Linalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis-Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10-3 µM-1 s-1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.
Chitinases catalyze the degradation of chitin, a ubiquitous polymer generated from the cell walls of fungi, shells of crustaceans, and cuticles of insects. They are gaining increasing attention in medicine, agriculture, food and drug industries, and environmental management. Their roles in the degradation of chitin for the production of industrially useful products and in the control of fungal pathogens and insect pests render them attractive for such purposes. However, chitinases have diverse sources, characteristics, and mechanisms of action that seem to restrain optimization procedures and render standardization techniques for enhanced practical applications complex. Hence, results of laboratory trials are not usually consistent with real-life applications. With the growing field of protein engineering, these complexities can be overcome by modifying or redesigning chitinases to enhance specific features required for specific applications. In this review, the variations in features and mechanisms of chitinases that limit their exploitation in biotechnological applications are compiled. Recent attempts to engineer chitinases for improved efficiency are also highlighted.
By inhibiting only two or three of 12 restriction enzymes, the series of [M(phen)(edda)] complexes [M(II) is Cu, Co, Zn; phen is 1,10-phenanthroline; edda is N,N'-ethylenediaminediacetate] exhibit DNA binding specificity. The Cu(II) and Zn(II) complexes could differentiate the palindromic sequences 5'-CATATG-3' and 5'-GTATAC-3', whereas the Co(II) analogue could not. This and other differences in their biological properties may arise from distinct differences in their octahedral structures. The complexes could inhibit topoisomerase I, stabilize or destabilize G-quadruplex, and lower the mitochondrial membrane potential of MCF7 breast cells. The pronounced stabilization of G-quadruplex by the Zn(II) complex may account for the additional ability of only the Zn(II) complex to induce cell cycle arrest in S phase. On the basis of the known action of anticancer compounds against the above-mentioned individual targets, we suggest the mode of action of the present complexes could involve multiple targets. Cytotoxicity studies with MCF10A and cisplatin-resistant MCF7 suggest that these complexes exhibit selectivity towards breast cancer cells over normal ones.
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.
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.
NADP(+)-dependent geraniol dehydrogenase (EC 1.1.1.183) is an enzyme that catalyzes the oxidation of geraniol to geranial. Stable, highly active cell-free extract was obtained from Polygonum minus leaves using polyvinylpolypyrrolidone, Amberlite XAD-4, glycerol, 2-mercaptoethanol, thiourea, and phenylmethylsulfonylfluoride in tricine-NaOH buffer (pH 7.5). The enzyme preparation was separated into two activity peaks, geraniol-DH I and II, by DEAE-Toyopearl 650M column chromatography at pH 7.5. Both isoenzymes were purified to homogeneity in three chromatographic steps. The geraniol-DH isoenzymes were similar in molecular mass, optimal temperature, and pH, but the isoelectric point, substrate specificity, and kinetic parameters were different. The K(m) values for geraniol of geraniol-DH I and II appeared to be 0.4 mM and 0.185 mM respectively. P. minus geraniol-DHs are unusual among geraniol-DHs in view of their thermal stability and optimal temperatures, and also their high specificity for allylic alcohols and NADP(+).
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.
Type A chitinases (EC 3.2.1.14), GH family 18, attack chitin ((1 → 4)-2-acetamido-2-deoxy-β-D-glucan) and chito-oligosaccharides from the reducing end to catalyze release of chitobiose (N,N'-diacetylchitobiose) via hydrolytic cleavage of N-acetyl-β-D-glucosaminide (1 → 4)-β-linkages and are thus "exo-chitobiose hydrolases." In this study, the chitinase type A from Serratia marcescens (SmaChiA) was used as a template for identifying two novel exo-chitobiose hydrolase type A enzymes, FbalChi18A and MvarChi18A, originating from the marine organisms Ferrimonas balearica and Microbulbifer variabilis, respectively. Both FbalChi18A and MvarChi18A were recombinantly expressed in Escherichia coli and were confirmed to exert exo-chitobiose hydrolase activity on chito-oligosaccharides, but differed in temperature and pH activity response profiles. Amino acid sequence comparison of the catalytic β/α barrel domain of each of the new enzymes showed individual differences, but ~69% identity of each to that of SmaChiA and highly conserved active site residues. Superposition of a model substrate on 3D structural models of the catalytic domain of the enzymes corroborated exo-chitobiose hydrolase type A activity for FbalChi18A and MvarChi18A, i.e., substrate attack from the reducing end. A main feature of both of the new enzymes was the presence of C-terminal 5/12 type carbohydrate-binding modules (SmaChiA has no C-terminal carbohydrate binding module). These new enzymes may be useful tools for utilization of chitin as an N-acetylglucosamine donor substrate via chitobiose.
