention to study PAHs biodegradation pathways and their toxicity towards humans and the environment. They found that the major mechanism responsible for the ecological recovery of PAH-contaminated sites happened to be from the microbial degradation process. However, there are a few limitations faced by the PAHs degrading bacteria where the bacteria die due to extremely polluted areas. This leads the researchers to utilize genetic engineering to produce enzymes that can withstand and survive in extreme environments. Recent information and technology such as path sources, properties and biochemical pathways by means to produce the simplest and less harmful components in polluted ecosystems are discussed in this review. In-depth studies in regards to bacteria biocatalysis involving bacterialproduced-enzymes to degrade PAHs help develop new methods to enhance the bioremediation effectiveness in the future

Expression, characterization, and rational design of chalcone synthase from Physcomitrella patens

Flavanoids are plant secondary metabolites synthesized by polyketide synthase (PKS) in plants, fungi and bacteria. The members of the chalcone synthase (CHS) superfamily, also known as the type III PKSs, function as the key entry enzyme of the flavonoid biosynthesis. Flavanoids are found in liverworts and mosses (bryophyte) that are thought to be the land plants ancestor. Although hundred of CHSs genes from various plant species have been successfully clone, expressed and studied, limited information is available on the CHS from the bryophytes. The model moss, Physcomitrella patens is currently the only bryophyte whose genome has been sequenced and its genome contains at least 17 putative type III PKS genes. Among them, a CHS gene was shown to be basal to all plant type III PKS genes in phylogenetic trees. P. patens CHS exhibited similar kinetic properties and substrate preference profiles as those of higher plants. This suggest that the P. Patens CHS may exhibit similar mechanism as the other land plants CHSs. Due to the unavailability of the P. Patens CHS crystal structure, the main aim of this work is to gain some insight on the structure and mechanism of the P. Patens CHS. Apart form the unavailability of P. patens CHS crystal structure, the major bottleneck of this work is to obtain the P. patens CHS due to the slow plant growth rate and low (less than 1%) extraction yield. Therefore, to speed up the process of obtaining the enzyme, the P. patens CHS has been successfully cloned and expressed in E. coli strain BL21 (DE3) plysS. However, the formation of inclusion bodies has become a major disadvantage of this approach. As alternative, P. patens CHS was secreted into the medium using a bacteriocin release protein expression vector. Secretion of P. patens CHS into the culture media was achieved by co-expression with a psW1 plasmid encoding bacteriocin release protein in E. coli Tuner (DE3) plysS. The optimized conditions were incubation of cells for 20 hours with 40 ng/ml mitomycin C at OD600 induction time of 0.5 was found to be the best condition for chalcone synthase secretion. The recombinant P. patens CHS was purified to 1.78-fold with 88.1% yield and specific activity of 4.26 U/mg by affinity chromatography technique using Ni 2+ Sepharose Fast Flow resin. The enzyme optimum pH and temperature were 7.0 and 30 °C, respectively. In addition, the enzyme was found to be stable up to 50 °C. Several crystallization attempts of P. patens CHS was carried out using vapour diffusion techniques, however, X-ray data processing was unable to be performed due to the weak diffraction spots obtained from the P. Patens crystals. Weak diffraction spots obtained might be due to poor crystal packing, the presence of impurities, nonspecific aggregations and crystal damaged by X-ray beam. Consequently, to study the structure and mechanism of P. patens CHS, combination of homology modeling and site-directed mutagenesis was conducted. The P. patens CHS structure was built using the M. sativa CHS crystal structure as a template. Based on the overall assessment, the quality of the modeled structure was comparable to template. Through the modeled PpCHS structure, showed the same four catalytic residues of the active site conserved in other CHS superfamily. Due to the function of the catalytic Cys170 in the first binding (initiation reaction) of the starter molecule on the catalytic active site, it was targeted as a critical residue. To investigate the effect of Cys170 substitutions towards the P. patens enzyme activity, two mutants were constructed, C170 R and C170S. From the mutant‟s data analysis, it could be concluded that the catalytic residue Cys170 plays an important role in the binding of substrate onto the active site. Substitution of Cys170 has lead to several changes in its interaction among the other residues resides in the active site cavity. The changes affect the binding capacity, cavity volume of the active site and the overall protein structure volume which decreased the production of reaction products. Apart, the mutant‟s physical properties such as temperature, pH and stability were found to be affected as well

