Remote protein homology detection and fold recognition refer to detection of structural homology in proteins where there are small or no similarities in the sequence. To detect protein structural classes from protein primary sequence information, homology-based methods have been developed, which can be divided to three types: discriminative classifiers, generative models for protein families and pairwise sequence comparisons. Support Vector Machines (SVM) and Neural Networks (NN) are two popular discriminative methods. Recent studies have shown that SVM has fast speed during training, more accurate and efficient compared to NN. We present a comprehensive method based on two-layer classifiers. The 1st layer is used to detect up to superfamily and family in SCOP hierarchy using optimized binary SVM classification rules. It used the kernel function known as the Bio-kernel, which incorporates the biological information in the classification process. The 2nd layer uses discriminative SVM algorithm with string kernel that will detect up to protein fold level in SCOP hierarchy. The results obtained were evaluated using mean ROC and mean MRFP and the significance of the result produced with pairwise t-test was tested. Experimental results show that our approaches significantly improve the performance of remote protein homology detection and fold recognition for all three different version SCOP datasets (1.53, 1.67 and 1.73). We achieved 4.19% improvements in term of mean ROC in SCOP 1.53, 4.75% in SCOP 1.67 and 4.03% in SCOP 1.73 datasets when compared to the result produced by well-known methods. The combination of first layer and second layer of BioSVM-2L performs well in remote homology detection and fold recognition even in three different versions of datasets.
This paper describes the development and application of multilocus sequencing typing (MLST) and multi-virulence locus sequencing typing (MVLST) methods in determining the genetic variation and relatedness of 43 Vibrio cholerae strains of different serogroups isolated from various sources in Malaysia. The MLST assay used six housekeeping genes (dnaE, lap, recA, gyrB, cat and gmd), while the MVLST assay incorporated three virulence genes (ctxAB, tcpA and tcpI) and three virulence-associated genes (hlyA, toxR and rtxA). Our data showed that the dnaE and rtxA genes were the most conserved genes in V. cholerae O1 strains. Among the 12 studied genes, transitional substitutions that led to silent mutations were observed in all, except for gmd and hlyA, while non-synonymous substitutions occurred more frequently in virulence and virulence-associated genes. Five V. cholerae O1 strains were found to be the El Tor variant O1 strains because they harboured the classical ctxB gene. In addition, the classical ctxB gene was also observed in O139 V. cholerae. A total of 29 MLST types were observed, and this assay could differentiate V. cholerae within the non-O1/non-O139 serogroups. A total of 27 MVLST types were obtained. MVLST appeared to be more discriminatory than MLST because it could differentiate V. cholerae strains from two different outbreaks and could separate the toxigenic from the non-toxigenic subtypes. Although the O1 V. cholerae strains were closely related, the combined MLST and MVLST analyses differentiated the strains isolated from different localities. In conclusion, sequence-based analysis in this study provided a better understanding of mutation points and the type of mutations in V. cholerae. The MVLST assay is useful to characterise O1 V. cholerae strains, while combined analysis may improve the discriminatory power and is suitable for the local epidemiological study of V. cholerae.
This work reviews the stripping off, role of water molecules in activity, and flexibility of immobilized Candida antarctica lipase B (CALB). Employment of CALB in ring opening polyester synthesis emphasizing on a polylactide is discussed in detail. Execution of enzymes in place of inorganic catalysts is the most green alternative for sustainable and environment friendly synthesis of products on an industrial scale. Robust immobilization and consequently performance of enzyme is the essential objective of enzyme application in industry. Water bound to the surface of an enzyme (contact class of water molecules) is inevitable for enzyme performance; it controls enzyme dynamics via flexibility changes and has intensive influence on enzyme activity. The value of pH during immobilization of CALB plays a critical role in fixing the active conformation of an enzyme. Comprehensive selection of support and protocol can develop a robust immobilized enzyme thus enhancing its performance. Organic solvents with a log P value higher than four are more suitable for enzymatic catalysis as these solvents tend to strip away very little of the enzyme surface bound water molecules. Alternatively ionic liquid can work as a more promising reaction media. Covalent immobilization is an exclusively reliable technique to circumvent the leaching of enzymes and to enhance stability. Activated polystyrene nanoparticles can prove to be a practical and economical support for chemical immobilization of CALB. In order to reduce the E-factor for the synthesis of biodegradable polymers; enzymatic ring opening polyester synthesis (eROPS) of cyclic monomers is a more sensible route for polyester synthesis. Synergies obtained from ionic liquids and immobilized enzyme can be much effective eROPS.
