Development of an enzyme-based fiber optic biosensor for detection of haloalkanes

Purpose – The main purpose of this study is to demonstrate the development of an enzyme-based sensor for haloalkane detection. Haloalkane is a toxic compound that is found as genotoxic impurities in pharmaceutical products and contaminants in waste. The need to investigate the genotoxic level in pharmaceutical manufacturing is very crucial because of its toxicity effects on human health. The potential of mini protein as an alternative bioreceptor was explored with the aim to be more effective and stable under extreme conditions. Design/methodology/approach – Mini proteins of haloalkane dehalogenase (HLD) were computationally designed and experimentally validated. Tapered multimode fiber (TMMF) was bio-functionalized with a bioreceptor either native (positive control) or mini protein. The absorbance-based sensor resulting from the binding interaction of mini protein with haloalkane was monitored through a wavelength range of 200-1,300 nm. Findings – An increment of the UV absorption is observed at 325 nm when haloalkane interacted with the immobilized bioreceptors, native or mini protein. Both biosensors displayed a continuous response over the range of 5-250 mM haloalkane. They also had the capability to detect haloalkanes below 1 min and with an operational stability of up to seven days without significant loss of sensitivity. Practical implications – The results indicate the potential viability of the enzyme HLD-based sensor to monitor the existence of haloalkane in both pharmaceutical and environmental products. Originality/value – The paper describes an outcome of experimental work on TMMF-based biosensor coated with HLD for label-free haloalkane detection. Mini protein can be used as an effective bioreceptor with some structural modification to improve functionality and stability

In silico design of potentially functional artificial metallo-haloalkane dehalogenase containing catalytic zinc

Artificial metalloenzymes are unique as they combine the good features of homogeneous and enzymatic catalysts, and they can potentially improve some difficult catalytic assays. This study reports a method that can be used to create an artificial metal-binding site prior to proving it to be functional in a wet lab. Haloalkane dehalogenase was grafted into a metal-binding site to form an artificial metallo-haloalkane dehalogenase and was studied for its potential functionalities in silico. Computational protocols regarding dynamic metal docking were studied using native metalloenzymes and functional artificial metalloenzymes. Using YASARA Structure, a simulation box covering template structure was created to be filled with water molecules followed by one mutated water molecule closest to the metal-binding site to metal ion. A simple energy minimization step was subsequently run using an AMBER force field to allow the metal ion to interact with the metal-binding residues. Long molecular dynamic simulation using YASARA Structure was performed to analyze the stability of the metal-binding site and the distance between metal-binding residues. Metal ions fluctuating around 2.0 Å across a 20 ns simulation indicated a stable metal-binding site. Metal-binding energies were predicted using FoldX, with a native metalloenzyme (carbonic anhydrase) scoring 18.0 kcal/mol and the best mutant model (C1a) scoring 16.4 kcal/mol. Analysis of the metal-binding site geometry was performed using CheckMyMetal, and all scores for the metalloenzymes and mutant models were in an acceptable range. Like native metalloenzymes, the metal-binding site of C1a was supported by residues in the second coordination shell to maintain a more coordinated metal-binding site. Short-chain multihalogenated alkanes (1,2-dibromoethane and 1,2,3-trichloropropane) were able to dock in the active site of C1a. The halides of the substrate were in contact with both the metal and halide-stabilizing residues, thus indicating a better stabilization of the substrate. The simple catalytic mechanism proposed is that the metal ion interacted with halogen and polarized the carbon–halogen bond, thus making the alpha carbon susceptible to attack by nucleophilic hydroxide. The interaction between halogen in the metal ion and halide-stabilizing residues may help to improve the stabilization of the substrate–enzyme complex and reduce the activation energy. This study reports a modified dynamic metal-docking protocol and validation tests to verify the metal-binding site. These approaches can be applied to design different kinds of artificial metalloenzymes or metal-binding sites

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.

