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.
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 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.
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.
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.
A new silica-gel nanospheres (SiO2NPs) composition was formulated, followed by biochemical surface functionalization to examine its potential in urea biosensor development. The SiO2NPs were basically synthesized based on sol-gel chemistry using a modified Stober method. The SiO2NPs surfaces were modified with amine (-NH2) functional groups for urease immobilization in the presence of glutaric acid (GA) cross-linker. The chromoionophore pH-sensitive dye ETH 5294 was physically adsorbed on the functionalized SiO2NPs as pH transducer. The immobilized urease determined urea concentration reflectometrically based on the colour change of the immobilized chromoionophore as a result of the enzymatic hydrolysis of urea. The pH changes on the biosensor due to the catalytic enzyme reaction of immobilized urease were found to correlate with the urea concentrations over a linear response range of 50-500 mM (R2 = 0.96) with a detection limit of 10 mM urea. The biosensor response time was 9 min with reproducibility of less than 10% relative standard deviation (RSD). This optical urea biosensor did not show interferences by Na+, K+, Mg2+ and NH4+ ions. The biosensor performance has been validated using urine samples in comparison with a non-enzymatic method based on the use of p-dimethylaminobenzaldehyde (DMAB) reagent and demonstrated a good correlation between the two different methods (R2 = 0.996 and regression slope of 1.0307). The SiO2NPs-based reflectometric urea biosensor showed improved dynamic linear response range when compared to other nanoparticle-based optical urea biosensors.
A simple visual ethanol biosensor based on alcohol oxidase (AOX) immobilised onto polyaniline (PANI) film for halal verification of fermented beverage samples is described. This biosensor responds to ethanol via a colour change from green to blue, due to the enzymatic reaction of ethanol that produces acetaldehyde and hydrogen peroxide, when the latter oxidizes the PANI film. The procedure to obtain this biosensor consists of the immobilization of AOX onto PANI film by adsorption. For the immobilisation, an AOX solution is deposited on the PANI film and left at room temperature until dried (30 min). The biosensor was constructed as a dip stick for visual and simple use. The colour changes of the films have been scanned and analysed using image analysis software (i.e., ImageJ) to study the characteristics of the biosensor's response toward ethanol. The biosensor has a linear response in an ethanol concentration range of 0.01%-0.8%, with a correlation coefficient (r) of 0.996. The limit detection of the biosensor was 0.001%, with reproducibility (RSD) of 1.6% and a life time up to seven weeks when stored at 4 °C. The biosensor provides accurate results for ethanol determination in fermented drinks and was in good agreement with the standard method (gas chromatography) results. Thus, the biosensor could be used as a simple visual method for ethanol determination in fermented beverage samples that can be useful for Muslim community for halal verification.
A novel method for the rapid modification of fullerene for subsequent enzyme attachment to create a potentiometric biosensor is presented. Urease was immobilized onto the modified fullerene nanomaterial. The modified fullerene-immobilized urease (C60-urease) bioconjugate has been confirmed to catalyze the hydrolysis of urea in solution. The biomaterial was then deposited on a screen-printed electrode containing a non-plasticized poly(n-butyl acrylate) (PnBA) membrane entrapped with a hydrogen ionophore. This pH-selective membrane is intended to function as a potentiometric urea biosensor with the deposition of C60-urease on the PnBA membrane. Various parameters for fullerene modification and urease immobilization were investigated. The optimal pH and concentration of the phosphate buffer for the urea biosensor were 7.0 and 0.5 mM, respectively. The linear response range of the biosensor was from 2.31 × 10-3 M to 8.28 × 10-5 M. The biosensor's sensitivity was 59.67 ± 0.91 mV/decade, which is close to the theoretical value. Common cations such as Na+, K+, Ca2+, Mg2+ and NH4+ showed no obvious interference with the urea biosensor's response. The use of a fullerene-urease bio-conjugate and an acrylic membrane with good adhesion prevented the leaching of urease enzyme and thus increased the stability of the urea biosensor for up to 140 days.
