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.
The initial assumption that viewed inclusion bodies as a hindrance to the efficient production of protein is no longer held due to the emergence of catalytically active inclusion bodies (CatIBs). Recent studies revealed their potential to be used in free form or immobilized as biocatalysts. The curiosity to acquire suitable catalysts has remained the measure of concern for researchers and industrialists. Numerous processes and production in various sectors of food industries, petroleum, pharmaceutical, cosmetics, and many others are still searching for a robust catalyst with outstanding features such as recyclability, resistance to pH, as well as temperature. CatIBs are forms of inclusion bodies that possess catalytic activity, which can improve catalysis efficiency, stability, and recyclability. One of the advantages of CatIBs is their potential to be used as catalysts for numerous bioprocesses when generated by an enzyme. These aggregates can efficiently be used as a replacement for traditional enzyme immobilization. This review tends to focus on the possibility of its application in various processes. The novelty of this review is that it considered the production of CatIBs both from artificial and natural perspectives, as well as how to improve it. Inclusion bodies' immobilization may provide an efficient alternative in the area of biocatalysis, and hence it will improve industrial sectors and substantially provide a means of achieving excellent performance in the near future.
Ultrasound technology has emerged as a promising tool for enhancing enzymatic biodiesel production, yet the cavitation effect induced can compromise enzyme stability. This study explored the efficiency of polyols in enhancing lipase stability under ultrasound conditions to further improve biodiesel yield. The incorporation of sorbitol resulted in the highest fatty acid methyl ester (FAME) content in the ultrasound-assisted biodiesel production catalyzed by Eversa® Transform 2.0 among the investigated polyols. Furthermore, sorbitol enhanced the stability of the lipase, allowing it to tolerate up to 100 % ultrasound amplitude, compared to 60 % amplitude in its absence. Enzyme activity assays revealed that sorbitol preserved 99 % of the lipase activity, in contrast to 84 % retention observed without sorbitol under an 80 % ultrasound amplitude. Circular dichroism (CD) and fluorescence spectroscopy analyses confirmed that sorbitol enhanced lipase rigidity and preserved its conformational structure under ultrasound exposure. Furthermore, employing a stepwise methanol addition strategy in ultrasound-assisted reactions with sorbitol achieved an 81.2 wt% FAME content in 8 h with only 0.2 wt% enzyme concentration. This promising result highlights the potential of sorbitol as a stabilizing agent in ultrasound-assisted enzymatic biodiesel production, offering a viable approach for enhancing biodiesel yield and enzyme stability in industrial applications.
Over the past decade, nanotechnology has been developed and employed across various entities. Among the numerous nanostructured material types, enzyme-incorporated nanomaterials have shown great potential in various fields, as an alternative to biologically derived as well as synthetically developed hybrid structures. The mechanism of incorporating enzyme onto a nanostructure depends on several factors including the method of immobilization, type of nanomaterial, as well as operational and environmental conditions. The prospects of enzyme-incorporated nanomaterials have shown promising results across various applications, such as biocatalysts, biosensors, drug therapy, and wastewater treatment. This is due to their excellent ability to exhibit chemical and physical properties such as high surface-to-volume ratio, recovery and/or reusability rates, sensitivity, response scale, and stable catalytic activity across wide operating conditions. In this review, the evolution of enzyme-incorporated nanomaterials along with their impact on our society due to its state-of-the-art properties, and its significance across different industrial applications are discussed. In addition, the weakness and future prospects of enzyme-incorporated nanomaterials were also discussed to guide scientists for futuristic research and development in this field.
