Polyhydroxyalkanoate (PHA) is a promising candidate for use as an alternative bioplastic to replace petroleum-based plastics. Our understanding of PHA synthase PhaC is poor due to the paucity of available three-dimensional structural information. Here we present a high-resolution crystal structure of the catalytic domain of PhaC from Chromobacterium sp. USM2, PhaCCs-CAT. The structure shows that PhaCCs-CAT forms an α/β hydrolase fold comprising α/β core and CAP subdomains. The active site containing Cys291, Asp447 and His477 is located at the bottom of the cavity, which is filled with water molecules and is covered by the partly disordered CAP subdomain. We designated our structure as the closed form, which is distinct from the recently reported catalytic domain from Cupriavidus necator (PhaCCn-CAT). Structural comparison showed PhaCCn-CAT adopting a partially open form maintaining a narrow substrate access channel to the active site, but no product egress. PhaCCs-CAT forms a face-to-face dimer mediated by the CAP subdomains. This arrangement of the dimer is also distinct from that of the PhaCCn-CAT dimer. These findings suggest that the CAP subdomain should undergo a conformational change during catalytic activity that involves rearrangement of the dimer to facilitate substrate entry and product formation and egress from the active site.

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BackgroundOver the last 20 years in biotechnology, the production of recombinant proteins has been a crucial bioprocess in both biopharmaceutical and research arena in terms of human health, scientific impact and economic volume. Although logical strategies of genetic engineering have been established, protein overexpression is still an art. In particular, heterologous expression is often hindered by low level of production and frequent fail due to opaque reasons. The problem is accentuated because there is no generic solution available to enhance heterologous overexpression. For a given protein, the extent of its solubility can indicate the quality of its function. Over 30% of synthesized proteins are not soluble. In certain experimental circumstances, including temperature, expression host, etc., protein solubility is a feature eventually defined by its sequence. Until now, numerous methods based on machine learning are proposed to predict the solubility of protein merely from its amino acid sequence. In spite of the 20 years of research on the matter, no comprehensive review is available on the published methods.ResultsThis paper presents an extensive review of the existing models to predict protein solubility in Escherichia coli recombinant protein overexpression system. The models are investigated and compared regarding the datasets used, features, feature selection methods, machine learning techniques and accuracy of prediction. A discussion on the models is provided at the end.ConclusionsThis study aims to investigate extensively the machine learning based methods to predict recombinant protein solubility, so as to offer a general as well as a detailed understanding for researches in the field. Some of the models present acceptable prediction performances and convenient user interfaces. These models can be considered as valuable tools to predict recombinant protein overexpression results before performing real laboratory experiments, thus saving labour, time and cost.

