Strategies to immobilize the individual enzymes are crucial for enhancing catalytic applicability and require a controlled immobilization process. Herein, protocol for immobilizing Candida rugosa lipase (CRL) onto modified magnetic silica derived from oil palm leaves ash (OPLA) was optimized for the effects of concentration of CRL, immobilization time, and temperature, monitored by titrimetric and spectrometric methods. XRD and TGA-DTG spectrometric observations indicated that OPLA-silica was well coated over magnetite (SiO2-MNPs) and CRLs were uniformly bound by covalent bonds to SiO2-MNPs (CRL/Gl-A-SiO2-MNPs). The optimized immobilization protocol showed that in the preparation of CRL/Gl-A-SiO2-MNPs, CRL with 68.3 mg/g protein loading and 74.6 U/g specific activity was achieved using 5 mg/mL of CRL, with an immobilization time of 12 h at 25 °C. The present work also demonstrated that acid-pretreated OPLA is a potential source of renewable silica, envisioning its applicability for practical use in enzymatic catalysis on solid support.
There is an array of methodologies to prepare nanocellulose (NC) and its fibrillated form (CNF) with enhanced physicochemical characteristics. However, acids, bases or organosolv treatments on biomass are far from green, and seriously threaten the environment. Current approach to produce NC/CNF from biomass should be revised and embrace the concept of sustainability and green chemistry. Although hydrothermal process, high-pressure homogenization, ball milling technique, deep eutectic solvent treatment, enzymatic hydrolysis etc., are the current techniques for producing NC, the route designs remain imperfect. Herein, this review highlights the latest methodologies in the pre-processing and isolating of NC/CNF from lignocellulose biomass, by largely focusing on related papers published in the past two years till date. This article also explores the latest advancements in environmentally friendly NC extraction techniques that cooperatively use ball milling and enzymatic hydrolytic routes as an eco-efficient way to produce NC/CNF, alongside the potential applications of the nano-sized celluloses.
In recent biomedical research, the area of cancer and infectious diseases has a leading position in the utilization of medicinal plants as a source of drug discovery. Malaysia has a diversity and a large number of underutilized fruits that are rich in phenolic compounds. Artoarpus altilis consider an underutilized fruit that is rich in phenolic compounds. Methanol extracts of A. altilis have been previously found to contain a high content of antioxidant phytochemicals. The purpose of the study was to evaluate the cytotoxicity and toxicological effect of methanol fruit extracts against MCF-7 cells. To determine the least concentration that might kill or suppress the growth of the cancer cells was in a concentration-dependent manner approach. The variation in the cytotoxic activity among the extracts was indicated by determining the IC50 of each extract against cells at 72 h. The IC50 of the samples was measured using a trypan blue exclusion assay. The methanol extract of the pulp part showed the least inhibition concentration of 15.40±0.91 μg/mL on MCF-7 cells. In the study, the molecular mechanism of methanol extracts-induced apoptosis and cell cycle arrested in human cancer cells were investigated in a time-dependent-manners approach by using flow cytometry. The treated cells were stained with nexin to detect early and late apoptosis and with propidium iodide (PI) for cell cycle arrest associated with the DNA fragmentation, various cell arrests occurred at G1/S, S, and G2/M phases. Lastly, the gene expression analysis by (RT-qPCR) method was carried out by analyzing the expression of the gene of interest for the quantification of mRNA levels. Results after cells treated with IC50 were revealed by upregulating anti-apoptotic genes/downregulated of pro-apoptotic BCL-2 gene expressions were triggered the treated cells into CASPASE-3, intrinsic and extrinsic pathways. These findings suggest that the methanol extracts of three parts of A. altilis fruit have potential anticancer activity against MCF-7 cells mainly the pulp part of the fruit.
This review aims to briefly describe the potential role of dehalogenase-producing halophilic bacteria in decontamination of organohalide pollutants. Hypersaline habitats pose challenges to life because of low water activity (water content) and is considered as the largest and ultimate sink for pollutants due to naturally and anthropogenic activities in which a substantial amount of ecological contaminants are organohalides. Several such environments appear to host and support substantial diversity of extremely halophilic and halotolerant bacteria as well as halophilic archaea. Biodegradation of several toxic inorganic and organic compounds in both aerobic and anaerobic conditions are carried out by halophilic microbes. Therefore, remediation of polluted marine/hypersaline environments are the main scorching issues in the field of biotechnology. Although many microbial species are reported as effective pollutants degrader, but little has been isolated from marine/hypersaline environments. Therefore, more novel microbial species with dehalogenase-producing ability are still desired.
