Candida rugosa lipase supported on biomass-based nanocellulose-chitosan composite for synthesis of butyl butyrate

Abstract

The current practices of large-scale oil palm plantations such as passive dumping and open burning of unwanted matured oil palm fronds leaves (OPFL) are aesthetically displeasing and contributes to poor regional air quality along with increased health problems. Improper disposal of such large quantities of agricultural biomass is environmentally challenging and is unsustainable in the long run. In this regard, further research into development of new technological applications for OPFL warrants attention of the scientific community. In this study, OPFL were bleached, alkaline treated and acid-hydrolyzed to obtain the purified nanocellulose (NC). X-Ray diffractogram revealed the extracted NC was crystalline with a crystallinity index of 70.2%. This indicates its suitability as nano-fillers for the preparation of chitosan/nanocellulose (CS-NC) supports to immobilize Candida rugosa lipase (CRL) in the production of CRL/CS-NC biocatalysts. Characterization of CRL/CS-NC using FTIR-ATR, TGA, FESEM, XRD, Raman spectroscopy and fluorescence optical microscopy revealed that the CRL molecules were successfully bound to the surface of the CS-NC supports via imine bonds formed through a Schiff base mechanism. The results indicated that CS was highly hydrogen bonded to the NC. The optimum protocol to immobilize CRL onto the CS-NC supports was assessed for factors namely reaction temperature, concentration of glutaraldehyde and pH of buffer, to yield the highest conversion of butyl butyrate in 3 h of incubation. A maximum percent conversion of butyl butyrate at 88% was achieved using an immobilization temperature of 25°C, 0.3% concentration of glutaraldehyde and buffer at pH 7. The efficacy of CRL/CS-NC was compared with the free CRL for conditions viz. incubation time, temperature, molar ratio butanol:butyric acid, stirring rate and enzyme loading. Under optimum conditions (3 h, 50°C, molar ratio of acid/alcohol of 1:2, 200 rpm and 3 mg/mL CRL/CS-NC), the lipase successfully synthesized 90.2% of butyl butyrate as compared to 62.9% by the free CRL (3 h, 40°C, molar ratio of acid/alcohol of 1:2, 200 rpm and 5 mg/mL CRL). Thermal stability of CRL/CS-NC was improved by 1.5-fold over the free CRL, with the biocatalyst reusable for up to 8 successive esterification cycles. FTIR-ATR and NMR analyses on purified butyl butyrate confirmed that the ester was successfully synthesized. Kinetic assessments showed the CRL/CS-NC catalyzed esterification process followed a ping-pong bi-bi mechanism model (Vmax 4.5 mM min-1) with butanol inhibition (Ki,B 69.05 mM) and showed a greater preference for butyric acid (Km,A 155.52 mM) over butanol (Km,B 917.78 mM). In conclusion, NC obtained from OPFL was suitable as raw material for the preparation of a highly functional CS-NC support. Activity of CRL/CS-NC was improved for rapid and high–yield synthesis of butyl butyrate. Hence, the developed CRL/CS-NC was a possible practical substitute to the homogenous acid catalyst in the synthesis of butyl butyrate.