Latent polyphenol oxidase (LPPO), an enzyme responsible for the browning reaction of sago starches during processing and storage, was investigated. The enzyme was effectively extracted and partially purified from the pith using combinations of nonionic detergents. With Triton X-114 and a temperature-induced phase partitioning method, the enzyme showed a recovery of 70% and purification of 4. 1-fold. Native PAGE analysis of the partially purified LPPO revealed three activity bands when stained with catechol and two bands with pyrogallol. The molecular masses of the enzymes were estimated by SDS-PAGE to be 37, 45, and 53 kDa. The enzyme showed optimum pH values of 4.5 with 4-methylcatechol as a substrate and 7.5 with pyrogallol. The LPPO was highly reactive toward diphenols and triphenols. The activity of the enzyme was greatly enhanced in the presence of trypsin, SDS, ethanol, and linoleic acid.
Trimeresurus purpureomaculatus venom acetylcholinesterase has been partially purified by Sephadex G-200 gel filtration chromatography and DEAE Sephacel ion exchange chromatography. The enzyme has a mol. wt of 58,600. It was strongly inhibited by physostigmine salicylate and edrophonium chloride and exhibited substrate inhibition at high substrate concentration. The content of acetylcholinesterase in Trimeresurus purpureomaculatus venom was estimated to be much less than 0.3%.
The aim of this study was to investigate the influence of some environmental factors on bacterial metabolism. Fermentative hydrogen production by C. acetobutylicum, using glucose as the substrate. The effect of initial pH (4-8), inoculum size (1-20% (v/v)) and glucose concentration (1-30 g L(-1)) on hydrogen production were studied. The optimum cultivation temperature for hydrogen production was at 30 degrees C. The results show that substrate concentration and inoculum size resulted in hydrogen yield (Y(P/S)) of 391 mL g(-1) glucose utilized with maximum hydrogen productivity of 77.5 mL/L/h. Higher substrate concentration or inoculum size adversely affects hydrogen production, which decreases hydrogen yield by 15% to 334 mL g(-1) glucose utilized when 30% (v/v) inoculum size was used. The use of 30 g L(-1) substrate concentration resulted in a 75% decrease to 97 mL g(-1) glucose supplied. Concluded that proper Xo/So enhanced the hydrogen production.
A broad substrate specificity enzyme that can act on a wide range of substrates would be an asset in industrial application. T1 lipase known to have broad substrate specificity in its native form apparently exhibits the same active sites as polyhydroxylalkanoate (PHA) depolymerase. PhaZ6Pl is one of the PHA depolymerases that can degrade semicrystalline 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. A structural study on PhaZ6Pl contains no lid in its structure and therefore T1 lipase was designed with removal of its lid region. BSLA lipase was chosen as the reference protein for T1 lipase modification since it contains no lid. Initially, structures of both enzymes were compared via protein-protein superimposition in 3D-space and the location of the lid region of T1 lipase was highlighted. A total of three variants of T1 lipase without lid were successfully designed by referring to BSLA lipase (a lipase without lid). The ability of T1 lipase without lid variants in degrading P(3HB) was investigated quantitatively. All the variants showed activity towards the substrate which confirmed that T1 lipase without lid is indeed able to degrade P(3HB). In addition, D2 was recorded to have the highest activity amongst other variants. Results obtained in this study highlighted the fact that native T1 lipase is a versatile hydrolase enzyme which does not only record triglyceride degradation but also P(3HB) by simply removing the lid region.
Bioregeneration of mono-amine modified silica gel (MAMS) adsorbent loaded with Acid Orange 7 (AO7), Acid Yellow 9 (AY9) and Acid Red 14 (AR14), respectively, was investigated under two different operational conditions, namely absence/presence of sucrose/bacto-peptone as the co-substrate and different biomass acclimation concentrations. The results revealed that the AY9- and AR14-loaded MAMS adsorbents could almost be completely bioregenerated but only in the presence of co-substrate whereas the bioregeneration of AO7-loaded MAMS could achieve up to 71% in the absence of the co-substrate. These differences could be related to the structural properties of the investigated azo dyes. In addition, the results showed that the bioregeneration duration of AO7-loaded MAMS could be progressively shortened by using biomass acclimated to increasingly higher AO7 concentration. However, the bioregeneration efficiencies were found to be relatively unchanged under different biomass acclimation concentrations.
