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.
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.
While a group of oral commensals have been implicated in the aetiology of chronic periodontitis; the asaccharolytic Gram negative anaerobe Porphyromonas gingivalis is most commonly reported to be associated with severe forms of the disease. Although a variety of human tissues can produce a number of peptidylarginine deiminase (PAD), enzymes that convert peptide bound arginine residues to citrulline, P. gingivalis is one of the few prokaryotes known to express PAD. Protein and peptide citrullination are important in the development of rheumatoid arthritis and in recent years a number of authors have suggested a possible link between periodontitis and rheumatoid arthritis (RA). Indeed, some have linked P. gingivalis directly to RA via the action of PAD. Accordingly, the prime purpose of this study was to further characterise PAD in P. gingivalis cells particular emphasis on substrate specificity, using arginine containing peptides and RA relevant proteins.
Dehalogenases continue to garner interest of the scientific community due to their potential applications in bioremediation of halogen-contaminated environment and in synthesis of various industrially relevant products. Example of such enzymes is DehL, an L-2-haloacid dehalogenase (EC 3.8.1.2) from Rhizobium sp. RC1 that catalyses the specific cleavage of halide ion from L-2-halocarboxylic acids to produce the corresponding D-2-hydroxycarboxylic acids. Recently, the catalytic residues of DehL have been identified and its catalytic mechanism has been fully elucidated. However, the enantiospecificity determinants of the enzyme remain unclear. This information alongside a well-defined catalytic mechanism are required for rational engineering of DehL for substrate enantiospecificity. Therefore, using quantum mechanics/molecular mechanics and molecular mechanics Poisson-Boltzmann surface area calculations, the current study theoretically investigated the molecular basis of DehL enantiospecificity. The study found that R51L mutation cancelled out the dehalogenation activity of DehL towards it natural substrate, L-2-chloropropionate. The M48R mutation, however introduced a new activity towards D-2-chloropropionate, conveying the possibility of inverting the enantiospecificity of DehL from L-to d-enantiomer with a minimum of two simultaneous mutations. The findings presented here will play important role in the rational design of DehL dehalogenase for improving substrate utility.
Dehalogenases are of high interest due to their potential applications in bioremediation and in synthesis of various industrial products. DehL is an L-2-haloacid dehalogenase (EC 3.8.1.2) that catalyses the cleavage of halide ion from L-2-halocarboxylic acid to produce D-2-hydroxycarboxylic acid. Although DehL utilises the same substrates as the other L-2-haloacid dehalogenases, its deduced amino acid sequence is substantially different (<25%) from those of the rest L-2-haloacid dehalogenases. To date, the 3D structure of DehL is not available. This limits the detailed understanding of the enzyme's reaction mechanism. The present work predicted the first homology-based model of DehL and defined its active site. The monomeric unit of the DehL constitutes α/β structure that is organised into two distinct structural domains: main and subdomains. Despite the sequence disparity between the DehL and other L-2-haloacid dehalogenases, its structural model share similar fold as the experimentally solved L-DEX and DehlB structures. The findings of the present work will play a crucial role in elucidating the molecular details of the DehL functional mechanism.
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.
Box-Wilson design (BWD) model was applied to determine the optimum values of influencing parameters in anaerobic fermentation to produce hydrogen using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564). The main focus of the study was to find the optimal relationship between the hydrogen yield and three variables including initial substrate concentration, initial medium pH and reaction temperature. Microbial growth kinetic parameters for hydrogen production under anaerobic conditions were determined using the Monod model with incorporation of a substrate inhibition term. The values of micro(max) (maximum specific growth rate) and K, (saturation constant) were 0.398 h(-1) and 5.509 g L(-1), respectively, using glucose as the substrate. The experimental substrate and biomass-concentration profiles were in good agreement with those obtained by the kinetic-model predictions. By varying the conditions of the initial substrate concentration (1-40 g L(-1)), reaction temperature (25-40 degrees C) and initial medium pH (4-8), the model predicted a maximum hydrogen yield of 3.24 mol H2 (mol glucose)(-1). The experimental data collected utilising this design was successfully fitted to a second-order polynomial model. An optimum operating condition of 10 g L(-1) initial substrate concentration, 37 degrees C reaction temperature and 6.0 +/- 0.2 initial medium pH gave 80% of the predicted maximum yield of hydrogen where as the experimental yield obtained in this study was 77.75% exhibiting a close accuracy between estimated and experimental values. This is the first report to predict bio-hydrogen yield by applying Box-Wilson Design in anaerobic fermentation while optimizing the effects of environmental factors prevailing there by investigating the effects of environmental factors.
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.
Transporters mediate the movement of compounds across the membranes that separate the cell from its environment and across the inner membranes surrounding cellular compartments. It is estimated that one third of a proteome consists of membrane proteins, and many of these are transport proteins. Given the increase in the number of genomes being sequenced, there is a need for computational tools that predict the substrates that are transported by the transmembrane transport proteins. In this paper, we present TranCEP, a predictor of the type of substrate transported by a transmembrane transport protein. TranCEP combines the traditional use of the amino acid composition of the protein, with evolutionary information captured in a multiple sequence alignment (MSA), and restriction to important positions of the alignment that play a role in determining the specificity of the protein. Our experimental results show that TranCEP significantly outperforms the state-of-the-art predictors. The results quantify the contribution made by each type of information used.
