Over the past decade, L-homophenylalanine is extensively used in the pharmaceutical industry as a precursor for production of angiotensin-converting enzyme (ACE) inhibitor, which possesses significant clinical application in the management of hypertension and congestive heart failure (CHF). A number of chemical methods have been reported thus far for the synthesis of L-homophenylalanine. However, chemical methods generally suffer from process complexity, high cost, and environmental pollution. On the other hand, enantiomerically pure L-homophenylalanine can be obtained elegantly and efficiently by employing biocatalytic methods, where it appears to be the most attractive process in terms of potential industrial applications, green chemistry and sustainability. Herein we review the biocatalytic synthesis of vital L-homophenylalanine as potentially useful intermediate in the production of pharmaceutical drugs in environmentally friendly conditions, using membrane bioreactor for sustainable biotransformation process. One envisages the future prospects of developing an integrated membrane bioreactor system with improved performance for L-homophenylalanine production.
Sago pith residue is one of the most abundant lignocellulosic biomass which can serve as an alternative cheap substrate for fermentable sugars production. This residue is the fibrous waste left behind after the starch extraction process and contains significant amounts of starch (58%), cellulose (23%), hemicellulose (9.2%) and lignin (3.9%). The conversion of sago pith residue into fermentable sugars is commonly performed using cellulolytic enzymes or known as cellulases. In this study, crude cellulases were produced by two local isolates, Trichoderma asperellum UPM1 and Aspergillus fumigatus, UPM2 using sago pith residue as substrate. A. fumigatus UPM2 gave the highest FPase, CMCase and β-glucosidase activities of 0.39, 23.99 and 0.78 U/ml, respectively, on day 5. The highest activity of FPase, CMCase and β-glucosidase by T. asperellum UPM1 was 0.27, 12.03 and 0.42 U/ml, respectively, on day 7. The crude enzyme obtained from A. fumigatus UPM2 using β-glucosidase as the rate-limiting enzyme (3.9, 11.7 and 23.4 IU) was used for the saccharification process to convert 5% (w/v) sago pith residue into reducing sugars. Hydrolysis of sago pith residue using crude enzyme containing β-glucosidase with 23.4 IU, produced by A. fumigatus UPM2 gave higher reducing sugars production of 20.77 g/l with overall hydrolysis percentage of 73%.
An easy and efficient strategy to prepare betulinic acid esters with various anhydrides was used by the enzymatic synthesis method. It involves lipase-catalyzed acylation of betulinic acid with anhydrides as acylating agents in organic solvent. Lipase from Candida antarctica immobilized on an acrylic resin (Novozym 435) was employed as a biocatalyst. Several 3-O-acyl-betulinic acid derivatives were successfully obtained by this procedure. The anticancer activity of betulinic acid and its 3-O-acylated derivatives were then evaluated in vitro against human lung carcinoma (A549) and human ovarian (CAOV3) cancer cell lines. 3-O-glutaryl-betulinic acid, 3-O-acetyl-betulinic acid, and 3-O-succinyl-betulinic acid showed IC(50)<10 microg/ml against A549 cancer cell line tested and showed better cytotoxicity than betulinic acid. In an ovarian cancer cell line, all betulinic acid derivatives prepared showed weaker cytotoxicity than betulinic acid.