Raw oil palm frond leaves as cost-effective substrate for cellulase and xylanase productions by Trichoderma asperellum UC1 under solid-state fermentation

Production of cellulases and xylanase by a novel Trichoderma asperellum UC1 (GenBank accession no. MF774876)under solid state fermentation (SSF)of raw oil palm frond leaves (OPFL)was optimized. Under optimum fermentation parameters (30 °C, 60–80% moisture content, 2.5 × 106 spores/g inoculum size)maximum CMCase, FPase, β-glucosidase and xylanase activity were recorded at 136.16 IU/g, 26.03 U/g, 130.09 IU/g and 255.01 U/g, respectively. Cellulases and xylanase were produced between a broad pH range of pH 6.0–12.0. The enzyme complex that comprised of four endo-β-1,4-xylanases and endoglucanases, alongside exoglucanase and β-glucosidase showed thermophilic and acidophilic characteristics at 50–60 °C and pH 3.0–4.0, respectively. Glucose (16.87 mg/g)and fructose (18.09 mg/g)were among the dominant sugar products from the in situ hydrolysis of OPFL, aside from cellobiose (105.92 mg/g)and xylose (1.08 mg/g). Thermal and pH stability tests revealed that enzymes CMCase, FPase, β-glucosidase and xylanase retained 50% residual activities for up to 15.18, 4.06, 17.47 and 15.16 h of incubation at 60 °C, as well as 64.59, 25.14, 68.59 and 19.20 h at pH 4.0, respectively. Based on the findings, it appeared that the unique polymeric structure of raw OPFL favored cellulases and xylanase productions

Enzymatic properties and mutational studies of chalcone synthase from Physcomitrella patens

PpCHS is a member of the type III polyketide synthase family and catalyses the synthesis of the flavonoid precursor naringenin chalcone from p-coumaroyl-CoA. Recent research reports the production of pyrone derivatives using either hexanoyl-CoA or butyryl-CoA as starter molecule. The Cys-His-Asn catalytic triad found in other plant chalcone synthase predicted polypeptides is conserved in PpCHS. Site directed mutagenesis involving these amino acids residing in the active-site cavity revealed that the cavity volume of the active-site plays a significant role in the selection of starter molecules as well as product formation. Substitutions of Cys 170 with Arg and Ser amino acids decreased the ability of the PpCHS to utilize hexanoyl-CoA as a starter molecule, which directly effected the production of pyrone derivatives (products). These substitutions are believed to have a restricted number of elongations of the growing polypeptide chain due to the smaller cavity volume of the mutant’s active site

Identification of potential dual -targets anti- toxoplasma gondii compounds through structure-based virtual screening and in-vitro studies.

Toxoplasma gondii is the etiologic agent of toxoplasmosis, a disease which can lead to morbidity and mortality of the fetus and immunocompromised individuals. Due to the limited effectiveness or side effects of existing drugs, the search for better drug candidates is still ongoing. In this study, we performed structure-based screening of potential dual-targets inhibitors of active sites of T. gondii drug targets such as uracil phosphoribosyltransferase (UPRTase) and adenosine kinase (AK). First screening of virtual compounds from the National Cancer Institute (NCI) was performed via molecular docking. Subsequently, the hit compounds were tested in-vitro for anti- T. gondii effect using cell viability assay with Vero cells as host to determine cytotoxicity effects and drug selectivities. Clindamycin, as positive control, showed a selectivity index (SI) of 10.9, thus compounds with SI > 10.9 specifically target T. gondii proliferation with no significant effect on the host cells. Good anti- T. gondii effects were observed with NSC77468 (7-ethoxy-4-methyl-6,7-dihydro-5H-thiopyrano[2,3-d]pyrimidin-2-amine) which showed SI values of 25. This study showed that in-silico selection can serve as an effective way to discover potentially potent and selective compounds against T. gondii