Biomass from oil palm frond leaves (OPFL) is an excellent reservoir of lignocellulosic material which full potential remains untapped. This study aimed to statistically optimize the covalent immobilization of Candida rugosa lipase (CRL) onto a ternary support comprised of OPFL derived nanocellulose (NC) and montmorillonite (MMT) in alginate (ALG) (CRL-ALG/NC/MMT). The coarser topology and the presence of characteristic spherical globules in the field emission scanning electron micrographs and atomic force micrographs, respectively, supported the existence of CRL on ALG/NC/MMT. In addition, amide peaks at 3478 and 1640 cm-1 in the fourier transform infrared spectra affirmed that CRL was covalently bonded to ALG/NC/MMT. The optimized Taguchi Design-assisted immobilization of CRL onto ALG/NC/MMT (7 h of immobilization, 35℃, pH 5, 7 mg/mL protein loading) gave a production yield of 92.89 % of ethyl levulinate (EL), as proven by gas chromatography-mass spectrometric ([M] +m/z 144, C7H12O3), FTIR and nuclear magnetic resonance (CAS-539-88-8) data. A higher optimal reaction temperature (50℃) and the reusability of CRL-ALG/NC/MMT for up to 9 esterification cycles substantiated the appreciable structural rigidification of the biocatalyst by ALG/NC/MMT, which improved the catalytic activity and thermal stability of the lipase.
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.
To overcome drawbacks in the conventional chemical route to synthesize eugenyl benzoate, immobilized Rhizomucor miehei lipase (RML) as the biocatalyst was proposed. The RML conjugated to a hybrid support consisting of biopolymers, chitosan (CS) and chitin nanowhiskers (CNWs). 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDAC) was used as the crosslinker to bind the lipase. Immobilization of RML was the highest on crosslinked CS/CNWs which gave a protein loading of ∼8.12 mg/g, corresponding to specific and residual activity of 537 U/g and 137%, respectively. Fourier transform infrared spectroscopy, thermogravimetric analysis-differential thermogravimetry, field emission scanning electron and atomic force microscopy of RML-CS/CNWs revealed that RML was successfully attached to the surface of crosslinked CS/CNWs. Under an optimized condition, the highest yield of eugenyl benzoate (56.3%) was attained after 5 h using 3 mg/mL of RML-CS/CNWs with molar ratio of eugenol: benzoic acid of 3:1, as compared to only 47.3% for the free RML. Analyses of FTIR and NMR on purified eugenyl benzoate affirmed that the ester was successfully produced in the enzymatic esterification. Therefore, the use of the RML-CS/CNWs biocatalysts appears promising to afford good yields of eugenyl benzoate within a relatively shorter reaction time.
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.
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 demonstration of the structure-properties relationship of shape-dependent photocatalysts remains a challenge today. Herein, one-dimensional (1-D)-like titania (TiO2), as a model photocatalyst, has been synthesized under a strong magnetic field in the presence of a magnetically responsive liquid crystal as the structure-aligning agent to demonstrate the relationship between a well-aligned structure and its photocatalytic properties. The importance of the 1-D-like TiO2 and its relationship with the electronic structures that affect the electron-hole recombination and the photocatalytic activity need to be clarified. The synthesis of 1-D-like TiO2 with liquid crystal as the structure-aligning agent was carried out using the sol-gel method under a magnetic field (0.3 T). The mixture of liquid crystal, 4'-pentyl-4-biphenylcarbonitrile (5CB), tetra-n-butyl orthotitanate (TBOT), 2-propanol, and water, was subjected to slow hydrolysis under a magnetic field. The TiO2-5CB took a well-aligned whiskerlike shape when the reaction mixture was placed under the magnetic field, while irregularly shaped TiO2-5CB particles were formed when no magnetic field was applied. It shows that the strong interaction between 5CB and TBOT during the hydrolysis process under a magnetic field controls the shape of titania. The intensity of the emission peaks in the photoluminescence spectrum of 1-D-like TiO2-5CB was lowered compared with the TiO2-5CB synthesized without the magnetic field, suggesting the occurrence of electron transfer from 5CB to the 1-D-like TiO2-5CB during ultraviolet irradiation. Apart from that, direct current electrical conductivity and Hall effect studies showed that the 1-D-like TiO2 composite enhanced electron mobility. Thus, the recombination of electrons and holes was delayed due to the increase in electron mobility; hence, the photocatalytic activity of the 1-D-like TiO2 composite in the oxidation of styrene in the presence of aqueous hydrogen peroxide under UV irradiation was enhanced. This suggests that the 1-D-like shape of TiO2 composite plays an important role in its photocatalytic activity.
In this study, nanocellulose (NC) was successfully extracted from oil palm frond leaves (OPFL) using a combination of bleaching, alkaline treatment and acid hydrolysis. X-ray diffractogram revealed the extracted NC was crystalline with a crystallinity index of 70.2%. This indicates its suitability as nano-fillers for preparing the chitosan/nanocellulose (CS-NC) supports to immobilize Candida rugosa lipase (CRL) to produce the CRL/CS-NC biocatalysts. FTIR, FESEM and TGA characterizations of the CRL/CS-NC confirm the CRLs were successfully conjugated to the CS-NC supports. The air-dried CS-NC supports gave satisfactory immobilization of the CRLs (5.2mg/g) with the resultant CRL/CS-NCs catalysed conversions of ≥80% of butyl butyrate within 6h. Time course reaction profile revealed that 76.3% butyl butyrate conversion was achieved at 4h immobilization time using 3mg/mL of CRL/CS-NCs. NMR analyses on the purified butyl butyrate confirmed that the ester was successfully synthesized.
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.