In view of several disadvantages as well as adverse effects associated with the use of chemical processes for producing esters, alternative techniques such as the utilization of enzymes on multi-walled carbon nanotubes (MWCNTs), have been suggested. In this study, the oxidative MWCNTs prepared using a mixture of HNO3 and H2SO4 (1:3 v/v) were used as a supportive material for the immobilization of Candida rugosa lipase (CRL) through physical adsorption process. The resulting CRL-MWCNTs biocatalysts were utilized for synthesizing geranyl propionate, an important ester for flavoring agent as well as in fragrances. Enzymatic esterification of geraniol with propionic acid was carried out using heptane as a solvent and the efficiency of CRL-MWCNTs as a biocatalyst was compared with the free CRL, considering the incubation time, temperature, molar ratio of acid:alcohol, presence of desiccant as well as its reusability. It was found that the CRL-MWCNTs resulted in a 2-fold improvement in the percentage of conversion of geranyl propionate when compared with the free CRL, demonstrating the highest yield of geranyl propionate at 6h at 55°C, molar ratio acid: alcohol of 1:5 and with the presence of 1.0g desiccant. It was evident that the CRL-MWCNTs biocatalyst could be reused for up to 6 times before a 50% reduction in catalytic efficiency was observed. Hence, it appears that the facile physical adsorption of CRL onto F-MWCNTs has improved the activity and stability of CRL as well as served as an alternative method for the synthesis of geranyl propionate.
Carboxylesterases (CEs) are members of prominent esterase, and as their name imply, they catalyze the cleavage of ester linkages. By far, a considerable number of novel CEs have been identified to investigate their exquisite physiological and biochemical properties. They are abundant enzymes in nature, widely distributed in relatively broad temperature range and in various sources; both macroorganisms and microorganisms. Given the importance of these enzymes in broad industries, interest in the study of their mechanisms and structural-based engineering are greatly increasing. This review presents the current state of knowledge and understanding about the structure and functions of this ester-metabolizing enzyme, primarily from bacterial sources. In addition, the potential biotechnological applications of bacterial CEs are also encompassed. This review will be useful in understanding the molecular basis and structural protein of bacterial CEs that are significant for the advancement of enzymology field in industries.
In this work, lipase from Candida rugosa was immobilized onto chitosan/graphene oxide beads. This was to provide an enzyme-immobilizing carrier with excellent enzyme immobilization activity for an enzyme group requiring hydrophilicity on the immobilizing carrier. In addition, this work involved a process for the preparation of an enzymatically active product insoluble in a reaction medium consisting of lauric acid and oleyl alcohol as reactants and hexane as a solvent. This product enabled the stability of the enzyme under the working conditions and allowed the enzyme to be readily isolated from the support. In particular, this meant that an enzymatic reaction could be stopped by the simple mechanical separation of the "insoluble" enzyme from the reaction medium. Chitosan was incorporated with graphene oxide because the latter was able to enhance the physical strength of the chitosan beads by its superior mechanical integrity and low thermal conductivity. The X-ray diffraction pattern showed that the graphene oxide was successfully embedded within the structure of the chitosan. Further, the lipase incorporation on the beads was confirmed by a thermo-gravimetric analysis. The lipase immobilization on the beads involved the functionalization with coupling agents, N-hydroxysulfosuccinimide sodium (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), and it possessed a high enzyme activity of 64 U. The overall esterification conversion of the prepared product was 78% at 60 °C, and it attained conversions of 98% and 88% with commercially available lipozyme and novozyme, respectively, under similar experimental conditions.
Immobilization of cross-linked tannase on pristine multiwalled carbon nanotubes (MWCNT) was successfully performed. Cross-linking of tannase molecules was made through glutaraldehyde. The immobilized tannase exhibited significantly improved pH, thermal, and recycling stability. The optimal pH for both free and immobilized tannase was observed at pH 5.0 with optimal operating temperature at 30°C. Moreover, immobilized enzyme retained greater biocatalytic activities upon 10 repeated uses compared to free enzyme in solution. Immobilization of tannase was accomplished by strong hydrophobic interaction most likely between hydrophobic amino acid moieties of the glutaraldehyde-cross-linked tannase to the MWCNT.