A thermophilic lipolytic bacterium identified as Bacillus sp. L2 via 16S rDNA was previously isolated from a hot spring in Perak, Malaysia. Bacillus sp. L2 was confirmed to be in Group 5 of bacterial classification, a phylogenically and phenotypically coherent group of thermophilic bacilli displaying very high similarity among their 16S rRNA sequences (98.5-99.2%). Polymerase chain reaction (PCR) cloning of L2 lipase gene was conducted by using five different primers. Sequence analysis of the L2 lipase gene revealed an open reading frame (ORF) of 1251 bp that codes for 417 amino acids. The signal peptides consist of 28 amino acids. The mature protein is made of 388 amino acid residues. Recombinant lipase was successfully overexpressed with a 178-fold increase in activity compared to crude native L2 lipase. The recombinant L2 lipase (43.2 kDa) was purified to homogeneity in a single chromatography step. The purified lipase was found to be reactive at a temperature range of 55-80 °C and at a pH of 6-10. The L2 lipase had a melting temperature (Tm) of 59.04 °C when analyzed by circular dichroism (CD) spectroscopy studies. The optimum activity was found to be at 70 °C and pH 9. Lipase L2 was strongly inhibited by ethylenediaminetetraacetic acid (EDTA) (100%), whereas phenylmethylsulfonyl fluoride (PMSF), pepstatin-A, 2-mercaptoethanol and dithiothreitol (DTT) inhibited the enzyme by over 40%. The CD spectra of secondary structure analysis showed that the L2 lipase structure contained 38.6% α-helices, 2.2% ß-strands, 23.6% turns and 35.6% random conformations.
Fishes are rich sources of structurally diverse bioactive compounds. In recent years, much attention has been paid to the existence of peptides with biological activities and proteins derived from foods that might have beneficial effects for humans. Antioxidant and antimicrobial peptides isolated from fish sources may be used as functional ingredients in food formulations to promote consumer health and improve the shelf life of food products. This paper presents an overview of the antioxidant and antimicrobial peptides derived from various fishes. In addition, we discuss the extraction of fish proteins, enzymatic production, and the techniques used to isolate and characterize these compounds. Furthermore, we review the methods used to assay the bioactivities and their applications in food and nutraceuticals.
Matched MeSH terms: Fish Proteins/isolation & purification*; Fish Proteins/pharmacology*
Molecular pathogenesis of chronic myeloid leukemia (CML) is well established and molecular monitoring for patients with CML has become an important practice in the management of patients on imatinib therapy. In the present study, we report the use of RQ-PCR method for detection of BCR-ABL fusion gene for our CML cases. We performed a two-step RQ-PCR on bone marrow aspirates or peripheral blood of 37 CML patients. Quantitative expression of BCR-ABL fusion gene was carried out relative to the expression of a housekeeping gene as endogenous control to compensate for uneven cell numbers, RNA quality, or variations in reverse transcription efficiencies. Twenty-four of these patients were pre-treated with hydroxyurea or alpha interferon prior to the imatinib therapy. Their BCR-ABL fusion gene levels were monitored for 18 months. All samples processed were evaluable. The PCR amplification efficiency of the ABL gene is 90.5% (0.2158) and the BCR-ABL gene, 93.4% (0.1573).