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In this paper, we report the cloning and characterization of three Paenibacillus azotofixans DNA regions containing genes involved in nitrogen fixation. Sequencing analysis revealed the presence of nifB1H1D1K1 gene organization in the 4,607-bp SacI DNA fragment. This is the first report of linkage of a nifB open reading frame upstream of the structural nif genes. The second (nifB2H2) and third (nifH3) nif homologues are confined within the 6,350-bp HindIII and 2,840-bp EcoRI DNA fragments, respectively. Phylogenetic analysis demonstrated that NifH1 and NifH2 form a monophyletic group among cyanobacterial NifH proteins. NifH3, on the other hand, clusters among NifH proteins of the highly divergent methanogenic archaea.Nitrogen fixation-related genes have been highly conserved throughout evolution even though they are widely distributed among eubacteria and archaea (4,7,11,13,15). In terms of their physical and biochemical properties, the mechanisms of the nitrogen fixation process are very similar among these organisms. The conventional dinitrogenase is composed of an ␣ 2 ␤ 2 tetramer; the ␣ and ␤ subunits are encoded by the nifD and nifK genes, respectively. Also included in the nitrogenase complex is nitrogenase reductase, which is encoded by the nifH gene. In most diazotrophs, the nifHDK genes are contiguous. Sequence and mutational analyses of nitrogen fixation-related genes of various diazotrophs indicate that the arrangement of nif and associated genes differs considerably among these organisms. Examples of organisms with a noncontiguous arrangement of structural nif genes are Frankia sp. strain FaC1, Bradyrhizobium japonicum, and Rhizobium sp. strain Irc78 (2,14).Paenibacillus azotofixans ATCC 35681 is a gram-positive, facultatively anaerobic diazotroph that falls into a broad cluster of nitrogen fixers in rRNA group 3; this cluster also includes P. macerans and P. polymyxa (3). Diazotrophic strains of P. azotofixans were shown to possess the ability to fix atmospheric dinitrogen with high efficiency (8,25,29). In contrast to the majority of diazotrophs, their ability to fix nitrogen is not affected by the presence of nitrate (29).PCR amplification of the nifH gene fragment. The objective of identifying DNA fragments containing nif homologues was achieved by using the 380-bp nifH gene as a homologous probe. Alignment of NifH polypeptide sequences from representative diazotrophs was performed using ClustalX software (9). Based on these sequence alignments, nifH-degenerate oligonucleotides (5Ј-TAY GGN AAR GGN GGN ATN GGN AA-3Ј and 5Ј-GCR AAN CCN CCR CAN ACN ACR TC-3Ј) were designed as primers.Chromosomal DNA (40 ng/ml) was PCR amplified in a 50-l reaction volume containing 1ϫ PCR buffer (Promega), a 1 mM concentration of each primer, a 0.2 mM concentration of each deoxynucleoside triphosphate, 1.5 mM MgCl 2 , and 2.0 U of Weiss Taq DNA polymerase (Promega). The following PCR parameters were used: 94°C for 5 min; 30 thermal cycles of 94°C for 30s, 45°C for 30s, and 72°C for 30s; and a final extension step at ...

Background: Moringa oleifera belongs to plant family, Moringaceae and popularly called -wonderful tree‖, for it is used traditionally to cure many diseases including cancer in Africa and Asia, however, there is limited knowledge on cytotoxic activity of Moringa oleifera seeds on MCF7 breast cancer cell. The present study evaluated antiproliferative effect on MCF7 of the seed. Materials and Methods: Seeds of Moringa oleifera were grinded to powder and its phytochemicals were extracted using water and 80% ethanol solvents, part of the ethanolic extract were sequentially partitioned to fractions with four solvents (hexane, dichloromethane, chloroform, and n-butanol). Antiproliferative effects on MCF7 of the samples were determined. Finally, potent samples that significantly inhibited MCF7 growth were tested on MCF 10A. Results: Crude water extract, hexane and dichloromethane fractions of the seeds inhibited the proliferation of MCF7 with the following IC 50 values 280 µg/ml, 130 µg/ml and 26 µg/ml respectively, however, of the 3 samples, only hexane fraction had minimal cytotoxic effect on MCF 10A (IC 50 > 400µg/ml). Conclusion: Moringa oleifera seed has antiproliferative effect on MCF7.

Clinacanthus nutans is an essential medicinal plant that had been used in various local remedies to treat many illnesses. A study had been conducted to determine the phenolic, flavonoid, antioxidant activities and phytochemical compounds of C. nutans in different locations. C. nutans were harvested from eight locations and the leaves were extracted with 80 % methanol by maceration process. Then, the phytochemical screening using Gas Chromatography-Mass Spectrometry (GC-MS), 2,2 diphenyl-2-picrylhydrazyl hydrate (DPPH) assay method, total phenolic content by Folin-Ciocalteu's assay method and total flavonoid content by aluminium chloride (AlCl 3 ) were carried out. The C. nutans extracts showed higher antioxidant activities than phenolic and flavonoid content. The neutral pH sandy clay soil from location KKK (Kuala Ketil, Kedah, Malaysia) had higher antioxidant activities (58.0 %), phenolic (44.1 mg GA.100 g -1 ) and flavonoid content (30.8 mg QE.100 g -1 ) compared to other locations. The GC-MS analysis showed the presence of phytochemicals constituents of 20 compounds. The results revealed that environmental factors (light intensity, temperature and soil characteristics) of eight locations were responsible for variations of phenolic, flavonoids, antioxidants and GC-MS analysis in C. nutans. The findings of this study will provide baseline data for future breeding programs for commercial cultivation.

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