Enzymatic reduction of carbon dioxide (CO2 ) to methanol (CH3 OH) can be accomplished using a designed set-up of three oxidoreductases utilizing reduced pyridine nucleotide (NADH) as cofactor for the reducing equivalents electron supply. For this enzyme system to function efficiently a balanced regeneration of the reducing equivalents during reaction is required. Herein, we report the optimization of the enzymatic conversion of formaldehyde (CHOH) to CH3 OH by alcohol dehydrogenase, the final step of the enzymatic redox reaction of CO2 to CH3 OH, with kinetically synchronous enzymatic cofactor regeneration using either glucose dehydrogenase (System I) or xylose dehydrogenase (System II). A mathematical model of the enzyme kinetics was employed to identify the best reaction set-up for attaining optimal cofactor recycling rate and enzyme utilization efficiency. Targeted process optimization experiments were conducted to verify the kinetically modeled results. Repetitive reaction cycles were shown to enhance the yield of CH3 OH, increase the total turnover number (TTN) and the biocatalytic productivity rate (BPR) value for both system I and II whilst minimizing the exposure of the enzymes to high concentrations of CHOH. System II was found to be superior to System I with a yield of 8 mM CH3 OH, a TTN of 160 and BPR of 24 μmol CH3 OH/U · h during 6 hr of reaction. The study demonstrates that an optimal reaction set-up could be designed from rational kinetics modeling to maximize the yield of CH3 OH, whilst simultaneously optimizing cofactor recycling and enzyme utilization efficiency.
Short-chain fructooligosaccharides (scFOSs) can be produced from the levan hydrolysis using levanase. Levanase from Bacillus lehensis G1 (rlevblg1) is an enzyme that specifically converts levan to scFOSs. However, the use of free levanase presents a lack of stability and reusability, thus hindering the synthesis of scFOSs for continuous reactions. Here, CLEAs for rlevblg1 were prepared and characterized. Cross-linked levanase aggregates using glutaraldehyde (CLLAs-ga) and bovine albumin serum (CLLAs-ga-bsa) showed the best activity recovery of 92.8% and 121.2%, respectively. The optimum temperature of CLLAs-ga and CLLAs-ga-bsa was increased to 35 °C and 40 °C, respectively, from its free rlevblg1 (30 °C). At high temperature (50 °C), the half-life of CLLAs-ga-bsa was higher than that of free rlevblg1 and CLLAs-ga. Both CLLAs exhibited higher stability at pH 9 and pH 10. Hyperactivation of CLLAs-ga-bsa was achieved with an effectiveness factor of more than 1 and with improved catalytic efficiency. After 3 h reaction, CLLAs-ga-bsa produced the highest total scFOSs yield of 35.4% and total sugar of 60.4% per gram levan. Finally, the reusability of CLLAs for 8 cycles with more than 50% activity retained makes them as a potential synthetic catalyst to be explored for scFOSs synthesis.
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%.
The substitution of the oxyanion Q114 with Met and Leu was carried out to investigate the role of Q114 in imparting enantioselectivity on T1 lipase. The mutation improved enantioselectivity in Q114M over the wild-type, while enantioselectivity in Q114L was reduced. The enantioselectivity of the thermophilic lipases, T1, Q114L and Q114M correlated better with log p as compared to the dielectric constant and dipole moment of the solvents. Enzyme activity was good in solvents with log p < 3.5, with the exception of hexane which deviated substantially. Isooctane was found to be the best solvent for the esterification of (R,S)-ibuprofen with oleyl alcohol for lipases Q114M and Q114L, to afford E values of 53.7 and 12.2, respectively. Selectivity of T1 was highest in tetradecane with E value 49.2. Solvents with low log p reduced overall lipase activity and dimethyl sulfoxide (DMSO) completely inhibited the lipases. Ester conversions, however, were still low. Molecular sieves employed as desiccant were found to adversely affect catalysis in the lipase variants, particularly in Q114M. The higher desiccant loading also increased viscosity in the reaction and further reduced the efficiency of the lipase-catalyzed esterifications.
