Deletion studies for elucidating the role of Streptomyces griseus ChiC non-catalytic residues

The soil bacterium, Streptomyces griseus, produces an antifungal chitinase (SgChiC) which has a smaller catalytic domain (in addition to a chitin binding domain) when compared with its counterparts from plants. Here, we carried out rational deletion of residues distant from the active site residues in the catalytic domain from 205 to 49 amino acid residues. The truncated residues were reconstructed and its 3-dimendional model predicted by homology modeling. In an insilico binding study, tri-N-acetyl glucosamine ((GlCNAc)3) was observed to bind to the active site of the truncated model similarly as in the wild type catalytic domain. This suggests that the variant model of SgChiC with a truncated catalytic domain possibly retains its chitinolytic properties. Further analysis of the simulation results revealed an increase in conformational space and flexibility of the reconstructed model over the less dynamic structure of the wild-type model. This suggests that the deleted residues played a role in the compactness and rigidity of the domain. Experimental assays to investigate the hydrolytic and kinetic properties of this truncated variant are currently been carried out. Outcomes of this study will reveal the relationship between the architecture of the ChiC domain and its function. This will guide future design studies for the enhancement of its functional properties and consequently its efficiency as a biocontrol agent.

Thermostable lipases and their dynamics of improved enzymatic properties

Thermal stability is one of the most desirable characteristics in the search for novel lipases. The search for thermophilic microorganisms for synthesising functional enzyme biocatalysts with the ability to withstand high temperature, and capacity to maintain their native state in extreme conditions opens up new opportunities for their biotechnological applications. Thermophilic organisms are one of the most favoured organisms, whose distinctive characteristics are extremely related to their cellular constituent particularly biologically active proteins. Modifications on the enzyme structure are critical in optimizing the stability of enzyme to thermophilic conditions. Thermostable lipases are one of the most favourable enzymes used in food industries, pharmaceutical field, and actively been studied as potential biocatalyst in biodiesel production and other biotechnology application. Particularly, there is a trade-off between the use of enzymes in high concentration of organic solvents and product generation. Enhancement of the enzyme stability needs to be achieved for them to maintain their enzymatic activity regardless the environment. Various approaches on protein modification applied since decades ago conveyed a better understanding on how to improve the enzymatic properties in thermophilic bacteria. In fact, preliminary approach using advanced computational analysis is practically conducted before any modification is being performed experimentally. Apart from that, isolation of novel extremozymes from various microorganisms are offering great frontier in explaining the crucial native interaction within the molecules which could help in protein engineering. In this review, the thermostability prospect of lipases and the utility of protein engineering insights into achieving functional industrial usefulness at their high temperature habitat are highlighted. Similarly, the underlying thermodynamic and structural basis that defines the forces that stabilize these thermostable lipase is discussed

Integrative structural and computational biology of phytases for the animal feed industry

Resistance to high temperature, acidic pH and proteolytic degradation during the pelleting process and in the digestive tract are important features of phytases as animal feed. The integration of insights from structural and in silico analyses into factors affecting thermostability, acid stability, proteolytic stability, catalytic efficiency and specific activity, as well as N-glycosylation, could improve the limitations of marginal stable biocatalysts with trade-offs between stability and activity. Synergistic mutations give additional benefits to single substitutions. Rigidifying the flexible loops or inter-molecular interactions by reinforcing non-bonded interactions or disulfide bonds, based on structural and roof mean square fluctuation (RMSF) analyses, are contributing factors to thermostability. Acid stability is normally achieved by targeting the vicinity residue at the active site or at the neighboring active site loop or the pocket edge adjacent to the active site. Extending the positively charged surface, altering protease cleavage sites and reducing the affinity of protease towards phytase are among the reported contributing factors to improving proteolytic stability. Remodeling the active site and removing steric hindrance could enhance phytase activity. N-glycosylation conferred improved thermostability, proteases degradation and pH activity. Hence, the integration of structural and computational biology paves the way to phytase tailoring to overcome the limitations of marginally stable phytases to be used in animal feeds

Ability of T1 lipase to degrade amorphous P(3HB): structural and functional study

An enzyme with broad substrate specificity would be an asset for industrial application. T1 lipase apparently has the same active site residues as polyhydroxyalkanoates (PHA) depolymerase. Sequences of both enzymes were studied and compared, and a conserved lipase box pentapeptide region around the nucleophilic serine was detected. The alignment of 3-D structures for both enzymes showed their active site residues were well aligned with an RMSD value of 1.981 Å despite their sequence similarity of only 53.8%. Docking of T1 lipase with P(3HB) gave forth high binding energy of 5.4 kcal/mol, with the distance of 4.05 Å between serine hydroxyl (OH) group of TI lipase to the carbonyl carbon of the substrate, similar to the native PhaZ7 Pl . This suggests the possible ability of T1 lipase to bind P(3HB) in its active site. The ability of T1 lipase in degrading amorphous P(3HB) was investigated on 0.2% (w/v) P(3HB) plate. Halo zone was observed around the colony containing the enzyme which confirms that T1 lipase is indeed able to degrade amorphous P(3HB). Results obtained in this study highlight the fact that T1 lipase is a versatile hydrolase enzyme which does not only record triglyceride degradation activity but amorphous P(3HB) degradation activity as well

