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.

Proteome analysis of Escherichia coli periplasmic proteins in response to over-expression of recombinant human interferon α2b

The periplasmic proteome of recombinant E. coli cells expressing human interferon-α2b (INF-α2b) was analysed by 2D-gel electrophoresis to find the most altered proteins. Of some unique up- and down-regulated proteins in the proteome, ten were identified by MS. The majority of the proteins belonged to the ABC transporter protein family. Other affected proteins were ones involved in the regulation of transcription such as DNA-binding response regulator, stress-related proteins and ecotin. Thus, the production of INF-α2b acts as a stress on the cells and results in the induction of various transporters and stress related proteins

Current progress in production of flavonoids using systems and synthetic biology platforms

Flavonoid is an industrially-important compound due to its high pharmaceutical and cosmeceutical values. However, conventional methods in extracting and synthesizing flavonoids are costly, laborious and not sustainable due to small amount of natural flavonoids, large amounts of chemicals and space used. Biotechnological production of flavonoids represents a viable and sustainable route especially through the use of metabolic engineering strategies in microbial production hosts. In this review, we will highlight recent strategies for the improving the production of flavonoids using synthetic biology approaches in particular the innovative strategies of genetically-encoded biosensors for in vivo metabolite analysis and high-throughput screening methods using fluorescence-activated cell sorting (FACS). Implementation of transcription factor based-biosensor for microbial flavonoid production and integration of systems and synthetic biology approaches for natural product development will also be discussed

Computational design of artificial metallo-haloalkane dehalogenase

Haloalkane dehalogenases (HLDs) can catalyze conversion of some toxic haloalkanes to corresponding harmless alcohols. The limiting factors of native HLDs are their slow product releasing step, low activity against non-natural substrates and synthetic substrates. By creating an artificial metallo-HLD might provide solutions for these problems as metalloenzymes can provide certain advantages like high turnover rate, better stabilization of substrate-enzyme docking and broader substrate specificity. Metallo-HLDs are expected to carry out hydrolysis of haloalkane in 1 step catalysis and with higher KM. Nowadays, computational studies have been improved and commonly used by researchers to validate some structural designs before engineer the proteins in the lab. Computational studies using molecular dynamic simulation software and online molecular tools had largely increase the rate of success in protein engineering. In this work, through the computational design starting from template and metal binding site selection, in silico mutation, in silico metal docking, several validation of metal binding site and in silico docking of substrates had successful created 2 model of metallo-HLDs. These computational approaches had been validated using native metalloenzyme and functional artificial metalloenzyme as positive controls

Engineering T1 lipase for degradation of poly-(R)-3-hydroxybutyrate

Enzymes with broad substrate specificities that can act on a wide range of substrates would be valuable for industrial applications. T1 lipase is known to have broad substrate specificity in its native form, with active site residues that are similar to polyhydroxylalkanoate (PHA) depolymerase (PhaZ). PhaZ6 from Pseudomonas lemoignei (PhaZ6Pl) is one of PhaZs that can degrade semicrystalline poly-(R)-3-hydroxybutyrate [P(3HB)]. The objective of this study is to enable T1 lipase to degrade semicrystalline P(3HB) similar to PhaZ6Pl while maintaining its native function. Structural analyses on PhaZ6Pl built structure revealed that it does not contain a lid, as opposed to T1 lipase. Therefore, T1 lipase were designed by removing its lid region. This was performed by using Bacillus subtilis lipase A (BSLA) as the reference for T1 lipase modification as the latter does not have a lid region and that its structure fits almost perfectly with T1 lipase based on their superimposed structures. A total of three variants of T1 lipase without lid were successfully designed, namely D1 (without α6–loop–α7), D2 (without α6) and D3 (α6 and loop) in the lid region. All the variants showed PHA depolymerase activity towards P(3HB), with D2 variant exhibiting the highest activity amongst other variants. Further analysis on D2 showed that it was able to maintain its native hydrolytic activity towards olive oil, albeit with decrement in its catalytic efficiency. Results obtained in this study highlighted the fact that native T1 lipase is a versatile hydrolase enzyme which does not only perform triglyceride degradation but also P(3HB) degradation by simply removing the helix 6 which was specifically proven to affect catalytic activity and substrate specificity of the enzyme

Thermostable lipases

The stability of biocatalysts is an important criterion when dealing with bioprocesses at high temperature in order to sustain its operational activity throught the processes. Much efforts has been focused on the screening of microorganisms harboring intrinsically stable biocatalysts. This chapter presents an overview of the issues involving screening, growth and production, purification and characterization of wild-type and recombinant enzymes with emphisis on thermostable lipases. High temperature, using olive oil as the sole carbon source, dictated the isolation of thermophilic lipolytic bacteria Geobacillus sp. strain T1 and Bacillus spp. strain 42 and strain L2.Tryptone and casamino acid were the best nitrogen sources, while corn oil and Tween 60 were the best substrates for the production of strain 42 lipase and L2 lipase, respectively. Molecular expression of thermophilic genes in mesophilic host not only reduced the exposure of recombinant enzymes to denaturing environment but facilitate protein purification and expression in bulk quantity in a shorter time. These lipases exhibited optimum temperature and pH of 70-80C and 7-9, respectively. These valuable properties create various potential industrial applications, particularly palm-based industry with respect to its high activity and substrate solubility at high temperature