A mutant of the lipase from Geobacillus sp. strain T1 with a phenylalanine to leucine substitution at position 16 was overexpressed in Escherichia coli strain BL21(De3)pLysS. The crude enzyme was purified by two-step affinity chromatography with a final recovery and specific activity of 47.4 and 6,315.8 U/mg, respectively. The molecular weight of the purified F16L lipase was approximately 43 kDa by 12% SDS-PAGE analysis. The F16L lipase was demonstrated to be a thermophilic enzyme due its optimum temperature at 70 °C and showed stability over a temperature range of 40-60 °C. The enzyme exhibited an optimum pH 7 in phosphate buffer and was relatively stable at an alkaline pH 8-9. Metal ions such as Ca(2+), Mn(2+), Na(+), and K(+) enhanced the lipase activity, but Mg(2+), Zn(2+), and Fe(2+) inhibited the lipase. All surfactants tested, including Tween 20, 40, 60, 80, Triton X-100, and SDS, significantly inhibited the lipolytic action of the lipase. A high hydrolytic rate was observed on long-chain natural oils and triglycerides, with a notable preference for olive oil (C18:1; natural oil) and triolein (C18:1; triglyceride). The F16L lipase was deduced to be a metalloenzyme because it was strongly inhibited by 5 mM EDTA. Moderate inhibition was observed in the presence of PMSF at a similar concentration, indicating that serine residues are involved in its catalytic action. Further, the activity was not impaired by water-miscible solvents, including methanol, ethanol, and acetone.
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.
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.
Kojic acid monooleate is a fatty acid derivative of kojic acid which can be widely used as a skin whitening agent in a cosmetic applications. In avoiding any possible harmful effects from chemically synthesized product, the enzymatic synthesis appears to be the best way to satisfy the consumer demand nowadays. The ability of immobilized lipase from Rhizomucor meihei (lipozyme RMIM) to catalyze the direct esterification of kojic acid and oleic acid was investigated. Response Surface Methodology (RSM) and 5-level-4-factor central composite rotatable were employed to evaluate the effects of synthesis parameters such as enzyme amount (0.1-0.4 g), temperature (30-60 degrees C), substrate molar ratio (1-4 mmol, kojic acid:oleic acid) and reaction time (24-48 h) on percentage molar conversion to kojic acid monooleate. Analysis of the product using TLC, GC and FTIR showed the presence of kojic acid monooleate. The optimal conditions for the enzymatic reaction were obtained after analysis with backward elimination using 0.17 g of enzyme and 4 mmol of substrate at 52.50 degrees C for 42 h. Under these conditions the esterification percentage was 37.21%. The results demonstrated that response surface methodology can be applied effectively to optimize the lipase-catalysed synthesis of kojic acid monooleate. The optimum conditions can be used to scale up the process.
Depolymerase is an enzyme that plays an important role in the hydrolysis of polyhydroxyalkanoates [PHAs]. In the current study, Burkholderia cepacia DP1 was obtained from Penang, Malaysia in which the enzyme was purified using ion exchange and gel filtration (Superdex-75) column chromatography. The molecular mass of the enzyme was estimated to be 53.3 kDa using SDS-PAGE. The enzyme activity was increased to 36.8 folds with the recovery of 16.3% after purification. The enzyme activity was detected between pH 6.0-10 and at 35-55 °C with pH 6.0 and 45 °C facilitating the maximum activity. Depolymerase was inactivated by Tween-20, Tween-80, SDS and PMSF, but insensitive to metal ions (Mg2+, Ca2+, K+, Na2+, Fe3+) and organic solvents (methanol, ethanol, and acetone). The apparent Km values of the purified P(3HB) depolymerase enzyme for P(3HB) and P(3HB-co-14%3HV) were 0.7 mg/ml and 0.8 mg/ml, respectively. The Vmax values of the purified enzyme were 10 mg/min and 8.89 mg/min for P(3HB) and P(3HB-co-14%3HV), respectively. The current study discovered a new extracellular poly(3-hydroxybutyrate) [P(3HB)] depolymerase enzyme from Burkholderia cepacia DP1 isolated and purified to homogeneity from the culture supernatant. To the best of our knowledge, this is the first report demonstrating the purification and biochemical characterization of P(3HB) depolymerase enzyme from genus Burkholderia.