An integral approach to decoding both culturable and uncultured microorganisms' metabolic activity involves the whole genome sequencing (WGS) of individual/complex microbial communities. WGS of culturable microbes, amplicon sequencing, metagenomics, and single-cell genome analysis are selective techniques integrating genetic information and biochemical mechanisms. These approaches transform microbial biotechnology into a quick and high-throughput culture-independent evaluation and exploit pollutant-degrading microbes. They are windows into enzyme regulatory bioremediation pathways (i.e., dehalogenase) and the complete bioremediation process of organohalide pollutants. While the genome sequencing technique is gaining the scientific community's interest, it is still in its infancy in the field of pollutant bioremediation. The techniques are becoming increasingly helpful in unraveling and predicting the enzyme structure and explore metabolic and biodegradation capabilities.
The high dependency and surplus use of agrochemical products have liberated enormous quantities of toxic halogenated pollutants into the environment and threaten the well-being of humankind. Herein, this study performed molecular docking, molecular dynamic (MD) simulations, molecular mechanics-Poisson Boltzmann Surface Area (MM-PBSA) calculations on the DehH2 from Bacillus thuringiensis, to identify the order of which the enzyme degrades different substrates, haloacids, haloacetate and chlorpyrifos. The study discovered that the DehH2 favored the degradation of haloacids and haloacetates (-3.3 - 4.6 kcal/mol) and formed three hydrogen bonds with Asp125, Arg201 and Lys202. Despite the inconclusive molecular docking result, chlorpyrifos was consistently shown to be the least favored substrate of the DehH2 in MD simulations and MM-PBSA calculations. Results of MD simulations revealed the DehH2-haloacid- (RMSD 0.15 - 0.25 nm) and DehH2-haloacetates (RMSF 0.05 - 0.25 nm) were more stable, with the DehH2-L-2CP complex being the most stable while the least was the DehH2-chlorpyrifos (RMSD 0.295 nm; RMSF 0.05 - 0.59 nm). The Molecular Mechanics Poisson-Boltzmann Surface Area calculations showed the DehH2-L-2CP complex (-24.27 kcal/mol) having the lowest binding energy followed by DehH2-MCA (-22.78 kcal/mol), DehH2-D-2CP (-21.82 kcal/mol), DehH2-3CP (-21.11 kcal/mol), DehH2-2,2-DCP (-18.34 kcal/mol), DehH2-2,3-DCP (-8.34 kcal/mol), DehH2-TCA (-7.62 kcal/mol), while chlorpyrifos was unable to spontaneously bind to DehH2 (+127.16 kcal/mol). In a nutshell, the findings of this study offer valuable insights into the rational tailoring of the DehH2 for expanding its substrate specificity and catalytic activity in the near future.Communicated by Ramaswamy H. Sarma.
Oil palm leaves (OPL) silica (SiO2) can replace the energy-intensive, commercially produced SiO2. Moreover, the agronomically sourced biogenic SiO2 is more biocompatible and cost-effective enzyme support, which properties could be improved by the addition of magnetite (Fe3O4) and graphene oxide (GO) to yield better ternary support to immobilize enzymes, i.e., Candida rugosa lipase (CRL). This study aimed to optimize the Candida rugosa lipase (CRL immobilization onto the ternary OPL-silica-magnetite (Fe3O4)-GO (SiO2/Fe3O4/GO) support, for use as biocatalyst for ethyl valerate (EV) production. Notably, this is the first study detailing the CRL/SiO2/Fe3O4/GO biocatalyst preparation for rapid and high yield production of ethyl valerate (EV). AFM and FESEM micrographs revealed globules of CRL covalently bound to GL-A-SiO2/Fe3O4/GO; similar to Raman and UV-spectroscopy results. FTIR spectra revealed amide bonds at 3478 cm-1 and 1640 cm-1 from covalent interactions between CRL and GL-A-SiO2/Fe3O4/GO. Optimum immobilization conditions were 4% (v/v) glutaraldehyde, 8 mg/mL CRL, at 16 h stirring in 150 mM NaCl at 30 °C, offering 24.78 ± 0.26 mg/g protein (specific activity = 65.24 ± 0.88 U/g). The CRL/SiO2/Fe3O4/GO yielded 77.43 ± 1.04 % of EV compared to free CRL (48.75 ± 0.70 %), verifying the suitability of SiO2/Fe3O4/GO to hyperactivate and stabilize CRL for satisfactory EV production.
l-2-Haloacid dehalogenase (DehL) from Rhizobium sp. RC1 is a stereospecific enzyme that acts exclusively on l-isomers of 2-chloropropionate and dichloroacetate. The amino acid sequence of this enzyme is substantially different from those of other l-specific dehalogenases produced by other organisms. DehL has not been crystallised, and hence its three-dimensional structure is unavailable. Herein, we review what is known concerning DehL and tentatively identify the amino acid residues important for catalysis based on a comparative structural and sequence analysis with well-characterised l-specific dehalogenases.