Secretory expression of thermostable T1 lipase through bacteriocin release protein

The extracellular production of T1 lipase was performed by co-expression of pJL3 vector encoding bacteriocin release protein in prokaryotic system. Secretory expression was optimized by considering several parameters, including host strains, inducer (IPTG) concentration, media, induction at A600 nm, temperature, and time of induction. Among the host strains tested, Origami B excreted out 18,100 U/ml of lipase activity into culture medium when induced with 50 μM IPTG for 12 h. The Origami B harboring recombinant plasmid pGEX/T1S and pJL3 vector was chosen for further study. IPTG at 0.05 mM, YT medium, induction at A600 nm of 1.25, 30 °C, and 32 h of induction time were best condition for T1 lipase secretion with Origami B as a host

High level expression of thermostable lipase from Geobacillus sp. strain T1

A thermostable extracellular lipase of Geobacillus sp. strain T1 was cloned in a prokaryotic system. Sequence analysis revealed an open reading frame of 1,251 bp in length which codes for a polypeptide of 416 amino acid residues. The polypeptide was composed of a signal peptide (28 amino acids) and a mature protein of 388 amino acids. Instead of Gly, Ala was substituted as the first residue of the conserved pentapeptide Gly-X-Ser-X-Gly. Successful gene expression was obtained with pBAD, pRSET, pET, and pGEX as under the control of araBAD, T7, T7 lac, and tac promoters, respectively. Among them, pGEX had a specific activity of 30.19 Umg−1 which corresponds to 2927.15 Ug−1 of wet cells after optimization. The recombinant lipase had an optimum temperature and pH of 65°C and pH 9, respectively. It was stable up to 65°C at pH 7 and active over a wide pH range (pH 6–11). This study presents a rapid cloning and overexpression, aimed at improving the enzyme yield for successful industrial application

A thermoalkaliphilic lipase of Geobacillus sp. T1

A thermoalkaliphilic T1 lipase gene of Geobacillus sp. strain T1 was overexpressed in pGEX vector in the prokaryotic system. Removal of the signal peptide improved protein solubility and promoted the binding of GST moiety to the glutathione-Sepharose column. High-yield purification of T1 lipase was achieved through two-step affinity chromatography with a final specific activity and yield of 958.2 U/mg and 51.5%, respectively. The molecular mass of T1 lipase was determined to be approximately 43 kDa by gel filtration chromatography. T1 lipase had an optimum temperature and pH of 70°C and pH 9, respectively. It was stable up to 65°C with a half-life of 5 h 15 min at pH 9. It was stable in the presence of 1 mM metal ions Na+, Ca2+, Mn2+, K+ and Mg2+ , but inhibited by Cu2+, Fe3+ and Zn2+. Tween 80 significantly enhanced T1 lipase activity. T1 lipase was active towards medium to long chain triacylglycerols (C10–C14) and various natural oils with a marked preference for trilaurin (C12) (triacylglycerol) and sunflower oil (natural oil). Serine and aspartate residues were involved in catalysis, as its activity was strongly inhibited by 5 mM PMSF and 1 mM Pepstatin. The T m for T1 lipase was around 72.2°C, as revealed by denatured protein analysis of CD spectra

Geobacillus zalihae sp. nov., a thermophilic lipolytic bacterium isolated from palm oil mill effluent in Malaysia

<p>Abstract</p> <p>Background</p> <p>Thermophilic <it>Bacillus </it>strains of phylogenetic <it>Bacillus </it>rRNA group 5 were described as a new genus <it>Geobacillus</it>. Their geographical distribution included oilfields, hay compost, hydrothermal vent or soils. The members from the genus <it>Geobacillus </it>have a growth temperatures ranging from 35 to 78°C and contained iso-branched saturated fatty acids (iso-15:0, iso-16:0 and iso-17:0) as the major fatty acids. The members of <it>Geobacillus </it>have similarity in their 16S rRNA gene sequences (96.5–99.2%). Thermophiles harboring intrinsically stable enzymes are suitable for industrial applications. The quest for intrinsically thermostable lipases from thermophiles is a prominent task due to the laborious processes via genetic modification.</p> <p>Results</p> <p>Twenty-nine putative lipase producers were screened and isolated from palm oil mill effluent in Malaysia. Of these, isolate T1^Twas chosen for further study as relatively higher lipase activity was detected quantitatively. The crude T1 lipase showed high optimum temperature of 70°C and was also stable up to 60°C without significant loss of crude enzyme activity. Strain T1^Twas a Gram-positive, rod-shaped, endospore forming bacterium. On the basic of 16S rDNA analysis, strain T1^Twas shown to belong to the <it>Bacillus </it>rRNA group 5 related to <it>Geobacillus thermoleovorans </it>(DSM 5366^T) and <it>Geobacillus kaustophilus </it>(DSM 7263^T). Chemotaxonomic data of cellular fatty acids supported the affiliation of strain T1^Tto the genus <it>Geobacillus</it>. The results of physiological and biochemical tests, DNA/DNA hybridization, RiboPrint analysis, the length of lipase gene and protein pattern allowed genotypic and phenotypic differentiation of strain T1^Tfrom its validly published closest phylogenetic neighbors. Strain T1^Ttherefore represents a novel species, for which the name <it>Geobacillus zalihae </it>sp. nov. is proposed, with the type strain T1^T(=DSM 18318^T; NBRC 101842^T).</p> <p>Conclusion</p> <p>Strain T1^Twas able to secrete extracellular thermostable lipase into culture medium. The strain T1^Twas identified as <it>Geobacillus zalihae </it>T1^Tas it differs from its type strains <it>Geobacillus kaustophilus </it>(DSM 7263^T) and <it>Geobacillus thermoleovorans </it>(DSM 5366^T) on some physiological studies, cellular fatty acids composition, RiboPrint analysis, length of lipase gene and protein profile.</p