Amylase is an important and indispensable enzyme that plays a pivotal role in the field of biotechnology. It is produced mainly from microbial sources and is used in many industries. Industrial sectors with top-down and bottom-up approaches are currently focusing on improving microbial amylase production levels by implementing bioengineering technologies. The further support of energy consumption studies, such as those on thermodynamics, pinch technology, and environment-friendly technologies, has hastened the large-scale production of the enzyme. Herein, the importance of microbial (bacteria and fungi) amylase is discussed along with its production methods from the laboratory to industrial scales.
Type I pullulanase from Anoxybacillus sp. SK3-4 (PulASK) is an unusual debranching enzyme that specifically hydrolyzes starch α-1,6 linkages at long branches producing oligosaccharides (≥G8), but is nonreactive against short branches; thus, incapable of producing reducing sugars (G1-G7). We report on the effects of both single and co-immobilization of PulASK on product specificity. PulASK was purified and immobilized through covalent attachment to three epoxides (ReliZyme EP403/M, Immobead IB-150P, and Immobead IB-150A) and an amino-epoxide (ReliZyme HFA403/M) activated supports. Following immobilization, all PulASK derivatives were active on both short and long branches in starch producing reducing sugars (predominantly maltotriose) and oligosaccharides (≥G8), respectively, a feature that is absent in the free enzyme. This study also demonstrated that co-immobilization of PulASK and α-amylase from Anoxybacillus sp. SK3-4 (TASKA) on ReliZyme HFA403/M significantly changed the product specificity compared to the free enzymes alone or individually immobilized enzymes. In conclusion, individual or co-immobilization caused changes in the product specificity, presumably due to changes in the enzyme binding pocket caused by the influence of carrier surface properties (hydrophobic or hydrophilic) and the lengths of the spacer arms.
The β-1,6-glucanases are ubiquitous enzymes which appear to be implicated in the morphogenesis and have the ability to become virulence factor in plant-fungal symbiotic interaction. To our knowledge, no report on ß-1,6-glucanases purification from Trichoderma longibrachiatum has been made, although it has been proven to have a significant effect as a biocontrol agent for several diseases. Therefore, the aim of this study was to purify β-1,6- glucanase from T. longibrachiatum T28, with an assessment on the physicochemical properties and substrate specificity. β-1,3-glucanase enzyme, from the culture filtrate of T. longibrachiatum T28, was successively purified through precipitation with 80% acetone, followed by anionexchange chromatography on Neobar AQ and chromatofocusing on a Mono P HR 5/20 column. (One β-1,6-glucanase) band at 42kDa in size was purified, as shown by the SDS-PAGE. The physicochemical evaluation showed an optimum pH of 5 and optimum temperature of 50°C for enzyme activity with an ability to maintain 100% enzyme stability. Enzyme activity was slightly reduced by 10-20% in the presence of 20 mM of Zn2+, Ca2+, Co2+, Mg2+, Cu2+, Mn2+ and Fe2+. The highest β-1,6-glucanase hydrolysis activity was obtained on pustulan due to the similarity of β-glucosidic bonds followed by laminarin, glucan and cellulose. Therefore, it can be concluded that the characterization of ß-1,6-glucanase secreted by T. longibrachiatum in term of molecular weight, responsed to selected physicochemical factors and the substrate specificity are approximately identical to other Trichoderma sp.