The enzymatic production of fatty acids from vegetable oils is becoming a preferred method due to its mild conditions, simplicity, and scalability. This review analyzes studies on enzymatic hydrolysis, exploring various feedstocks, lipases, reaction conditions, and conversion yields. However, a key limitation is the longer reaction time compared to conventional methods. This limitation is primarily due to the immiscibility of triacylglycerols (TAGs) with water at low temperatures and pressures, as well as the lower activity of enzymes compared to chemical catalysts. To overcome these issues, chemical additives are identified as the most effective process intensification strategy. They are easy to implement, cause less damage to lipases, and are more efficient than mechanical methods. The impact of various chemical additives was thoroughly examined for potential improvements in the enzymatic hydrolysis of vegetable oils. A synergistic combination of chemical additives comprising ionic liquids (ILs) and polyols, along with ultrasound, as well as the consideration of immobilization techniques were explored. Overall, this review highlights the potential of chemical additives and their synergistic feasibility in enhancing the enzymatic performance of lipase-catalyzed hydrolysis reactions.
Type I pullulanase from Anoxybacillus sp. SK3-4 (PulASK) is an unusual debranching enzyme that specifically hydrolyzes starch α-1,6 linkages at long branches producing oligosaccharides (≥G8), but is nonreactive against short branches; thus, incapable of producing reducing sugars (G1-G7). We report on the effects of both single and co-immobilization of PulASK on product specificity. PulASK was purified and immobilized through covalent attachment to three epoxides (ReliZyme EP403/M, Immobead IB-150P, and Immobead IB-150A) and an amino-epoxide (ReliZyme HFA403/M) activated supports. Following immobilization, all PulASK derivatives were active on both short and long branches in starch producing reducing sugars (predominantly maltotriose) and oligosaccharides (≥G8), respectively, a feature that is absent in the free enzyme. This study also demonstrated that co-immobilization of PulASK and α-amylase from Anoxybacillus sp. SK3-4 (TASKA) on ReliZyme HFA403/M significantly changed the product specificity compared to the free enzymes alone or individually immobilized enzymes. In conclusion, individual or co-immobilization caused changes in the product specificity, presumably due to changes in the enzyme binding pocket caused by the influence of carrier surface properties (hydrophobic or hydrophilic) and the lengths of the spacer arms.
The present study reports the effects of three commercial immobilized lipases namely Novozyme 435 from Candida antarctica lipase B (CALB), Lipozyme TL IM from Thermomyces lanuginosus and Lipozyme RM IM from Rhizomucor miehei on the production of trimethylolpropane (TMP) ester from high oleic palm methyl ester (HO-PME) and TMP. The TMP ester is a promising base oil for biolubricants that are easily biodegradable and non-toxic to humans and the environment. Enzymatic catalysts are insensitive to free fatty acid (FFA) content, hence able to mitigate the side reactions and consequently reduce product separation cost. The potential of these enzymes to produce TMP ester in a solvent-free medium was screened at various reaction time (8, 23, 30 and 48 h), operating pressure (0.1, 0.3 and 1.0 mbar) and enzyme dosage (1, 3, 5 and 10% w/w). The reaction was conducted at a constant temperature of 70 °C and a molar ratio of 3.9:1 (HO-PME: TMP). Novozyme 435 produced the highest yield of TMP ester of 95.68 ± 3.60% under the following conditions: 23 h reaction time, 0.1 mbar operating pressure and 5% w/w of enzyme dosage. The key lubrication properties of the produced TMP ester are viscosity index (208 ± 2), pour point (- 30 ± - 2 °C), cloud point (- 15 ± - 2 °C), onset thermal degradation temperature (427.8 °C), and oxidation stability, RPVOT (42 ± 4 min). The properties of the TMP ester produced from the enzymatic transesterification are comparable to other vegetable oil-based biolubricants produced by chemical transesterification.
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.