beta-Galactosidase (EC. 3.2.1.23) from ripe carambola (Averrhoa carambola L. cv. B10) fruit was fractionated through a combination of ion exchange and gel filtration chromatography into four isoforms, viz. beta-galactosidase I, II, III and IV. This beta-galactosidases had apparent native molecular masses of 84, 77, 58 and 130 kDa, respectively. beta-Galactosidase I, the predominant isoform, was purified to electrophoretic homogeneity; analysis of the protein by SDS-PAGE revealed two subunits with molecular masses of 48 and 36 kDa. N-terminal amino acid sequence of the respective polypeptides shared high similarities albeit at different domains, with the deduced amino acid sequence of certain plant beta-galactosidases, thus, explaining the observed low similarity between the two subunits. beta-Galactosidase I was probably a heterodimer that have glycoprotein properties and a pI value of 7.2, with one of the potential glycosylation sites appeared to reside within the 48-kDa-polypeptide. The purified beta-galactosidase I was substantially active in hydrolyzing (1-->4)beta-linked spruce and a mixture of (1-->3)beta- and (1-->6)beta-linked gum arabic galactans. This isoform also had the capability to solubilize and depolymerize structurally intact pectins as well as to modify alkaline-soluble hemicelluloses, reflecting in part changes that occur during ripening.
The conversion of aldehydes to valuable alkanes via cyanobacterial aldehyde deformylating oxygenase is of great interest. The availability of fossil reserves that keep on decreasing due to human exploitation is worrying, and even more troubling is the combustion emission from the fuel, which contributes to the environmental crisis and health issues. Hence, it is crucial to use a renewable and eco-friendly alternative that yields compound with the closest features as conventional petroleum-based fuel, and that can be used in biofuels production. Cyanobacterial aldehyde deformylating oxygenase (ADO) is a metal-dependent enzyme with an α-helical structure that contains di‑iron at the active site. The substrate enters the active site of every ADO through a hydrophobic channel. This enzyme exhibits catalytic activity toward converting Cn aldehyde to Cn-1 alkane and formate as a co-product. These cyanobacterial enzymes are small and easy to manipulate. Currently, ADOs are broadly studied and engineered for improving their enzymatic activity and substrate specificity for better alkane production. This review provides a summary of recent progress in the study of the structure and function of ADO, structural-based engineering of the enzyme, and highlight its potential in producing biofuels.
Interests in Acinetobacter haemolyticus lipases are showing an increasing trend concomitant with growth of the enzyme industry and the widening search for novel enzymes and applications. Here, we present a structural model that reveals the key catalytic residues of lipase KV1 from A. haemolyticus. Homology modeling of the lipase structure was based on the structure of a carboxylesterase from the archaeon Archaeoglobus fulgidus as the template, which has a sequence that is 58% identical to that of lipase KV1. The lipase KV1 model is comprised of a single compact domain consisting of seven parallel and one anti-parallel β-strand surrounded by nine α-helices. Three structurally conserved active-site residues, Ser165, Asp259, and His289, and a tunnel through which substrates access the binding site were identified. Docking of the substrates tributyrin and palmitic acid into the pH 8 modeled lipase KV1 active sites revealed an aromatic platform responsible for the substrate recognition and preference toward tributyrin. The resulting binding modes from the docking simulation correlated well with the experimentally determined hydrolysis pattern, for which pH 8 and tributyrin being the optimum pH and preferred substrate. The results reported herein provide useful insights into future structure-based tailoring of lipase KV1 to modulate its catalytic activity.
The synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaC(Cs)). PhaC(Cs) showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. An in vitro assay of recombinant PhaC(Cs) expressed in Escherichia coli showed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strain C. necator (307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaC(Cs) was 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC from C. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation in Escherichia coli expressing PhaC(Cs) of up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaC(Cs) is a naturally occurring, highly active PHA synthase with superior polymerizing ability.
SIB1 FKBP22 is a peptidyl prolyl cis-trans isomerase (PPIase) member from a psychrotrophic bacterium, Shewanella sp. SIB1, consisting of N- and C-domains responsible for dimerization and catalytic PPIase activity, respectively. This protein was assumed to be involved in cold adaptation of SIB1 cells through its dual activity of PPIase activity and chaperone like-function. Nevertheless, the catalytic inhibition by FK506 and its substrate specificity remain unknown. Besides, ability of SIB1 FKBP22 to inhibit phosphatase activity of calcinuerin is also interesting to be studied since it may reflect wider cellular functions of SIB1 FKBP22. In this study, we found that wild type (WT) SIB1 FKBP22 bound to FK506 with IC50 of 77.55 nM. This value is comparable to that of monomeric mutants (NNC-FKBP22, C-domain+ and V37R/L41R mutants), yet significantly higher than that of active site mutant (R142A). In addition, WT SIB1 FKBP22 and monomeric variants were found to prefer hydrophobic residues preceding proline. Meanwhile, R142A mutant has wider preferences on bulkier hydrophobic residues due to increasing hydrophobicity and binding pocket space. Surprisingly, in the absence of FK506, SIB1 FKBP22 and its variants inhibited, with the exception of N-domain, calcineurin phosphatase activity, albeit low. The inhibition of SIB1 FKBP22 by FK506 is dramatically increased in the presence of FK506. Altogether, we proposed that local structure at substrate binding pocket of C-domain plays crucial role for the binding of FK506 and peptide substrate preferences. In addition, C-domain is essential for inhibition, while dimerization state is important for optimum inhibition through efficient binding to calcineurin.