This work reviews the stripping off, role of water molecules in activity, and flexibility of immobilized Candida antarctica lipase B (CALB). Employment of CALB in ring opening polyester synthesis emphasizing on a polylactide is discussed in detail. Execution of enzymes in place of inorganic catalysts is the most green alternative for sustainable and environment friendly synthesis of products on an industrial scale. Robust immobilization and consequently performance of enzyme is the essential objective of enzyme application in industry. Water bound to the surface of an enzyme (contact class of water molecules) is inevitable for enzyme performance; it controls enzyme dynamics via flexibility changes and has intensive influence on enzyme activity. The value of pH during immobilization of CALB plays a critical role in fixing the active conformation of an enzyme. Comprehensive selection of support and protocol can develop a robust immobilized enzyme thus enhancing its performance. Organic solvents with a log P value higher than four are more suitable for enzymatic catalysis as these solvents tend to strip away very little of the enzyme surface bound water molecules. Alternatively ionic liquid can work as a more promising reaction media. Covalent immobilization is an exclusively reliable technique to circumvent the leaching of enzymes and to enhance stability. Activated polystyrene nanoparticles can prove to be a practical and economical support for chemical immobilization of CALB. In order to reduce the E-factor for the synthesis of biodegradable polymers; enzymatic ring opening polyester synthesis (eROPS) of cyclic monomers is a more sensible route for polyester synthesis. Synergies obtained from ionic liquids and immobilized enzyme can be much effective eROPS.
New acrylic microspheres were synthesised by photopolymerisation where the succinimide functional group was incorporated during the microsphere preparation. An optical biosensor for urea based on reflectance transduction with a large linear response range to urea was successfully developed using this material. The biosensor utilized succinimide-modified acrylic microspheres immobilized with a Nile blue chromoionophore (ETH 5294) for optical detection and urease enzyme was immobilized on the surface of the microspheres via the succinimide groups. No leaching of the enzyme or chromoionophore was observed. Hydrolysis of the urea by urease changes the pH and leads to a color change of the immobilized chromoionophore. When the color change was monitored by reflectance spectrophotometry, the linear response range of the biosensor to urea was from 0.01 to 1,000 mM (R2 = 0.97) with a limit of detection of 9.97 μM. The biosensor response showed good reproducibility (relative standard deviation = 1.43%, n = 5) with no interference by major cations such as Na+, K+, NH4+ and Mg2+. The use of reflectance as a transduction method led to a large linear response range that is better than that of many urea biosensors based on other optical transduction methods.
The influence of water activity and water content was investigated with farnesyl laurate synthesis catalyzed by Lipozyme RM IM. Lipozyme RM IM activity depended strongly on initial water activity value. The best results were achieved for a reaction medium with an initial water activity of 0.11 since it gives the best conversion value of 96.80%. The rate constants obtained in the kinetics study using Ping-Pong-Bi-Bi and Ordered-Bi-Bi mechanisms with dead-end complex inhibition of lauric acid were compared. The corresponding parameters were found to obey the Ordered-Bi-Bi mechanism with dead-end complex inhibition of lauric acid. Kinetic parameters were calculated based on this model as follows: V (max) = 5.80 mmol l(-1) min(-1) g enzyme(-1), K (m,A) = 0.70 mmol l(-1) g enzyme(-1), K (m,B) = 115.48 mmol l(-1) g enzyme(-1), K (i) = 11.25 mmol l(-1) g enzyme(-1). The optimum conditions for the esterification of farnesol with lauric acid in a continuous packed bed reactor were found as the following: 18.18 cm packed bed height and 0.9 ml/min substrate flow rate. The optimum molar conversion of lauric acid to farnesyl laurate was 98.07 ± 0.82%. The effect of mass transfer in the packed bed reactor has also been studied using two models for cases of reaction limited and mass transfer limited. A very good agreement between the mass transfer limited model and the experimental data obtained indicating that the esterification in a packed bed reactor was mass transfer limited.
Immobilized Candida antarctica lipase B-catalyzed esterification of xylitol and two fatty acids (capric and caproic acid) were studied in a solvent-free system. The Taguchi orthogonal array method based on three-level-four-variables with nine experiments was applied for the analysis and optimization of the reaction parameters including time, substrate molar ratio, amount of enzyme, and amount of molecular sieve. The obtained conversion was higher in the esterification of xylitol and capric acid with longer chain length. The optimum conditions derived via the Taguchi approach for the synthesis of xylitol caprate and xylitol caproate were reaction time, 29 and 18h; substrate molar ratio, 0.3 and 1.0; enzyme amount, 0.20 and 0.05g, and molecular sieve amount of 0.03g, respectively. The good correlation between the predicted conversions (74.18% and 61.23%) and the actual values (74.05% and 60.5%) shows that the model derived from the Taguchi orthogonal array can be used for optimization and better understanding of the effect of reaction parameters on the enzymatic synthesis of xylitol esters in a solvent-free system.