In this study, the authors investigated the effects of a single layer graphene as a coating layer on top of metal thin films such as silver, gold, aluminum and copper using finite-difference time domain method. To enhance the resolution of surface plasmon resonance (SPR) sensor, it is necessary to increase the SPR reflectivity and decrease the full-width-half maximum (FWHM) of the SPR curve so that there is minimum uncertainty in the determination of the resonance dip. Numerical data was verified with analytical and experimental data where all the data were in good agreement with resonance angle differing in <10% due to noise present in components such as humidity and temperature. In further analysis, reflectivity and FWHM were compared among four types of metal with various thin film thicknesses where graphene was applied on top of the metal layers, and data was compared against pure conventional metal thin films. A 60 nm-thick Au thin film results in higher performance with reflectivity of 92.4% and FWHM of 0.88° whereas single layer graphene-on-60 nm-thick Au gave reflectivity of 91.7% and FWHM of 1.32°. However, a graphene-on-40 nm-thick Ag also gave good performance with narrower FWHM of 0.88° and reflection spectra of 89.2%.
Polyhydroxyalkanoate (PHA) synthase, PhaC, is a key enzyme in the biosynthesis of PHA, a type of bioplastics with huge potential to replace petroleum-based plastics. While two structures have been determined, the exact mechanism remains unclear partly due to the absence of a tunnel for product passage. A model of the class I PhaC from Aquitalea sp. USM4, characterised with Km of 394 μM and kcat of 476 s-1 on 3-(R)-hydroxybutyryl-CoA, revealed a three-branched channel at the dimeric interface. Two of them are opened to the solvent and are expected to serve as the putative routes for substrate entrance and product exit, while the third is elongated in the class II PhaC1 model from Pseudomonas aeruginosa, indicating a role in accommodating the hydroxyalkanoate (HA) moiety of a HA-CoA substrate. Docking of the two tetrahedral intermediates, formed during the transfer of the growing PHA chain from the catalytic Cys to a new molecule of substrate and back to Cys, suggests a common elongation mechanism requiring the HA moiety of the ligand to rotate ~180°. Substrate specificity is determined in part by a bulky Phe/Tyr/Trp residue in the third branch in class I, which is conserved as Ala in class II to create room for longer substrates.
In this study, the substrate specificity and the inhibition kinetics of various types of insecticides to the acetylcholinesterase (AChE) from a local fish; Puntius schwanenfeldii were investigated. The substrate specificity determination was done using three thiocholine substrates, which were ATC, PTC and BTC. The results showed that he partially purified cholinesterase from Puntius schwanenfeldii that preferred ATC is a true AChE. The Km and Vmax values of AChE for these substrates were 16.61 mmol and 286.5 U/mg for ATC, 19.92 mmol and 245.3 U/mg for PTC, and 48.64 mmol and 219.6 U/mg for BTC, respectively. The IC50 values for the carbamates bendiocarb, carbaryl, propoxur, carbofuran and methomyl were 0.838, 7.045, 29.441, 1.411 and 8.335 mg/L, respectively, which were comparable to the IC50 values for carbamates from several AChE from fish.
In this study, transformation of BrCHS var 2 into B. rotunda cell suspension culture, followed by chalcone synthase enzymatic assay and HPLC analysis was conducted to investigate whether the substrate specificity for BrCHS var 2 is either cinnamoyl-CoA or p-coumaroyl-CoA. The HPLC profile showed an increase in the amount of pinocembrin chalcone when cinnamoyl-CoA and malonyl-CoA were added but not p-coumaroyl-CoA. Molecular docking was performed to explore the binding of cinnamoyl-CoA and p-coumaroyl-CoA to BrCHS var 2 receptor and the docking results showed that cinnamoyl-CoA formed numerous hydrogen bonds and more negative docked energy than p-coumaroyl-CoA. Cinnamoyl-CoA showed good interactions with Cys 164 to initiate the subsequent formation of pinocembrin chalcone, whereas the hydroxyl group of p-coumaroyl-CoA formed an unfavorable interaction with Gln 161 that caused steric hindrance to subsequent formation of naringenin chalcone. Docked conformation analysis results also showed that malonyl-CoA formed hydrogen bonding with Cys 164, His 303, and Asn 336 residues in BrCHS var 2. The results show that cinnamoyl-CoA is the preferred substrate for BrCHS var 2.