Azelaic acid (AzA) and its derivatives have been known to be effective in the treatment of acne and various cutaneous hyperpigmentary disorders. The esterification of azelaic acid with lauryl alcohol (LA) to produce dilaurylazelate using immobilized lipase B from Candida antarctica (Novozym 435) is reported. Response surface methodology was selected to optimize the reaction conditions. A well-fitting quadratic polynomial regression model for the acid conversion was established with regards to several parameters, including reaction time and temperature, enzyme amount, and substrate molar ratios. The regression equation obtained by the central composite design of RSM predicted that the optimal reaction conditions included a reaction time of 360 min, 0.14 g of enzyme, a reaction temperature of 46 °C, and a molar ratio of substrates of 1:4.1. The results from the model were in good agreement with the experimental data and were within the experimental range (R² of 0.9732).The inhibition zone can be seen at dilaurylazelate ester with diameter 9.0±0.1 mm activities against Staphylococcus epidermidis S273. The normal fibroblasts cell line (3T3) was used to assess the cytotoxicity activity of AzA and AzA derivative, which is dilaurylazelate ester. The comparison of the IC50 (50% inhibition of cell viability) value for AzA and AzA derivative was demonstrated. The IC50 value for AzA was 85.28 μg/mL, whereas the IC50 value for AzA derivative was more than 100 μg/mL. The 3T3 cell was still able to survive without any sign of toxicity from the AzA derivative; thus, it was proven to be non-toxic in this MTT assay when compared with AzA.
Tropical carnivorous plant, Nepenthes, locally known as "monkey cup", utilises its pitcher as a passive trap to capture insects. It then secretes enzymes into the pitcher fluid to digest the insects for nutrients acquisition. However, little is known about the microbiota and their activity in its pitcher fluid. Eighteen bacteria phyla were detected from the metagenome study in the Nepenthes pitcher fluid. Proteobacteria, Bacteroidetes and Actinobacteria are the dominant phyla in the Nepenthes pitcher fluid. We also performed culturomics approach by isolating 18 bacteria from the Nepenthes pitcher fluid. Most of the bacterial isolates possess chitinolytic, proteolytic, amylolytic, and cellulolytic and xylanolytic activities. Fifteen putative chitinase genes were identified from the whole genome analysis on the genomes of the 18 bacteria isolated from Nepenthes pitcher fluid and expressed for chitinase assay. Of these, six clones possessed chitinase activity. In conclusion, our metagenome result shows that the Nepenthes pitcher fluid contains vast bacterial diversity and the culturomic studies confirmed the presence of biocatalytic bacteria within the Nepenthes pitcher juice which may act in symbiosis for the turn over of insects trapped in the Nepenthes pitcher fluid.
The issue of environmental pollution has been worsened by the emergence of new contaminants whose morphology is yet to be fully understood . Several techniques have been adopted to mitigate the pollution effects of these emerging contaminants, and bioremediation involving plants, microbes, or enzymes has stood out as a cost-effective and eco-friendly approach. Enzyme-mediated bioremediation is a very promising technology as it exhibits better pollutant degradation activity and generates less waste. However, this technology is subject to challenges like temperature, pH, and storage stability, in addition to recycling difficulty as it is arduous to isolate them from the reaction media. To address these challenges, the immobilization of enzymes has been successfully applied to ameliorate the activity, stability, and reusability of enzymes. Although this has significantly increased the uses of enzymes over a wide range of environmental conditions and facilitated the use of smaller bioreactors thereby saving cost, it still comes with additional costs for carriers and immobilization. Additionally, the existing immobilization methods have their individual limitations. This review provides state-of-the-art information to readers focusing on bioremediation using enzymes. Different parameters such as: the sustainability of biocatalysts, the ecotoxicological evaluation of transformation contaminants, and enzyme groups used were reviewed. The efficacy of free and immobilized enzymes, materials and methods for immobilization, bioreactors used, challenges to large-scale implementation, and future research needs were thoroughly discussed.