Pichia pastoris as a host to overexpress the thermostable T1 lipase from Geobacillus zalihae

Pichia pastoris was known to be a good expression system in producing various heterologous proteins. The gene encoding thermostable Tl lipase from Geobacillus zalihae was cloned into pPICZαB and expressed in P. pastoris strains (GS115, X-33 and KM71H) under regulation of alcohol oxidase promoter. The expression of the gene in Escherichia coli system showed low expression level. Therefore, this study would highlight on the overexpression of the T1 lipase in yeast extracellularly. Recombinant X-33/pPICZαB/T1-2 (XPB2), GS115/pPICZαB/T1-5 (GPB5) and KM71H/pPICZαB/T1-7 (KPB7) were chosen for optimization in shake flask. Optimization strategies showed that these recombinants preferred YPTM medium with initial induction OD600nm = 7 cell biomass and 2% (v/v) methanol to provide optimal expression conditions. Hyper-resistant transformants at 3000 ug/mL zeocin gave better expression than 100 ug/mL zeocin selection. Time course study by using different inocula age showed that OD600nm = 7 expressed lipase at the highest level. The highest expression level was attained with GPB5 (88 U/ml), XPB2 (81 U/ml) and KPB7 (51 U/ml). Western Blot analysis confirmed that the molecular mass of recombinant Tl lipase was 45 kDa. In conclusion, thermostable Tl lipase was successfully overexpressed by using secretory P. pastoris system with two-fold higher than E. coli system

CsoR metalloregulatory protein: function, mechanism and relevance

Transition metals are required constituent in bacterial metabolism to assist in some enzymatic reactions. However, intracellular accumulations of these metal ions are harmful to the bacteria as it can trigger unnecessary redox reactions. To overcome this condition, metalloregulatory proteins assist organisms to adapt to sudden elevated and deprived metal ion concentration in the environment via metal homeostasis. CsoR protein is a copper(I) [Cu(I)] sensing operon repressor that is found to be present in all major classes of eubacteria. This metalloregulatory protein binds to the operator region in its apo state under Cu(I) limiting condition and detaches off from the regulatory region when it binds to the excess cytosolic Cu(I) ion, thus derepressing the expression of genes involved in Cu(I) homeostasis. CsoR proteins exist in dimeric and tetrameric states and form certain coordination geometries upon attachment with Cu(I). Certain CsoR proteins have also been found to possess the ability to bind to other types of metals with various binding affinities in some Gram positive bacteria. The role of this metalloregulatory protein in host pathogen interaction and its relation to bacterial virulence are also discussed

Genome sequence of Photobacterium sp. strain J15: an insight view for conservation genetics and molecular engineering

The genus Photobacterium was one of the earliest known bacterial taxa (Beijerinck 1889) and it comes from the family Vibrionaceae, one of the most widely distributed groups of prokaryotes that have radiated into hundreds of existing niches in the environment. However, the precise evolutionary history of this bacteria could not be infer due to the lack of whole genome sequencing data on Photobacterium available publicly. Photobacterium sp. strain J15 is a marine bacterium which was isolated from Tanjung Pelepas, Johor and it initially was studied for its lipase and asparaginase producing properties. Lipases has been used widely in the production of food, detergent and also in pharmaceutical industry while asparaginase on the other hand is on the World Health Organization’s List of Essential Medicines. Recently, it is also found out that Photobacterium species have the ability to produce DHAs, which is recommended for infant’s consumption. However, there is a limitation in the industrial use of these enzymes and it is mainly due to the high production cost which could be overcome by molecular technologies. Here we present the draft genome sequence of this Photobacterium sp. It was sequenced using Illumina technology and the genome sequence was then being assembled and the assembly produced 23 scaffolds with a final reading of 5,647,128 bp. However, there are 15,246 of unsequenced nucleotides which would be closed by using Sanger sequencing. The whole genome sequence and the future genome scale metabolic model of the Photobacterium sp. strain J15 is hope to uncover the mechanisms of this bacterium which will ease the molecular engineering applications besides being important in analysing the evolutionary biology and future genetics conservation of this interesting group of bacteria

Rosmarinic acid as pancreatic lipase inhibitor

The present invention pertains to rosmarinic acid for use in the inhibition of pancreatic lipase. The use of the present invention may be applied in medical and dietary methods for the reduction of body fat in mammals. The invention is in particular useful for the treatment of metabolic disorders such as obesity or diabetes