Novi bakteriocinogeni sojevi Lactobacillus plantarum i njihova diferencijacija analizom slijeda 16S rDNA, 16S-23S i 23S-5S međugenskih razmaknica, te analizom nasumično umnožene polimorfne DNA

Six strains of bacteriocinogenic Lactobacillus plantarum (TL1, RG11, RS5, UL4, RG14 and RI11) isolated from Malaysian foods were investigated for their structural bacteriocin genes. A new combination of plantaricin EF and plantaricin W bacteriocin structural genes was successfully amplified from all studied strains, suggesting that they were novel bacteriocin-producing L. plantarum strains. A four-base pair variable region was detected in the short 16S-23S intergenic spacer regions of the studied strains by a comparative analysis with 17 L. plantarum strains deposited in the GenBank, implying they were new genotypes. The studied L. plantarum strains were subsequently differentiated into four groups on the basis of the detected four-base pair variable region of the short 16S-23S intergenic spacer region. Further analysis of the DNA sequence of 23S-5S intergenic spacer region revealed only one type of 23S-5S intergenic spacer region present in the studied strains, indicating it was highly conserved among the studied L. plantarum strains. Three randomly amplified polymorphic DNA experiments using three different combinations of arbitrary primers successfully differentiated the studied L. plantarum strains from each other, confirming they were different strains. In conclusion, the studied L. plantarum strains were shown to be novel bacteriocin producers and high level of strain discrimination could be achieved with a combination of randomly amplified polymorphic DNA analysis and the analysis of the variable region of short 16S-23S intergenic spacer region present in L. plantarum strains.Ispitana je prisutnost strukturalnih bakteriocinskih gena u šest sojeva Lactobacillus plantarum (TL1, RG11, RS5, UL4, RG14 i RI11), izoliranih iz malezijske hrane. Nova kombinacija strukturalnih gena za plantaricin EF i plantaricin W uspješno je umnožena iz svih ispitanih sojeva, što upućuje na to da se radi o novim sojevima L. plantarum koji proizvode bakteriocine. Usporednom analizom sa 17 sojeva L. plantarum pohranjenih u GenBank otkrivena je varijabilna regija od četiri para baza u kratkim 16S-23S međugenskim razmaknicama tih sojeva, potvrđujući da se radi o novim genotipovima. Ispitani sojevi L. plantarum nakon toga su razvrstani u četiri skupine, na temelju uočene varijabilne regije od četiri para baza kratke 16S-23S međugenske razmaknice. Daljnjom analizom DNA slijeda 23S-5S međugenske razmaknice otkriven je samo jedan tip te razmaknice, pokazujući da je konzerviran u ispitanim sojevima L. plantarum. Trima pokusima nasumičnog umnožavanja polimorfne DNA, koristeći tri različite kombinacije proizvoljno odabranih početnica, uspješno su diferencirani ispitani sojevi L. plantarum, što potvrđuje da se radi o različitim sojevima. Dakle, može se zaključiti da novi sojevi L. plantarum proizvode bakteriocin i da se mogu uspješno razlikovati analizom nasumično umnožene polimorfne DNA i varijabilne regije kratke 16S-23S međugenske razmaknice

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