Plastic biodegradation has emerged as a sustainable approach and green alternative in handling the ever-increasing accumulation of plastic wastes in the environment. The complete biodegradation of polyethylene terephthalate is one of the most recent breakthroughs in the field of plastic biodegradation. Despite the success, the effective and complete biodegradation of a wide variety of plastics is still far from the practical implementation, and an on-going effort has been mainly devoted to the exploration of novel microorganisms and enzymes for plastic biodegradation. However, alternative strategies which enhance the existing biodegradation process should not be neglected in the continuous advancement of this field. Thus, this review highlights various strategies which have shown to improve the biodegradation of plastics, which include the pretreatment of plastics using UV irradiation, thermal, or chemical treatments to increase the susceptibility of plastics toward microbial action. Alternative pretreatment strategies are also suggested and compared with the existing techniques. Besides, the effects of additives such as pro-oxidants, natural polymers, and surfactants on plastic biodegradation are discussed. In addition, considerations governing the biodegradation performance, such as the formulation of biodegradation medium, cell-free biocatalysis, and physico-chemical properties of plastics, are addressed. Lastly, the challenges and future prospects for the advancement of plastic biodegradation are also highlighted.
Ionic liquids (ILs), a class of materials with unique physicochemical properties, have been used extensively in the fields of chemical engineering, biotechnology, material sciences, pharmaceutics, and many others. Because ILs are very polar by nature, they can migrate into the environment with the possibility of inclusion in the food chain and bioaccumulation in living organisms. However, the chemical natures of ILs are not quintessentially biocompatible. Therefore, the practical uses of ILs must be preceded by suitable toxicological assessments. Among different methods, the use of microorganisms to evaluate IL toxicity provides many advantages including short generation time, rapid growth, and environmental and industrial relevance. This article reviews the recent research progress on the toxicological properties of ILs toward microorganisms and highlights the computational prediction of various toxicity models.
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 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.
The discharge of an alarming number of recalcitrant pollutants from various industrial activities presents a serious threat to environmental sustainability and ecological integrity. Bioremediation has gained immense interest around the world due to its environmentally friendly and cost-effective nature. In contrast to physical and chemical methods, the use of microbial enzymes, particularly immobilized biocatalysts, has been demonstrated as a versatile approach for the sustainable mitigation of environmental pollution. Considerable attention is now devoted to developing novel enzyme engineering approaches and state-of-the-art bioreactor design for ameliorating the overall bio-catalysis and biodegradation performance of enzymes. This review discusses the contemporary and state of the art technical and scientific progress regarding applying oxidoreductase enzyme-based biocatalytic systems to remediate a vast number of pharmaceutically active compounds from water and wastewater bodies. A comprehensive insight into enzyme immobilization, the role of mediators, bioreactors designing, and transformation products of pharmaceuticals and their associated toxicity is provided. Additional studies are necessary to elucidate enzymatic degradation mechanisms, monitor the toxicity levels of the resulting degraded metabolites and optimize the entire bio-treatment strategy for technical and economical affordability.
Deep Eutectic Solvents (DESs) have recently emerged as a new generation of ionic liquids for lignocellulose pretreatment. However, DESs contain salt components which tend to inactivate cellulase in the subsequent saccharification process. To alleviate this problem, it is necessary to evaluate the applicability of the DESs-Cellulase system. This was accomplished in the present study by first studying the stability of cellulase in the presence of selected DESs followed by applicability evaluation based on glucose production, energy consumption and kinetic performance. Results showed that the cellulase was able to retain more than 90% of its original activity in the presence of 10% (v/v) for glycerol based DES (GLY) and ethylene glycol based DES (EG). Furthermore, both DESs system exhibited higher glucose percentage enhancement and lower energy consumption as compared to diluted alkali system. Among the two DESs studied, EG showed comparatively better kinetic performance.
