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.
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.
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.
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 current demands of sustainable green methodologies have increased the use of enzymatic technology in industrial processes. Employment of enzyme as biocatalysts offers the benefits of mild reaction conditions, biodegradability and catalytic efficiency. The harsh conditions of industrial processes, however, increase propensity of enzyme destabilization, shortening their industrial lifespan. Consequently, the technology of enzyme immobilization provides an effective means to circumvent these concerns by enhancing enzyme catalytic properties and also simplify downstream processing and improve operational stability. There are several techniques used to immobilize the enzymes onto supports which range from reversible physical adsorption and ionic linkages, to the irreversible stable covalent bonds. Such techniques produce immobilized enzymes of varying stability due to changes in the surface microenvironment and degree of multipoint attachment. Hence, it is mandatory to obtain information about the structure of the enzyme protein following interaction with the support surface as well as interactions of the enzymes with other proteins. Characterization technologies at the nanoscale level to study enzymes immobilized on surfaces are crucial to obtain valuable qualitative and quantitative information, including morphological visualization of the immobilized enzymes. These technologies are pertinent to assess efficacy of an immobilization technique and development of future enzyme immobilization strategies.
Carbon nanomaterials, due to their catalytic activity and high surface area, have potential as cell immobilization supports to increase the production of xylanase. Recombinant Kluyveromyces lactis used for xylanase production was integrated into a polymeric gel network with carbon nanomaterials. Carbon nanomaterials were pretreated before cell immobilization with hydrochloric acid (HCl) treatment and glutaraldehyde (GA) crosslinking, which contributes to cell immobilization performance. Carbon nanotubes (CNTs) and graphene oxide (GO) were further screened using a Plackett-Burman experimental design. Cell loading and agar concentration were the most important factors in xylanase production with low cell leakage. Under optimized conditions, xylanase production was increased by more than 400% compared to free cells. Immobilized cell material containing such high cell densities may exhibit new and unexplored beneficial properties because the cells comprise a large fraction of the component. The use of carbon nanomaterials as a cell immobilization support along with the entrapment method successfully enhances the production of xylanase, providing a new route to improved bioprocessing, particularly for the production of enzymes. KEY POINTS: • Carbon nanomaterials (CNTs, GO) have potential as cell immobilization supports. • Entrapment in a polymeric gel network provides space for xylanase production. • Plackett-Burman design screen for the most important factor for cell immobilization.
Nanobiocatalysis is a new frontier of emerging nanosized material support in enzyme immobilization application. This paper is about a comprehensive review on cellulose nanofibers (CNF), including their structure, surface modification, chemical coupling for enzyme immobilization, and potential applications. The CNF surface consists of mainly -OH functional group that can be directly interacted weakly with enzyme, and its binding can be improved by surface modification and interaction of chemical coupling that forms a strong and stable covalent immobilization of enzyme. The knowledge of covalent interaction for enzyme immobilization is important to provide more efficient interaction between CNF support and enzyme molecule. Enzyme immobilization onto CNF is having potential for improving enzymatic performance and production yield, as well as contributing toward green technology and sustainable sources.
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.
Trypsin has been immobilized by adsorption onto Amberlite XAD-7 beads. The Michaelis constant (Km) of the enzyme was increased about sevenfold following the immobilization. Its rate of penetration into the porous beads was determined by staining the beads, which had been split, with naphthol blue black. The extent of diffusional rate limitation of immobilized trypsin was related to the penetration depth of the enzyme into the beads. This can be controlled by manipulating the conditions during the preparation of the immobilized enzyme.
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.
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.
The present study explores the utilisation of a new raw material from lignocellulose biomass, Meranti wood sawdust (MWS) for high commercial value xylooligosaccharides (XOS) production using immobilised xylanase. The xylanase was immobilised by a combination of entrapment and covalent binding techniques. The hemicellulosic xylan from MWS was extracted using a standard chlorite delignification method. The production of total and derivatives of XOS from the degradation of the hemicellulosic xylan of MWS were compared to the production from the commercial xylan from Beechwood. The utilisation of the extracted xylan from MWS yielded 0.36 mg/mL of total XOS after 60 h of hydrolysis. During the hydrolysis reaction, the immobilised xylanase released a lower degree of polymerisation (DP) of XOS, mainly X2 and X3, which were the major products of xylan degradation by xylanase enzymes. The production of XOS with a lower DP from MWS demonstrated the biotechnological potential of the MWS in the future. The XOS production retained about 70% of its initial XOS production during the second cycle. This is also the first report on the utilisation of MWS wastes in enzymatic hydrolysis using immobilised xylanase for XOS production.