Response surface methodology (RSM) using a central composite design (CCD) was employed to optimize the conditions for extraction of serine protease from kesinai (Streblus asper) leaves. The effect of independent variables, namely temperature (42.5,47.5, X₁), mixing time (2-6 min, X₂), buffer content (0-80 mL, X₃) and buffer pH (4.5-10.5, X₄) on specific activity, storage stability, temperature and oxidizing agent stability of serine protease from kesinai leaves was investigated. The study demonstrated that use of the optimum temperature, mixing time, buffer content and buffer pH conditions protected serine protease during extraction, as demonstrated by low activity loss. It was found that the interaction effect of mixing time and buffer content improved the serine protease stability, and the buffer pH had the most significant effect on the specific activity of the enzyme. The most desirable conditions of 2.5 °C temperature, 4 min mixing time, 40 mL buffer at pH 7.5 was established for serine protease extraction from kesinai leaves.
A thermophilic Bacillus stearothermophilus F1 produces an extremely thermostable serine protease. The F1 protease sequence was used to predict its three-dimensional (3D) structure to provide better insights into the relationship between the protein structure and biological function and to identify opportunities for protein engineering. The final model was evaluated to ensure its accuracy using three independent methods: Procheck, Verify3D, and Errat. The predicted 3D structure of F1 protease was compared with the crystal structure of serine proteases from mesophilic bacteria and archaea, and led to the identification of features that were related to protein stabilization. Higher thermostability correlated with an increased number of residues that were involved in ion pairs or networks of ion pairs. Therefore, the mutants W200R and D58S were designed using site-directed mutagenesis to investigate F1 protease stability. The effects of addition and disruption of ion pair networks on the activity and various stabilities of mutant F1 proteases were compared with those of the wild-type F1 protease.
A binary mixture of starch-starch or starch with other biopolymers such as protein and non-starch polysaccharides could provide a new approach in producing starch-based food products. In the context of food processing, a specific adjustment in the rheological properties plays an important role in regulating production processing and optimizing the applicability, stability, and sensory of the final food products. This review examines various biopolymer mixtures based on starch and the influence of their interaction on physicochemical and rheological properties of the starch-based foods. It is evident that the physicochemical and rheological characteristics of the biopolymers mixture are highly dependent on the type of starch and other biopolymers that make them up mixing ratios, mixing procedure and presence of other food ingredients in the mixture. Understanding these properties will lead to improve the formulation of starch-based foods and minimize the need to resort to chemically modified starch.
An organic solvent tolerant lipase gene from Staphylococcus epidermidis AT2 was successfully cloned and expressed with pTrcHis2 in E. coli TOP10. Sequence analysis revealed an open reading frame (ORF) of 1,933 bp in length which coded for a polypeptide of 643 amino acid residues. The polypeptide comprised of a signal peptide (37 amino acids), pro-peptide and a mature protein of 390 amino acids. Expression of AT2 lipase resulted in an 18-fold increase in activity, upon the induction of 0.6 mM IPTG after a 10 h incubation period. Interestingly, this lipase was stable in various organic solvents (25% (v/v), mainly toluene, octanol, p-xylene and n-hexane). Literature shows that most of the organic solvent stable bacterial lipases were produced by Pseudomonas sp. and Bacillus sp., but very few from Staphylococcus sp. This lipase demonstrates great potential to be employed in various industrial applications.
Genes involved in the 4-aminobenzenesulfonate (4-ABS) degradation pathway of Hydrogenophaga sp. PBC were identified using transposon mutagenesis. The screening of 10,000 mutants for incomplete 4-ABS biotransformation identified four mutants with single transposon insertion. Genes with insertions that impaired the ability to utilize 4-ABS for growth included (1) 4-sulfocatechol 1,2-dioxygenase β-subunit (pcaH2) and 3-sulfomuconate cycloisomerase involved in the modified β-ketoadipate pathway; (2) 4-aminobenzenesulfonate 3,4-dioxygenase component (sadA) involved in aromatic ring hydroxylation; and (3) transposase gene homolog with a putative cis-diol dehydrogenase gene located downstream. The pcaH2 mutant strain accumulated brown metabolite during growth on 4-ABS which was identified as 4-sulfocatechol through thin layer chromatography and HPLC analyses. Supplementation of wild-type sadA gene in trans restored the 4-ABS degradation ability of the sadA mutant, thus supporting the annotation of its disrupted gene.