A virtual screening approach for identifying plants with anti H5N1 neuraminidase activity

Recent outbreaks of highly pathogenic and occasional drug-resistant influenza strains have highlighted the need to develop novel anti-influenza therapeutics. Here, we report computational and experimental efforts to identify influenza neuraminidase inhibitors from among the 3000 natural compounds in the Malaysian-Plants Natural-Product (NADI) database. These 3000 compounds were first docked into the neuraminidase active site. The five plants with the largest number of top predicted ligands were selected for experimental evaluation. Twelve specific compounds isolated from these five plants were shown to inhibit neuraminidase, including two compounds with IC50 values less than 92 μM. Furthermore, four of the 12 isolated compounds had also been identified in the top 100 compounds from the virtual screen. Together, these results suggest an effective new approach for identifying bioactive plant species that will further the identification of new pharmacologically active compounds from diverse natural-product resources

Characterization of polyhydroxyalkanoate production capacity, composition and weight synthesized by Burkholderia cepacia JC-1 from various carbon sources

Polyhydroxyalkanoates (PHA) are microbial polymers that have received widespread attention in recent decades as potential alternatives to some petrochemical-based plastics. However, widespread use of PHA is often impeded by its cost of production. Therefore, the search for and systematic investigation of versatile microbial PHA producers capable of using various carbon sources, even in the form of animal fats, for PHA biosynthesis is desirable. This study highlights the PHA production capacity, monomer composition and molecular weight synthesized by Burkholderia cepacia JC-1, a locally isolated strain from soil, from various carbon sources. In the category of simple sugars and plant oils, the use of glucose and palm oil at C:N ratio of 40 resulted in the highest accumulation of 52 wt% and 36 wt% poly(3-hydroxybutyrate) [P(3HB)] homopolymer and dry cell weight of 2.56 g/L and 3.17 g/L, respectively. Interestingly, B. cepacia JC-1 was able to directly utilize animal-derived lipid in the form of crude and extracted chicken fat, resulting in appreciable dry cell weight and PHA contents of up to 3.19 g/L and 47 wt% respectively, surpassing even that of palm oil in the group of triglycerides as substrates. The presence of antibiotics (streptomycin) in cultivation medium did not significantly affect cell growth and polymer production. The supply of sodium pentanoate as a co-substrate resulted in the incorporation of 3-hydroxyvalerate (3HV) monomer at fractions up to 37 mol%. The molecular weight of polymers produced from glucose, palm oil and chicken fat were in the range of 991–2118 kDa, higher than some reported studies involving native strains. The results from this study form an important basis for possible improvements in using B. cepacia JC-1 and crude chicken fats in solid form for PHA production in the future

Molecular strategies to increase keratinase production in heterologous expression systems for industrial applications

Keratinase is an important enzyme that can degrade recalcitrant keratinous wastes to form beneficial recyclable keratin hydrolysates. Keratinase is not only important as an alternative to reduce environmental pollution caused by chemical treatments of keratinous wastes, but it also has industrial significance. Currently, the bioproduction of keratinase from native keratinolytic host is considered low, and this hampers large-scale usage of the enzyme. Straightforward approaches of cloning and expression of recombinant keratinases from native keratinolytic host are employed to elevate the amount of keratinase produced. However, this is still insufficient to compensate for the lack of its large-scale production to meet the industrial demands. Hence, this review aimed to highlight the various sources of keratinase and the strategies to increase its production in native keratinolytic hosts. Molecular strategies to increase the production of recombinant keratinase such as plasmid selection, promoter engineering, chromosomal integration, signal peptide and propeptide engineering, codon optimization, and glycoengineering were also described. These mentioned strategies have been utilized in heterologous expression hosts, namely, Escherichia coli, Bacillus sp., and Pichia pastoris, as they are most widely used for the heterologous propagations of keratinases to further intensify the production of recombinant keratinases adapted to better suit the large-scale demand for them

Feather-degrading Bacillus cereus HD1: genomic analysis and its optimization for keratinase production and father degradation

Keratinase is an important enzyme that is used to degrade feather wastes produced by poultry industries and slaughterhouses that accumulate rapidly over time. The search for keratinase-producing microorganisms is important to potentially substitute physicochemical treatments of feather waste. In this study, the genome of Bacillus cereus HD1 and its keratinolytic prowess was investigated. The whole-genome shotgun size is 5,668,864 bp consisting of 6083 genes, 69 tRNAs, and 10 rRNAs. The genomic analyses revealed 15 potential keratinase genes and other enzymes that might assist keratin degradation, such as disulfide reductase and cysteine dioxygenase. The optimal conditions for feather degradation and keratinase production by B. cereus HD1 such as incubation time, pH, temperature, yeast extract, and glycerol concentrations were determined to be 5 days, pH 8, 37 °C, 0.05% (w/v), and 0.1% (v/v), respectively. Under optimized conditions, B. cereus HD1 exhibited feather degradation of 65%, with bacterial growth and maximum keratinase activity of 1.3 × 1011 CFU/mL and 41 U/mL, respectively, after 5 days of incubation in a feather basal medium. The findings obtained from this study may facilitate further research into utilizing B. cereus HD1 as a prominent keratinolytic enzymes production host and warrant potential biotechnological applications