The unique cellular enzymatic machinery of halophilic microbes allows them to thrive in extreme saline environments. That these microorganisms can prosper in hypersaline environments has been correlated with the elevated acidic amino acid content in their proteins, which increase the negative protein surface potential. Because these microorganisms effectively use hydrocarbons as their sole carbon and energy sources, they may prove to be valuable bioremediation agents for the treatment of saline effluents and hypersaline waters contaminated with toxic compounds that are resistant to degradation. This review highlights the various strategies adopted by halophiles to compensate for their saline surroundings and includes descriptions of recent studies that have used these microorganisms for bioremediation of environments contaminated by petroleum hydrocarbons. The known halotolerant dehalogenase-producing microbes, their dehalogenation mechanisms, and how their proteins are stabilized is also reviewed. In view of their robustness in saline environments, efforts to document their full potential regarding remediation of contaminated hypersaline ecosystems merits further exploration.
Inorganic biopolymer-based nanocomposites are useful for stabilizing lipases for enhanced catalytic performance and easy separation. Herein, we report the operational stability, regenerability, and thermodynamics studies of the ternary biogenic silica/magnetite/graphene oxide nanocomposite (SiO2/Fe3O4/GO) as a support for Candida rugosa lipase (CRL). The X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), field-electron scanning electron microscopy (FESEM), vibrating sample magnetometry (VSM), and nitrogen adsorption/desorption data on the support and biocatalyst corroborated their successful fabrication. XPS revealed the Fe3O4 adopted Fe2+ and Fe3+ oxidation states, while XRD data of GO yielded a peak at 2θ = 11.67°, with the SiO2/Fe3O4/GO revealing a high surface area (≈261 m2/g). The fourier transform infrared (FTIR) spectra affirmed the successful fabricated supports and catalyst. The half-life and thermodynamic parameters of the superparamagnetic immobilized CRL (CRL/SiO2/Fe3O4/GO) improved over the free CRL. The microwave-regenerated CRL/SiO2/Fe3O4/GO (≈82%) exhibited higher catalytic activity than ultrasonic-regenerated (≈71%) ones. Lower activation (Ea) and higher deactivation energies (Ed) were also noted for the CRL/SiO2/Fe3O4/GO (13.87 kJ/mol, 32.32 kJ/mol) than free CRL (15.26 kJ/mol, 27.60 kJ/mol). A peak at 4.28 min in the gas chromatograph-flame ionization detection (GC-FID) chromatogram of the purified ethyl valerate supported the unique six types of 14 hydrogen atoms of the ester (CAS: 539-82-2) in the proton nuclear magnetic resonance (1H-NMR) data. The results collectively demonstrated the suitability of SiO2/Fe3O4/GO in stabilizing CRL for improved operational stability and thermodynamics and permitted biocatalyst regenerability.
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 study reports the preparation of a composite consisting of magnetite coated with nanosilica extracted from oil palm leaves (OPL) ash as nanosupports for immobilization of Candida rugosa lipase (CRL) and its application for the synthesis of butyl butyrate. Results of immobilization parameters showed that ∼ 80% of CRL (84.5 mg) initially offered was immobilized onto the surface of the nanosupports to yield a maximum protein loading and specific activity of 67.5 ± 0.72 mg/g and 320.8 ± 0.42 U/g of support, respectively. Surface topography, morphology as well as information on surface composition obtained by Raman spectroscopy, atomic force microscopy, field emission scanning electron microscopy and transmission electron microscopy showed that CRL was successfully immobilized onto the nanosupports, affirming its biocompatibility. Under optimal conditions (3.5 mg/mL protein loading, at 45 ℃, 3 h and molar ratio 2:1 (1-butanol:n-butyric acid) the CRL/Gl-A-SiO2-MNPs gave a maximum yield of 94 ± 0.24% butyl butyrate as compared to 84 ± 0.32% in the lyophilized CRL. CRL/Gl-A-SiO2-MNPs showed an extended operational stability, retaining 50% of its initial activity after 17 consecutive esterification cycles. The results indicated that OPL derived nanosilica coated on magnetite can potentially be employed as carrier for lipase immobilization in replacement of the non-renewable conventionalsilica sources.