F-box proteins containing variable C-terminal domains make an essential part of SKP1-Cullin-Ring box-F box (SCF)
complex. SCF complex catalyzes the final step to link the ubiquitin tag with the target protein, destined for degradation,
through F-box protein that confer overall substrate specificity to the complex. In this study, we analyzed the role of
At2g02870, a Kelch containing F-box protein from Arabidopsis thaliana, by using reverse genetics strategy. At2g02870
loss of function mutant lines (at2g02870) were analyzed and compared with wild type plants for the expression of genes
and products of hydroperoxide lyase (HPL) branch of oxylipin pathway. We found that the at2g02870 plants have enhanced
expression of HPL pathway genes and produce more green leaf volatiles (GLV) than the wild type plants. Our results
suggested that the gene is involved in the regulation of HPL pathway, possibly through the degradation of enzymes or/
and the regulatory factors of the pathway.
A thrombin-like enzyme (termed albolabrase) was isolated in purified form from the venom of Cryptelytrops albolabris (white-lipped tree viper) using high performance anion ion exchange and gel filtration chromatography. The molecular mass of albolabrase was 33.7 kDa as determined by SDS-PAGE and 35.8 kDa as determined by Superose gel filtration chromatography. The N-terminal sequence was determined to be VVGGDECNINE which is homologous to many snake venom thrombin-like enzymes. Albolabrase exhibits both arginine ester hydrolase and arginine amidase activities and the enzyme is fastidious towards tripeptide chromogenic anilide substrates. The fibrinogen clotting activity was optimum at 3mg/mL bovine fibrinogen, and showed distinct species differences in the following decreasing order: bovine fibrinogen>dog fibrinogen≈human fibrinogen>goat fibrinogen. The enzyme failed to clot both rabbit and cat fibrinogens. Reversed-phase HPLC analysis on the breakdown products of fibrinogenolytic action of albolabrase indicated that the enzyme belongs to the AB class of snake venom thrombin-like enzyme. In the indirect ELISA, IgG anti-albolabrase reacted extensively with most crotalid venoms, except with Tropidolaemus wagleri and Calloselasma rhodostoma venoms. The double sandwich ELISA, however, showed that anti-albolabrase reacted strongly only with venoms from the Trimeresurus complex, and that the results support the proposed new taxonomy changes concerning the Trimeresurus complex.
Paenibacillus polymyxa β-glucosidase B (BglB), belongs to a GH family 1, is a monomeric enzyme that acts as an exo-β-glucosidase hydrolysing cellobiose and cellodextrins of higher degree of polymerization using retaining mechanism. A molecular dynamics (MD) simulation was performed at 300 K under periodic boundary condition for 5 ns using the complexes structure obtained from previous docking study, namely BglB-Beta-d-glucose and BglB-Cellobiose. From the root-mean-square deviation analysis, both enzyme complexes were reported to deviate from the initial structure in the early part of the simulation but it was stable afterwards. The root-mean-square fluctuation analysis revealed that the most flexible regions comprised of the residues from 26 to 29, 43 to 53, 272 to 276, 306 to 325 and 364 to 367. The radius of gyration analysis had shown the structure of BglB without substrate became more compact towards the end of the simulation compare to other two complexes. The residues His122 and Trp410 were observed to form stable hydrogen bond with occupancy higher than 10%. In conclusion, the behaviour of BglB enzyme towards the substrate binding was successfully explored via MD simulation approaches.