Sema4D, also known as CD100, is a protein belonging to class IV semaphorin. Its physiologic roles in the immune and nervous systems have been extensively explored. However, the roles of Sema4D have extended beyond these traditionally studied territories. Via interaction with its high affinity receptor Plexin-B1, Sema4D-Plexin-B1 involvement in tumor progression is strongly implied. Here, we critically review and delineate the Sema4D-Plexin-B1 interaction in many facets of tumor progression: tumor angiogenesis, regulation of tumor-associated macrophages and control of invasive growth. We correlate the in vitro and in vivo experimental data with the clinical study outcomes, and present a molecular mechanistic basis accounting for the intriguingly contradicting results from these recent studies.
Japanese encephalitis virus (JEV) is the most important cause of epidemic encephalitis worldwide but its origin is unknown. Epidemics of encephalitis suggestive of Japanese encephalitis (JE) were described in Japan from the 1870s onwards. Four genotypes of JEV have been characterised and representatives of each genotype have been fully sequenced. Based on limited information, a single isolate from Malaysia is thought to represent a putative fifth genotype. We have determined the complete nucleotide and amino acid sequence of Muar strain and compared it with other fully sequenced JEV genomes. Muar was the least similar, with nucleotide divergence ranging from 20.2 to 21.2% and amino acid divergence ranging from 8.5 to 9.9%. Phylogenetic analysis of Muar strain revealed that it does represent a distinct fifth genotype of JEV. We elucidated Muar signature amino acids in the envelope (E) protein, including E327 Glu on the exposed lateral surface of the putative receptor binding domain which distinguishes Muar strain from the other four genotypes. Evolutionary analysis of full-length JEV genomes revealed that the mean evolutionary rate is 4.35 × 10(-4) (3.4906 × 10(-4) to 5.303 × 10(-4)) nucleotides substitutions per site per year and suggests JEV originated from its ancestral virus in the mid 1500s in the Indonesia-Malaysia region and evolved there into different genotypes, which then spread across Asia. No strong evidence for positive selection was found between JEV strains of the five genotypes and the E gene has generally been subjected to strong purifying selection.
The stability of biocatalysts is an important criterion for a sustainable industrial operation economically. T1 lipase is a thermoalkalophilic enzyme derived from Geobacillus zalihae strain T1 (T1 lipase) that was isolated from palm oil mill effluent (POME) in Malaysia. We report here the results of high temperatures molecular dynamics (MD) simulations of T1 lipase in explicit solvent. We found that the N-terminal moiety of this enzyme was accompanied by a large flexibility and dynamics during temperature-induced unfolding simulations which preceded and followed by clear structural changes in two specific regions; the small domain (consisting of helices alpha3 and alpha5, strands beta1 and beta2, and connecting loops) and the main catalytic domain or core domain (consisting of helices alpha6- alpha9 and connecting loops which located above the active site) of the enzyme. The results suggest that the small domain of model enzyme is a critical region to the thermostability of this organism.
Extraribosomal functions of human ribosomal proteins (RPs) include the regulation of cellular growth and differentiation, and are inferred from studies that linked congenital disorders and cancer to the deregulated expression of RP genes. We have previously shown the upregulation and downregulation of RP genes in tumors of colorectal and nasopharyngeal carcinomas (NPCs), respectively. Herein, we show that a subset of RP genes for the large ribosomal subunit is differentially expressed among cell lines derived from the human nasopharyngeal epithelium. Three such genes (RPL27, RPL37a and RPL41) were found to be significantly downregulated in all cell lines derived from NPC tissues compared with a nonmalignant nasopharyngeal epithelial cell line. The expression of RPL37a and RPL41 genes in human nasopharyngeal tissues has not been reported previously. Our findings support earlier suspicions on the existence of NPC-associated RP genes, and indicate their importance in human nasopharyngeal organogenesis.