Biomass from oil palm frond leaves (OPFL) is an excellent reservoir of lignocellulosic material which full potential remains untapped. This study aimed to statistically optimize the covalent immobilization of Candida rugosa lipase (CRL) onto a ternary support comprised of OPFL derived nanocellulose (NC) and montmorillonite (MMT) in alginate (ALG) (CRL-ALG/NC/MMT). The coarser topology and the presence of characteristic spherical globules in the field emission scanning electron micrographs and atomic force micrographs, respectively, supported the existence of CRL on ALG/NC/MMT. In addition, amide peaks at 3478 and 1640 cm-1 in the fourier transform infrared spectra affirmed that CRL was covalently bonded to ALG/NC/MMT. The optimized Taguchi Design-assisted immobilization of CRL onto ALG/NC/MMT (7 h of immobilization, 35℃, pH 5, 7 mg/mL protein loading) gave a production yield of 92.89 % of ethyl levulinate (EL), as proven by gas chromatography-mass spectrometric ([M] +m/z 144, C7H12O3), FTIR and nuclear magnetic resonance (CAS-539-88-8) data. A higher optimal reaction temperature (50℃) and the reusability of CRL-ALG/NC/MMT for up to 9 esterification cycles substantiated the appreciable structural rigidification of the biocatalyst by ALG/NC/MMT, which improved the catalytic activity and thermal stability of the lipase.
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.
Spectroscopic and calorimetric methods were employed to assess the stability and the folding aspect of a novel recombinant alkaline-stable lipase KV1 from Acinetobacter haemolyticus under varying pH and temperature. Data on far ultraviolet-circular dichroism of recombinant lipase KV1 under two alkaline conditions (pH 8.0 and 12.0) at 40 °C reveal strong negative ellipticities at 208, 217, 222 nm, implying its secondary structure belonging to a α + β class with 47.3 and 39.0% ellipticity, respectively. Results demonstrate that lipase KV1 adopts its most stable conformation at pH 8.0 and 40 °C. Conversely, the protein assumes a random coil structure at pH 4.0 and 80 °C, evident from a strong negative peak at ∼ 200 nm. This blue shift suggests a general decline in enzyme activity in conjunction with the partially or fully unfolded state that invariably exposed more hydrophobic surfaces of the lipase protein. The maximum emission at ∼335 nm for pH 8.0 and 40 °C indicates the adoption of a favorable protein conformation with a high number of buried tryptophan residues, reducing solvent exposure. Appearance of an intense Amide I absorption band at pH 8.0 corroborates an intact secondary structure. A lower enthalpy value for pH 4.0 over pH 8.0 and 12.0 in the differential scanning calorimetric data corroborates the stability of the lipase at alkaline conditions, while a low Km (0.68 ± 0.03 mM) for tributyrin verifies the high affinity of lipase KV1 for the substrate. The data, herein offer useful insights into future structure-based tunable catalytic activity of lipase KV1.
The genera Anexodus Pascoe, 1866 and Pantilema Aurivillius, 1911 (Cerambycidae: Lamiinae: Morimopsini), both endemic to Borneo, are revised. Four species of Anexodus are recognized: A. aquilus Pascoe, 1886 (Malaysia: Sabah), A. sarawakensis Sudre, 1997 (Malaysia: Sarawak), A. syptakovaesp. n. (Malaysia: Sarawak), and A. tufisp. n. (Brunei). Pantilema is a monotypic genus containing P. angustum Aurivillius, 1911 (Malaysia: Sarawak) which is known only from the holotype. For the first time, genital structures are studied in these genera. An identification key for the species of Anexodus is provided and their intraspecific morphological variability and distributions are discussed.
The l-2-haloacid dehalogenases (EC 3.8.1.2) specifically cleave carbon-halogen bonds in the L-isomers of halogenated organic acids. These enzymes have potential applications for the bioremediation and synthesis of various industrial products. One such enzyme is DehL, the l-2-haloacid dehalogenase from Rhizobium sp. RC1, which converts the L-isomers of 2-halocarboxylic acids into the corresponding D-hydroxycarboxylic acids. However, its catalytic mechanism has not been delineated, and to enhance its efficiency and utility for environmental and industrial applications, knowledge of its catalytic mechanism, which includes identification of its catalytic residues, is required. Using ab initio fragment molecular orbital calculations, molecular mechanics Poisson-Boltzmann surface area calculations, and classical molecular dynamic simulation of a three-dimensional model of DehL-l-2-chloropropionic acid complex, we predicted the catalytic residues of DehL and propose its catalytic mechanism. We found that when Asp13, Thr17, Met48, Arg51, and His184 were individually replaced with an alanine in silico, a significant decrease in the free energy of binding for the DehL-l-2-chloropropionic acid model complex was seen, indicating the involvement of these residues in catalysis and/or structural integrity of the active site. Furthermore, strong inter-fragment interaction energies calculated for Asp13 and L-2-chloropropionic acid, and for a water molecule and His184, and maintenance of the distances between atoms in the aforementioned pairs during the molecular dynamics run suggest that Asp13 acts as the nucleophile and His184 activates the water involved in DehL catalysis. The results of this study should be important for the rational design of a DehL mutant with improved catalytic efficiency.