Currently, the chemically-assisted esterification to manufacture butyl butyrate employs corrosive homogeneous acid catalyst and liberates enormous quantities of hazardous by-products which complicate downstream treatment processes. This study aimed to identify the optimized esterification conditions, and the kinetic aspects of the enzyme-assisted synthesis of butyl butyrate using immobilized Candida rugosa lipase activated by chitosan-reinforced nanocellulose derived from raw oil palm leaves (CRL/CS-NC). The best process variables that gave the maximum conversion degree of butyl butyrate by CRL/CS-NC (90.2%) in just 3 h, as compared to free CRL (62.9%) are as follows: 50 °C, 1:2 M ratio of acid/alcohol, stirring rate of 200 rpm and a 3 mg/mL enzyme load. The enzymatic esterification followed the ping pong bi-bi mechanism with substrate inhibition, revealing a ˜1.1-fold higher Ki for CRL/CS-NC (55.55 mM) over free CRL (50.68 mM). This indicated that CRL/CS-NC was less inhibited by the substrates. Butanol was preferred over butyric acid as reflected by the higher apparent Michaelis-Menten constant of CRL/CS-NC for butanol (137 mM) than butyric acid (142.7 mM). Thus, the kinetics data conclusively showed that CRL/CS-NC (Vmax 0.48 mM min-1, Keff 0.07 min-1 mM-1) was catalytically more efficient than free CRL (Vmax 0.35 mM min-1, Keff 0.06 min-1 mM-1).
A green technology of biodiesel production focuses on the use of enzymes as the catalyst. In enzymatic biodiesel synthesis, suitable solvent system is very essential to reduce the inhibition effects of the solvent to the enzymes. This study produced ethanol-based biodiesel from a low-cost sludge palm oil (SPO) using locally-produced Candida cylindracea lipase from fermentation of palm oil mill effluent (POME) based medium. The optimum levels of ethanol-to-SPO molar ratio and enzyme loading were found to be 4:1 and 10 U/25 g of SPO respectively with 54.4% w/w SPO yield of biodiesel and 21.7% conversion of free fatty acid (FFA) into biodiesel. Addition of tert-butanol at 2:1 tert-butanol-to-SPO molar ratio into the ethanol-solvent system increased the yield of biodiesel to 71.6% w/w SPO and conversion of FFA into biodiesel to 28.8%. The SPO and ethanol have promising potential for the production of renewable biodiesel using enzymatic-catalyzed esterification and transesterification.
In the present study, catalytic pyrolysis of Chlorella vulgaris biomass was conducted to analyse the kinetic and thermodynamic performances through thermogravimetric approach. HZSM-5 zeolite, limestone (LS), bifunctional HZSM-5/LS were used as catalysts and the experiments were heated from 50 to 900 °C at heating rates of 10-100 °C/min. Iso-conversional model-free methods such as Flynn-Wall-Ozawa (FWO), Kissinger-Akahira-Sunose (KAS), Starink's, and Vyazovkin (V) were employed to evaluate the kinetic parameters meanwhile the thermodynamic parameters were determined by using FWO and KAS methods. The calculated EA values of non-catalytic and catalytic pyrolysis of HZSM-5 zeolite, LS, and bifunctional HZSM-5/LS were determined to be in the range of 156.16-158.10 kJ/mol, 145.26-147.84 kJ/mol, 138.81-142.06 kJ/mol, and 133.26 kJ/mol respectively. The results have shown that catalytic pyrolysis with the presence of bifunctional HZSM-5/LS resulted to a lower average EA and ΔH compared to HZSM-5, and LS which indicated less energy requirement in the process.
Bio-hydrogen production from wastewater using sludge as inoculum is a sustainable approach for energy production. This study investigated the influence of initial pH and temperature on bio-hydrogen production from dairy wastewater using pretreated landfill leachate sludge (LLS) as an inoculum. The maximum yield of 113.2 ± 2.9 mmol H2/g chemical oxygen demand (COD) (12.8 ± 0.3 mmol H2/g carbohydrates) was obtained at initial pH 6 and 37 °C. The main products of volatile fatty acids were acetate and butyrate with the ratio of acetate:butyrate was 0.4. At optimum condition, Gibb's free energy was estimated at -40 kJ/mol, whereas the activation enthalpy and entropy were 65 kJ/mol and 0.128 kJ/mol/l, respectively. These thermodynamic quantities suggest that bio-hydrogen production from dairy wastewater using pretreated LLS as inoculum was effective and efficient. In addition, genomic and bioinformatics analyses were performed in this study.