Esterification of succinic acid with oleyl alcohol catalyzed by immobilized Candida antarctica lipase B (Novozym 435) was investigated in this study. Response surface methodology (RSM) based on a five-level, four-variable central composite design (CCD) was used to model and analyze the reaction. A total of 21 experiments representing different combinations of the four parameters including temperature (35-65°C), time (30-450 min), enzyme amount (20-400 mg), and alcohol:acid molar ratio (1:1-8:1) were generated. A partial cubic equation could accurately model the response surface with a R(2) of 0.9853. The effect and interactions of the variables on the ester synthesis were also studied. Temperature was found to be the most significant parameter that influenced the succinate ester synthesis. At the optimal conditions of 41.1°C, 272.8 min, 20 mg enzyme amount and 7.8:1 alcohol:acid molar ratio, the esterification percentage was 85.0%. The model can present a rapid means for estimating the conversion yield of succinate ester within the selected ranges.
In order to characterize enzyme activity and stability corresponding to temperature effects, thermodynamic studies on commercial immobilized lipase have been carried out via enzymatic transesterification. An optimum temperature of 40 degrees C was obtained in the reaction. The decreasing reaction rates beyond the optimum temperature indicated the occurrence of reversible enzyme deactivation. Thermodynamic studies on lipase denaturation exhibited a first-order kinetics pattern, with considerable stability through time shown by the lipase as well. The activation and deactivation energies were 22.15 kJ mol(-1) and 45.18 kJ mol(-1), respectively, implying more energy was required for the irreversible denaturation of the enzyme to occur. At water content of 0.42%, the initial reaction rate and FAME yield displayed optimum values of 3.317 g/L min and 98%, respectively.
Synthesis of layered double hydroxides (LDHs) of Zn/Al-NO3- hydrotalcite (HIZAN) and Zn/Al-diocytyl sodium sulfosuccinate (DSS) nanocomposite (NAZAD) with a molar ratio of Zn/Al of 4:1 were carried out by coprecipitation through continuous agitation. Their structures were determined using X-ray diffractometer spectra, which showed that basal spacing for LDH synthesized by both methods was about 8.89 A. An expansion of layered structure of about 27.9 A was observed to accommodate the surfactant anion between the interlayer. This phenomenon showed that the intercalation process took place between the LDH interlayer. Lipase from Candida rugosa was immobilized onto these materials by physical adsorption method. It was found that the protein loading onto NAZAD is higher than HIZAN. The activity of immobilized lipase was investigated through esterification of oleic acid and 1-butanol in hexane. The effects of pore size, surface area, reaction temperature, thermostability of the immobilized lipases, storage stability in organic solvent, and leaching studies were investigated. Stability was found to be the highest in the nanocomposite NAZAD.
One of the potentials of carrier-free cross-linked enzyme aggregates (CLEA) immobilization is the ability to be separated and reuse. Yet, it might be impeded by the poor mechanical stability resulting low recyclability. CLEA of CGTase from Bacillus lehensis G1 (CGTase G1-CLEA) using chitosan (CS) as a cross-linker demonstrated high activity recovery however, displayed poor reusability. Therefore, the relationship between mechanical strength and reusability is studied by enhancing the CS mechanical properties and applying a new co-aggregation approach. Herein, CS was chemically cross-linked with glutaraldehyde (GA) and GA was introduced as a co-aggregant (coGA). CGTase G1-CLEA developed using an improved synthesized chitosan-glutaraldehyde (CSGA) cross-linker and a new coGA technique showed to increase its mechanical stability which retained 63.4% and 52.2%, respectively compared to using CS that remained 33.1% of their initial activity after stirred at 500 rpm. The addition of GA impacted the morphology and interaction consequently stabilizing the CLEAs durability in production of cyclodextrins. As a result, the reusability of CGTase G1-CLEA with CSGA and coGA increased by 56.6% and 42.8%, respectively compared to previous CLEA after 5 cycles for 2 h of reaction. This verifies that the mechanical strength of immobilized enzyme influences the improvement of its operational stability.