Bluetongue virus (BTV) is the 'type' species of the genus Orbivirus within the family Reoviridae. The BTV genome is composed of ten linear segments of double-stranded RNA (dsRNA), each of which codes for one of ten distinct viral proteins. Previous phylogenetic comparisons have evaluated variations in genome segment 3 (Seg-3) nucleotide sequence as way to identify the geographical origin (different topotypes) of BTV isolates. The full-length nucleotide sequence of genome Seg-3 was determined for thirty BTV isolates recovered in the eastern Mediterranean region, the Balkans and other geographic areas (Spain, India, Malaysia and Africa). These data were compared, based on molecular variability, positive-selection-analysis and maximum-likelihood phylogenetic reconstructions (using appropriate substitution models) to 24 previously published sequences, revealing their evolutionary relationships. These analyses indicate that negative selection is a major force in the evolution of BTV, restricting nucleotide variability, reducing the evolutionary rate of Seg-3 and potentially of other regions of the BTV genome. Phylogenetic analysis of the BTV-4 strains isolated over a relatively long time interval (1979-2000), in a single geographic area (Greece), showed a low level of nucleotide diversity, indicating that the virus can circulate almost unchanged for many years. These analyses also show that the recent incursions into south-eastern Europe were caused by BTV strains belonging to two different major-lineages: representing an 'eastern' (BTV-9, -16 and -1) and a 'western' (BTV-4) group/topotype. Epidemiological and phylogenetic analyses indicate that these viruses originated from a geographic area to the east and southeast of Greece (including Cyprus and the Middle East), which appears to represent an important ecological niche for the virus that is likely to represent a continuing source of future BTV incursions into Europe.
Soy protein isolate (SPI) gels were produced using single cross-linking agents (SCLA) of microbial transglutaminase (MTG) via incubation for 5 or 24 h (SCLA-MTG). When powdered SCLA-MTG gels were heated for 2 h with ribose (R2) (2 g/100 mL), dark brown gels were formed, and these were designated as combined cross-linking agent (CCLA) gels: MTG5(R2) and MTG24(R2). The results showed that the levels of Maillard-derived browning and cross-links of MTG5(R2) and MTG24(R2) gels were significantly (P < 0.05) lower than a control gel produced without MTG (SCLA-R2) even though the percentage of ribose remaining after heating of these gels was similar, indicating that a similar amount of ribose was consumed during heating. epsilon-(gamma-glutamyl)lysine bonds formed during incubation of SPI with MTG may have reduced the free amino group of SPI to take part in the Maillard reaction; nevertheless, ribose took part in the Maillard reaction and initiated the Maillard cross-linkings within the CCLA gels.
An all-atom level MD simulation in explicit solvent at high temperature is a powerful technique to increase our knowledge about the structurally important regions modulating thermal stability in thermenzymes. In this respect, two large-sized thermoalkalophilic enzymes from Bacillus stearothermophilus L1 (L1 lipase) and Geobacillus zalihae strain T1 (T1 lipase) are well-established representatives. In this paper, comparative results from temperature-induced MD simulations of both model systems at 300 K, 400 K and 500 K are presented and discussed with respect to identification of highly flexible regions critical to thermostability. From our MD simulation results, specific regions along the L1 lipase and T1 lipase polypeptide chain including the small domain and the main catalytic domain or core domain of both enzymes show a marked increase in fluctuations and dynamics followed by clear structural changes. Overall, the N-terminal moiety of both enzymes and their small domains exhibit hyper-sensitivity to thermal stress. The results appear to propose that these regions are critical in determining of the overall thermal stability of both organisms.
Chicken breast muscle powder (CBMP) and modified waxy cornstarch (MWCS) blends were prepared at different pH conditions (pH 4, 5, 6, 7, 8, and 9). The blends were characterized by light microscopy, frequency sweep, flow analysis, and freeze-thaw stability analysis. Light microscopy showed that the blend structure was coarse at pH conditions close to the isoelectric point of protein and became finer with increasing pH. Frequency sweep demonstrated that the blend was more liquid-like with relatively lower storage (G') and loss (G'') moduli as the pH was increased from pH 4 to pH 9. Flow analysis revealed that thixotropy behavior was evident in samples treated at pHs 4 and 5, whereas antithixotropy was shown by those adjusted to pHs 6, 7, 8, and 9. The CBMP-MWCS blends were found to show better freeze-thaw stability at pH 8 that could be attributed to the formation of a highly interactive network structure of CBMP and MWCS.