Increased scientific interest has led to the rise in biotechnological uses of halophilic and halotolerant microbes for hypersaline wastewater bioremediation. Hence, this study performed molecular docking, molecular dynamic (MD) simulations, and validation by Molecular Mechanic Poisson-Boltzmann Surface Area (MM-PBSA) calculations on the DehH2 from Bacillus thuringiensis H2. We aimed to identify the interactions of DehH2 with substrates haloacids, haloacetates, and chlorpyrifos under extreme salinity (35% NaCl). MD simulations revealed that DehH2 preferentially degraded haloacids and haloacetates (-6.3 to -4.7 kcal/mol) by forming three or four hydrogen bonds to the catalytic triad, Asp125, Arg201, and Lys202. Conversely, chlorpyrifos was the least preferred substrate in both MD simulations and MM-PBSA calculations. MD simulation results ranked the DehH2-L-2CP complex (RMSD □0.125-0.23 nm) as the most stable while the least was the DehH2-chlorpyrifos complex (RMSD 0.32 nm; RMSF 0.0 - 0.29). The order of stability was as follows: DehH2-L-2CP > DehH2-MCA > DehH2-D-2CP > DehH2-3CP > DehH2-2,2-DCP > DehH2-2,3-DCP > DehH2-TCA > DehH2-chlorpyrifos. The MM-PBSA calculations further affirmed the DehH2-L-2CP complex's highest stability with the lowest binding energy of -45.14 kcal/mol, followed closely by DehH2-MCA (-41.21 kcal/mol), DehH2-D-2CP (-31.59 kcal/mol), DehH2-3CP (-30.75 kcal/mol), DehH2-2,2- DCP (-29.72 kcal/mol), DehH2-2,3-DCP (-22.20 kcal/mol) and DehH2-TCA (-18.46 kcal/mol). The positive binding energy of the DehH2-chlorpyrifos complex (+180.57 kcal/mol) proved the enzyme's non-preference for the substrate. The results ultimately illustrated the unique specificity of the DehH2 to degrade the above-said pollutants under a hypersaline condition.Communicated by Ramaswamy H. Sarma.
The concentration, distribution, and risk assessment of parabens were determined in the surface water of the Terengganu River, Malaysia. Target chemicals were extracted via solid-phase extraction, followed by high-performance liquid chromatography analysis. Method optimization produced a high percentage recovery for methylparaben (MeP, 84.69 %), ethylparaben (EtP, 76.60 %), and propylparaben (PrP, 76.33 %). Results showed that higher concentrations were observed for MeP (3.60 μg/L) as compared with EtP (1.21 μg/L) and PrP (1.00 μg/L). Parabens are ubiquitously present in all sampling stations, with >99 % of detection. Salinity and conductivity were the major factors influencing the level of parabens in the surface water. Overall, we found no potential risk of parabens in the Terengganu River ecosystem due to low calculated risk assessment values (risk quotient
The chemical route of producing geranyl propionate involves the use of toxic chemicals, liberation of unwanted by-products as well as problematic separation process. In view of such problems, the use of Rhizomucor miehei lipase (RML) covalently bound onto activated chitosan-graphene oxide (RML-CS/GO) support is suggested. Following analyses using Fourier transform infrared spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, and thermogravimetry, properties of the RML-CS/GO were characterized. A response surface methodological approach using a 3-level-four-factor (incubation time, temperature, substrate molar ratio, and stirring rate) Box-Behnken design was used to optimize the experimental conditions to maximize the yield of geranyl propionate. Results revealed that 76 ± 0.02% of recovered protein had yielded 7.2 ± 0.04 mg g(-1) and 211 ± 0.3% U g(-1) of the maximum protein loading and esterification activity, respectively. The actual yield of geranyl propionate (49.46%) closely agreed with the predicted value (49.97%) under optimum reaction conditions (temperature: 37.67°C, incubation time: 10.20 hr, molar ratio (propionic acid:geraniol): 1:3.28, and stirring rate: 100.70 rpm) and hence, verifying the suitability of this approach. Since the method is performed under mild conditions, the RML-CS/GO biocatalyst may prove to be an environmentally benign alternative for producing satisfactory yield of geranyl propionate.