Site-directed mutagenesis of the oxyanion-containing amino acid Q114 in the recombinant thermophilic T1 lipase previously isolated from Geobacillus zalihae was performed to elucidate its role in the enzyme's enantioselectivity and reactivity. Substitution of Q114 with a hydrophobic methionine to yield mutant Q114M increased enantioselectivity (3.2-fold) and marginally improved reactivity (1.4-fold) of the lipase in catalysing esterification of ibuprofen with oleyl alcohol. The improved catalytic efficiency of Q114L was concomitant with reduced flexibility in the active site while the decreased enantioselectivity of Q114L could be directly attributed to diminished electrostatic repulsion of the substrate carboxylate ion that rendered partial loss in steric hindrance and thus enantioselectivity. The highest E-values for both Q114L (E-value 14.6) and Q114M (E-value 48.5) mutant lipases were attained at 50°C, after 12-16h, with a molar ratio of oleyl alcohol to ibuprofen of 1.5:1 and at 2.0% (w/v) enzyme load without addition of molecular sieves. Pertinently, site-directed mutagenesis on the Q114 oxyanion of T1 resulted in improved enantioselectivity and such approach may be applicable to other lipases of the same family. We demonstrated that electrostatic repulsion phenomena could affect flexibility/rigidity of the enzyme-substrate complex, aspects vital for enzyme activity and enantioselectivity of T1.
alpha-Galactosidase (EC 3.2.1.22) from ripe papaya (Carica papaya L.) fruit was fractionated by a combination of ion exchange and gel filtration chromatography into three forms, viz., alpha-galactosidase 1, 2 and 3. The predominant isoform, alpha-gal 2, was probably a tetramer with a native molecular mass of about 170 kDa and 52 kDa-sized subunits and an estimated pI of 7.3. The subunit's N-terminal amino acid sequence shared high identity (97%) with the deduced sequence of a papaya cDNA clone encoding a putative alpha-galactosidase PAG2 as well as with an Ajuga reptans L. GGT1 clone encoding a galactan: galactan galactosyltransferase (66%). During ripening, alpha-galactosidase activity increased concomitantly with firmness loss and this increase was largely ascribed to alpha-gal 2. The protein level of alpha-gal 2 as estimated by immunoblot was low in developing fruits and generally increased with ripening. alpha-Galactosidase 2 also had the ability to markedly catalyse increased pectin solubility and depolymerisation while the polymers were still structurally attached to the cell walls mimicking, in part, the changes that occur during ripening. The close correlation between texture changes, alpha-gal 2 activity and protein levels as well as capability to modify intact cell walls suggest that the enzyme might contribute to papaya fruit softening during ripening. The purported mechanism of alpha-gal 2 action as a softening enzyme was discussed in terms of its functional capacity as a glycanase or perhaps, as a transglycosylase.
Cytochrome P450s (CYPs) are important heme-containing proteins, well known for their monooxygenase reaction. The human cytochrome P450 4X1 (CYP4X1) is categorized as "orphan" CYP because of its unknown function. In recent studies it is found that this enzyme is expressed in neurovascular functions of the brain. Also, various studies have found the expression and activity of orphan human cytochrome P450 4X1 in cancer. It is found to be a potential drug target for cancer therapy. However, three-dimensional structure, the active site topology and substrate specificity of CYP4X1 remain unclear.
Oil palm trunk (OPT) is one of the most promising lignocellulosic bioresources. To develop effective biodegradation, thermophilic, anaerobic microorganisms were screened from bovine manure compost using fibrillated OPT (f-OPT) pretreated by wet disk milling as the substrate. One thermophilic, anaerobic bacterium, strain CL-2, whose 16S rDNA gene has 98.6% sequence identity with that of Caldicoprobacter faecale DSM 20678T, exhibited high degradation activity (32.7% reduction in total dry solids of f-OPT). Strain CL-2 did not use cellulose as a carbon source, but used hemicelluloses such as xylan, arabinoxylan, starch and pectin at 70 °C. Phylogenetic and morphologic analyses and the polysaccharide use suggest that CL-2 may be classified as a novel species of Caldicoprobacter, named Caldicoprobacter sp. CL-2. To characterize enzymatic activities of CL-2, extracellular enzymes were prepared from culture broth using beechwood xylan as the carbon source. The extracellular enzymes showed high xylanase activity, but low cellulase activity, suggesting that f-OPT degradation may depend on xylanase activity. To understand the xylanase system of CL-2, a major xylanase was cloned and characterized. The xylanase (CalXyn11A) had a modular structure consisting of a glycoside hydrolase (GH) family-11 domain and a family 36 carbohydrate-binding module. CalXyn11A did not show f-OPT degradation activity, but a strong synergistic effect was observed when CalXyn11A was added to the extracellular enzyme preparation. These results indicate that, rather than working alone, CalXyn11A has an important role in enhancing total lignocellulose degradation activity by cooperation with other GHs.