LML-type structured lipids are one type of medium- and long-chain triacylglycerols. LML was synthesized using immobilized Talaromyces thermophilus lipase (TTL)-catalyzed interesterification of tricaprylin and ethyl linoleate. The resin AB-8 was chosen, and the lipase/support ratio was determined to be 60 mg/g. Subsequently, the immobilized TTL with strict sn-1,3 regiospecificity was applied to synthesize LML. Under the optimized conditions (60 °C, reaction time 6 h, enzyme loading of 6% of the total weight of substrates, substrate of molar ratio of ethyl linoleate to tricaprylin of 6:1), Triacylglycerols with two long- and one medium-chain FAs (DL-TAG) content as high as 52.86 mol% was obtained. Scale-up reaction further verified the industrial potential of the established process. The final product contained 85.24 mol% DL-TAG of which 97 mol% was LML after purification. The final product obtained with the high LML content would have substantial potential to be used as functional oils.
Biocarriers are pivotal in enhancing the reusability of biocatalyst that would otherwise be less economical for industrial application. Ever since the induction of enzymatic technology, varied materials have been assessed for their biocompatibility with enzymes of distinct functionalities. Herein, cellulase was immobilized onto polymethacrylate particles (ICP) as the biocarrier grafted with ethylenediamine (EDA) and glutaraldehyde (GA). Carboxymethyl cellulose (CMC) was used as a model substrate for activity assay. Enzyme immobilization loading was determined by quantifying the dry weight differential of ICP (pre-& post-immobilization). Cellulase was successfully demonstrated to be anchored upon ICP and validated by FTIR spectra analysis. The optimal condition for cellulase immobilization was determined to be pH 6 at 20 °C. The maximum CMCase activity was achieved at pH 5 and 50 °C. Residual activity of ∼50% was retained after three iterations and dipped to ∼18% on following cycle. Also, ICP displayed superior pH adaptability as compared to free cellulase. The specific activity of ICP was 65.14 ± 1.11% relative to similar amount of free cellulase.
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.
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.
α-Amylase from Anoxybacillus sp. SK3-4 (ASKA) is a thermostable enzyme that produces a high level of maltose from starches. A truncated ASKA (TASKA) variant with improved expression and purification efficiency was characterized in an earlier study. In this work, TASKA was purified and immobilized through covalent attachment on three epoxide (ReliZyme EP403/M, Immobead IB-150P, and Immobead IB-150A) and an amino-epoxide (ReliZyme HFA403/M) activated supports. Several parameters affecting immobilization were analyzed, including the pH, temperature, and quantity (mg) of enzyme added per gram of support. The influence of the carrier surface properties, pore sizes, and lengths of spacer arms (functional groups) on biocatalyst performances were studied. Free and immobilized TASKAs were stable at pH 6.0-9.0 and active at pH 8.0. The enzyme showed optimal activity and considerable stability at 60 °C. Immobilized TASKA retained 50% of its initial activity after 5-12 cycles of reuse. Upon degradation of starches and amylose, only immobilized TASKA on ReliZyme HFA403/M has comparable hydrolytic ability with the free enzyme. To the best of our knowledge, this is the first report of an immobilization study of an α-amylase from Anoxybacillus spp. and the first report of α-amylase immobilization using ReliZyme and Immobeads as supports.
Cellulose nanomaterial with rod-like structure and highly crystalline order, usually formed by elimination of the amorphous region from cellulose during acid hydrolysis. Cellulose nanomaterial with the property of biocompatibility and nontoxicity can be used for enzyme immobilization. In this work, urease enzyme was used as a model enzyme to study the surface modification of cellulose nanomaterial and its potential for biosensor application. The cellulose nanocrystal (CNC) surface was modified using 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation to introduce the carboxyl group at C6 primary alcohol. The success of enzyme immobilization and surface modification was confirmed using chemical tests and measured using UV-Visible spectrophotometer. The immobilization strategy was then applied for biosensor application for urea detection. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques were used for electroanalytical characterization of the urea biosensor.