The non-stereospecific α-haloalkanoic acid dehalogenase DehE from Rhizobium sp. RC1 catalyzes the removal of the halide from α-haloalkanoic acid D,L-stereoisomers and, by doing so, converts them into hydroxyalkanoic acid L,D-stereoisomers, respectively. DehE has been extensively studied to determine its potential to act as a bioremediation agent, but its structure/function relationship has not been characterized. For this study, we explored the functional relevance of several putative active-site amino acids by site-specific mutagenesis. Ten active-site residues were mutated individually, and the dehalogenase activity of each of the 10 resulting mutants in soluble cell lysates against D- and L-2-chloropropionic acid was assessed. Interestingly, the mutants W34→A,F37→A, and S188→A had diminished activity, suggesting that these residues are functionally relevant. Notably, the D189→N mutant had no activity, which strongly implies that it is a catalytically important residue. Given our data, we propose a dehalogenation mechanism for DehE, which is the same as that suggested for other non-stereospecific α-haloalkanoic acid dehalogenases. To the best of our knowledge, this is the first report detailing a functional aspect for DehE, and our results could help pave the way for the bioengineering of haloalkanoic acid dehalogenases with improved catalytic properties.
The non-stereospecific α-haloalkanoic acid dehalogenase E (DehE) degrades many halogenated compounds but is ineffective against β-halogenated compounds such as 3-chloropropionic acid (3CP). Using molecular dynamics (MD) simulations and site-directed mutagenesis we show here that introducing the mutation S188V into DehE improves substrate specificity towards 3CP. MD simulations showed that residues W34, F37, and S188 of DehE were crucial for substrate binding. DehE showed strong binding ability for D-2-chloropropionic acid (D-2CP) and L-2-chloropropionic acid (L-2CP) but less affinity for 3CP. This reduced affinity was attributed to weak hydrogen bonding between 3CP and residue S188, as the carboxylate of 3CP forms rapidly interconverting hydrogen bonds with the backbone amide and side chain hydroxyl group of S188. By replacing S188 with a valine residue, we reduced the inter-molecular distance and stabilised bonding of the carboxylate of 3CP to hydrogens of the substrate-binding residues. Therefore, the S188V can act on 3CP, although its affinity is less strong than for D-2CP and L-2CP as assessed by Km. This successful alteration of DehE substrate specificity may promote the application of protein engineering strategies to other dehalogenases, thereby generating valuable tools for future bioremediation technologies.
Prostate cancer is an age-related disease that is linked to the inability of prostate cells to accumulate zinc following transformation. It is shown in the present study that the basal percentage of normal prostate cells expressing senescence-associated beta-galactosidase (SA-beta-gal) is higher than that of the cancer cells. In the presence of high zinc in the cell culture medium, the percentage of normal prostate cells expressing the SA-beta-gal increased but not that of the cancer cells. Increased intracellular zinc occurs in the prostate cancer cells treated with supraphysiologic concentration of zinc but it does not induce senescence or decrease the telomerase activities in these cells. Senescence, however, occurred when the prostate cancer cells DNA is damaged by irradiation. These findings suggest that prostate cancer cells are insensitive to the senescence-inducing effects of zinc but the cancer cells retain the capacity to undergo senescence through other pathways.
A two-level fractional factorial design (FFD) was used to determine the effects of six factors, i.e. substrate (domestic wastewater sludge - DWS) and co-substrate concentration (wheat flour - WF), temperature, initial pH, inoculum size and agitation rate on the production of cellulase enzyme by Trichoderma harzianum in liquid state bioconversion. On statistical analysis of the results from the experimental studies, optimum process conditions were found to be temperature 32.5 degrees C, substrate concentration (DWS) 0.75% (w/w), co-substrate (WF) concentration 2% (w/w), initial pH 5, inoculum size 2% (v/w) and agitation 175 rpm. Analysis of variance (ANOVA) showed a high coefficient of determination (R2) of 0.975. Cellulase activity reached 10.2 FPU/ml at day 3 during the fermentation process which indicated about 1.5-fold increase in production compared to the cellulase activity obtained from the results of design of experiment (6.9 FPU/ml). Biodegradation of DWS was also evaluated to verify the efficiency of the bioconversion process as a waste management method.