Cold-adapted enzymes are proteins produced by psychrophilic organisms that display a high catalytic efficiency at extremely low temperatures. Chitin consists of the insoluble homopolysaccharide β-(1, 4)-linked N-acetylglucosamine, which is the second most abundant biopolymer found in nature. Chitinases (EC 3.2.1.14) play an important role in chitin recycling in nature. Biodegradation of chitin by the action of cold-adapted chitinases offers significant advantages in industrial applications such as the treatment of chitin-rich waste at low temperatures, the biocontrol of phytopathogens in cold environments and the biocontrol of microbial spoilage of refrigerated food.
Maltogenic amylase (MAG1) from Bacillus lehensis G1 displayed the highest hydrolysis activity on β-cyclodextrin (β-CD) to produce maltose as a main product and exhibited high transglycosylation activity on malto-oligosaccharides with polymerization degree of three and above. These substrate and product specificities of MAG1 were elucidated from structural point of view in this study. A three-dimensional structure of MAG1 was constructed using homology modeling. Docking of β-CD and malto-oligosaccharides was then performed in the MAG1 active site. An aromatic platform in the active site was identified which is responsible in substrate recognition especially in determining the enzyme's preference toward β-CD. Molecular dynamics (MD) simulation showed MAG1 structure is most stable when docked with β-CD and least stable when docked with maltose. The docking analysis and MD simulation showed that the main subsites for substrate stabilization in the active site are -2, -1, +1 and +2. A bulky residue, Trp359 at the +2 subsite was identified to cause steric interference to the bound linear malto-oligosaccharides thus prevented it to occupy subsite +3, which can only be reached by a highly bent glucose molecule such as β-CD. The resulted modes of binding from docking simulation show a good correlation with the experimentally determined hydrolysis pattern. The subsite structure generated from this study led to a possible mode of action that revealed how maltose was mainly produced during hydrolysis. Furthermore, maltose only occupies subsite +1 and +2, therefore could not be hydrolyzed or transglycosylated by the enzyme. This important knowledge has paved the way for a novel structure-based molecular design for modulation of its catalytic activities.
Endo-β-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1 (Blg32) composed of 284 amino acids with a predicted molecular mass of 31.6 kDa is expressed in Escherichia coli and purified to homogeneity. Herein, Blg32 characteristics, substrates and product specificity as well as structural traits that might be involved in the production of sugar molecules are analysed. This enzyme functions optimally at the temperature of 70 °C, pH value of 8.0 with its catalytic activity strongly enhanced by Mn2+. Remarkably, the purified enzyme is highly stable in high temperature and alkaline conditions. It exhibits the highest activity on laminarin (376.73 U/mg) followed by curdlan and yeast β-glucan. Blg32 activity increased by 62% towards soluble substrate (laminarin) compared to insoluble substrate (curdlan). Hydrolytic products of laminarin were oligosaccharides with degree of polymerisation (DP) of 1 to 5 with the main product being laminaritriose (DP3). This suggests that the active site of Blg32 could recognise up to five glucose units. High concentration of Blg32 mainly produces glucose whilst low concentration of Blg32 yields oligosaccharides with different DP (predominantly DP3). A theoretical structural model of Blg32 was constructed and structural analysis revealed that Trp156 is involved in multiple hydrophobic stacking interactions. The amino acid was predicted to participate in substrate recognition and binding. It was also exhibited that catalytic groove of Blg32 has a narrow angle, thus limiting the substrate binding reaction. All these properties and knowledge of the subsites are suggested to be related to the possible mode of action of how Blg32 produces glucooligosaccharides.