Dimethyl adipate (DMA) was synthesized by immobilized Candida antarctica lipase B-catalyzed esterification of adipic acid and methanol. To optimize the reaction conditions of ester production, response surface methodology was applied, and the effects of four factors namely, time, temperature, enzyme concentration, and molar ratio of substrates on product synthesis were determined. A statistical model predicted that the maximum conversion yield would be 97.6%, at the optimal conditions of 58.5 degrees C, 54.0 mg enzyme, 358.0 min, and 12:1 molar ratio of methanol to adipic acid. The R(2) (0.9769) shows a high correlation between predicted and experimental values. The kinetics of the reaction was also investigated in this study. The reaction was found to obey the ping-pong bi-bi mechanism with methanol inhibition. The kinetic parameters were determined and used to simulate the experimental results. A good quality of fit was observed between the simulated and experimental initial rates.
Herein, an efficient epoxidation of 1-nonene is described. In a simple epoxidation system, commercially available Novozym 435, an immobilized Candida antarctica lipase B, and hydrogen peroxide (H(2)O(2)) were utilized to facilitate the in situ oxidation of phenylacetic acid to the corresponding peroxy acid which then reacted with 1-nonene to give 1-nonene oxide with high yield and selectivity. The aliphatic terminal alkene was epoxidised efficiently in chloroform to give an excellent yield (97%-99%) under the optimum reaction conditions, including temperature (35 °C), initial H(2)O(2) concentration (30%), H(2)O(2) amount (4.4 mmol), H(2)O(2) addition rate (one step), acid amount (8.8 mmol), and stirring speed (250 rpm). Interestingly, the enzyme was stable under the single-step addition of H(2)O(2) with a catalytic activity of 190.0 Ug-1. The entire epoxidation process was carried out within 12 h using a conventional water bath shaker.
Integrating polypyrrole-cellulose nanocrystal-based composites with glucose oxidase (GOx) as a new sensing regime was investigated. Polypyrrole-cellulose nanocrystal (PPy-CNC)-based composite as a novel immobilization membrane with unique physicochemical properties was found to enhance biosensor performance. Field emission scanning electron microscopy (FESEM) images showed that fibers were nanosized and porous, which is appropriate for accommodating enzymes and increasing electron transfer kinetics. The voltammetric results showed that the native structure and biocatalytic activity of GOx immobilized on the PPy-CNC nanocomposite remained and exhibited a high sensitivity (ca. 0.73 μA·mM(-1)), with a high dynamic response ranging from 1.0 to 20 mM glucose. The modified glucose biosensor exhibits a limit of detection (LOD) of (50 ± 10) µM and also excludes interfering species, such as ascorbic acid, uric acid, and cholesterol, which makes this sensor suitable for glucose determination in real samples. This sensor displays an acceptable reproducibility and stability over time. The current response was maintained over 95% of the initial value after 17 days, and the current difference measurement obtained using different electrodes provided a relative standard deviation (RSD) of 4.47%.
Enzyme immobilization has been known to be one of the methods to improve the stability and reusability of enzyme. In this study, a strategy to optimize laccase immobilization on polyethylene terephthalate grafted with maleic anhydride electrospun nanofiber mat (PET-g-MAH ENM) was developed. The development involves the screening and optimization processes of the crucial factors that influence the immobilization yield such as enzyme concentration, pH values, covalent bonding (CV) time, CV temperature, crosslinking (CL) time, CL temperature and glutaraldehyde concentration using two-level factorial design and Box-Behnken design (BBD), respectively. It was found that laccase concentration, pH values and glutaraldehyde concentration play important role in enhancing the immobilization yield of laccase on PET-g-MAH ENM in the screening process. Subsequently, the optimization result showed at 0.28 mg/ml laccase concentration, pH 3 and 0.45% (v/v) glutaraldehyde concentrations gave the highest immobilization yield at 87.64% which was 81.2% increment from the immobilization yield before optimization. Under the optimum condition, the immobilized laccase was able to oxidize 2, 2-azino-bis 3-ethylbenzothiazoline-6- sulfonic acid (ABTS) in a broad range of pH (pH 3-6) and temperature (20- 70 °C). Meanwhile, the kinetic parameters for Km and Vmax were 1.331 mM and 0.041 mM/min, respectively. It was concluded that the optimization of immobilized laccase on PET-g-MAH ENM enhance the performance of this biocatalyst.
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.
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.