Enzymes play a crucial role in various industrial sectors. These biocatalysts not only ensure sustainability and safety but also enhance process efficiency through their unique specificity. Lipases possess versatility as biocatalysts and find utilization in diverse bioconversion reactions. Presently, microbial lipases are gaining significant focus owing to the rapid progress in enzyme technology and their widespread implementation in multiple industrial procedures. This updated review presents new knowledge about various origins of microbial lipases, such as fungi, bacteria, and yeast. It highlights both the traditional and modern purification methods, including precipitation and chromatographic separation, the immunopurification technique, the reversed micellar system, the aqueous two-phase system (ATPS), and aqueous two-phase flotation (ATPF), moreover, delves into the diverse applications of microbial lipases across several industries, such as food, vitamin esters, textile, detergent, biodiesel, and bioremediation. Furthermore, the present research unveils the obstacles encountered in employing lipase, the patterns observed in lipase engineering, and the application of CRISPR/Cas genome editing technology for altering the genes responsible for lipase production. Additionally, the immobilization of microorganisms' lipases onto various carriers also contributes to enhancing the effectiveness and efficiencies of lipases in terms of their catalytic activities. This is achieved by boosting their resilience to heat and ionic conditions (such as inorganic solvents, high-level pH, and temperature). The process also facilitates the ease of recycling them and enables a more concentrated deposition of the enzyme onto the supporting material. Consequently, these characteristics have demonstrated their suitability for application as biocatalysts in diverse industries.
Acylglycerols are widely used in the food industry due to their antimicrobial, emulsifying and nutritional properties. This study investigated the key reaction parameters, mass transfer mechanisms, and kinetic features of lipase-catalyzed esterification in a solvent-free system. Taguchi method was further employed to explore the relationship between "reaction parameter - yield composition". The results revealed that the maximum selectivity were achieved fror MAG (57.72 %), DAG (82.67 %) and TAG (79.29 %) under different enzymatic conditions. Fatty acid-to-glycerol molar ratio had the greatest impact on DAG selectivity, contributing 38.08 % of total impact level. Mass transfer analysis showed that external mass transfer limitation could be effectively overcome at stirring speeds above 600 rpm. Kinetic analysis confirmed that the reaction followed the Ping-Pong BiBi mechanism with alcohol and acid inhibition (SSE = 0.000643). This work provided a theoretical basis for developing more efficient and selective catalytic processes, aiding in quality control, reactor design, and industrial scale-up.
A biotechnological route via enzymatic esterification was proposed as an alternative way to synthesize the problematic anti-oxidant eugenyl benzoate. The new method overcomes the well-known drawbacks of the chemical route in favor of a more sustainable reaction process. The present work reports a Box-Behnken design (BBD) optimization process to synthesize eugenyl benzoate by esterification of eugenol and benzoic acid catalyzed by the chitosan-chitin nanowhiskers supported Rhizomucor miehei lipase (RML-CS/CNWs). Effects of four reaction parameters: reaction time, temperature, substrate molar ratio of eugenol: benzoic acid and enzyme loading were assessed. Under optimum conditions, a maximum conversion yield as high as 66% at 50°C in 5h using 3mg/mL of RML-CS/CNWs, and a substrate molar ratio (eugenol: benzoic acid) of 3:1. Kinetic assessments revealed the RML-CS/CNWs catalyzed the reaction via a ping-pong bi-bi mechanism with eugenol inhibition, characterized by a Vmax of 3.83mMmin-1. The Michaelis-Menten constants for benzoic acid (Km,A) and eugenol (Km,B) were 34.04 and 138.28mM, respectively. The inhibition constant for eugenol (Ki,B) was 438.6mM while the turnover number (kcat) for the RML-CS/CNWs-catalyzed esterification reaction was 40.39min-1. RML-CS/CNWs were reusable up to 8 esterification cycles and showed higher thermal stability than free RML.
An alternative environmentally benign support was prepared from chitosan-chitin nanowhiskers (CS/CNWs) for covalent immobilization of Rhizomucor miehei lipase (RML) to increase the operational stability and recyclability of RML in synthesizing eugenyl benzoate. The CS/CNWs support and RML-CS/CNWs were characterized using X-ray diffraction, fluorescent microscopy, and Fourier transform infrared spectroscopy. Efficiency of the RML-CS/CNWs was compared to the free RML to synthesize eugenyl benzoate for parameters: reaction temperature, stirring rate, reusability, and thermal stability. Under optimal experimental conditions (50°C, 250 rpm, catalyst loading 3 mg/mL), a twofold increase in yield of eugenyl benzoate was observed for RML-CS/CNWs as compared to free RML, with the former achieving maximum yield of the ester at 62.1% after 5 hr. Results demonstrated that the strategy adopted to prepare RML-CS/CNWs was useful, producing an improved and prospectively greener biocatalyst that supported a sustainable process to prepare eugenyl benzoate. Moreover, RML-CS/CNWs are biodegradable and perform esterification reactions under ambient conditions as compared to the less eco-friendly conventional acid catalyst. This research provides a facile and promising approach for improving activity of RML in which the resultant RML-CS/CNWs demonstrated good operational stability for up to eight successive esterification cycles to synthesize eugenyl benzoate.
The chemical-catalyzed transesterification process to produce biofuels i.e. pentyl valerate (PeVa) is environmentally unfriendly, energy-intensive with tedious downstream treatment. The present work reports the use of Rhizomucor miehei lipase (RML) crosslinked onto magnetic chitosan/chitin nanoparticles (RML-CS/CH/MNPs). The approach used to immobilize RML onto the CS/CH/MNPs yielded RML-CS/CH/MNPs with an immobilized protein loading and specific activity of 7.6 mg/g and 5.0 U·g-1, respectively. This was confirmed by assessing data of field emission scanning electron microscopy, X-ray diffraction, thermal gravimetric analysis and Fourier transform infrared spectroscopy. A three-level-four-factor Box-Behnken design (incubation time, temperature, substrate molar ratio, and enzyme loading) was used to optimize the RML-CS/CH/MNP-catalyzed esterification synthesis of PeVa. Under optimum condition, the maximum yield of PeVa (97.8%) can be achieved in 5 h at 50 °C using molar ratio valeric acid:pentanol (1:2) and an enzyme load of 2 mg/mL. Consequently, operational stability experiments showed that the protocol adopted to prepare the CS/CH/MNP nanoparticles had increased the durability of RML. The RML-CS/CH/MNP could catalyze up to eight successive esterification cycles to produce PeVa. The study also demonstrated the functionality of CS/CH/MNP nanoparticles as an eco-friendly support matrix for improving enzymatic activity and operational stability of RML to produce PeVa.
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.
One-pot synthesis of sugar-functionalized oligomeric caprolactone was carried out by lipase-catalyzed esterification of ε-caprolactone (ECL) with methyl-d-glucopyranoside (MGP) followed by the elongation of functionalized oligomer chain. Functionalization was performed in a custom-fabricated glass reactor equipped with Rushton turbine impeller and controlled temperature at 60 °C using tert-butanol as reaction medium. The overall reaction steps include MGP esterification of ECL monomer and its subsequent elongation by free 6-hydroxyhexanoate monomer units. A ping-pong bi-bi mechanism without ternary complex was proposed for esterification of ECL and MGP with apparent values of kinetic constant, namely maximal velocity (Vmax ), Michaelis constant for MGP (KmMGP ), and Michaelis constant for ECL (KmECL ) at 3.848 × 10-3 M H-1 , 8.189 × 10-2 M, and 6.050 M, respectively. Chain propagation step of MGP-functionalized ECL oligomer exhibits the properties of living polymerization mechanism. Linear relationship between conversion (%) and number average molecular weight, Mn (g mol-1 ), of functionalized oligomer was observed. Synthesized functionalized oligomer showed narrow range of molecular weight from 1,400 to 1,600 g mol-1 with more than 90% conversion achieved. Structural analysis confirmed the presence of covalent bond between the hydroxyl group in MGP with carboxyl end group of ECL oligomer.