Enhancement of Candida Rugosa Lipase Catalytic efficacy via immobilization onto selected organic and inorganic supports

• Main Author: Nur Amalina Mohd Amin
• Format: Thesis
• Language: English
• Published: Universiti Sains Islam Malaysia 2015
• Subjects: Candida Rugosa Lipase Catalytic
• Online Access: http://ddms.usim.edu.my/handle/123456789/8882

In this study, Na-feldspar, nanoclay (Cloisite®30B) and kenaf core fibre have been used as enzyme immobilization supports. Through the enzyme screening procedure in the synthesis of butyl oleate, highest specific activity was acquired from Candida rugosa lipase (CRL) with 1.948 x 10-4 mmol ester/min/mg of protein compared to Candida antarctica, Candida cylindracea, Mucor javanicus, Mucor miehei and Aspergillus niger. Similarly, CRL exhibited the highest conversion of butyl oleate which was about 86.80%, thus it was selected as biocatalyst for further characterizations to suite industrial applications. Prior to immobilization, the physicochemical properties of selected support materials for lipase immobilization (Na-feldspar, nanoclay Cloisite®30B, kenaf core fibre) were characterized by using Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Energy Dispersive X-Ray (EDX), X-Ray Diffraction (XRD) and Physisorption of Nitrogen. Immobilization of CRL onto the supports via physical adsorption method showed that the highest percentage of immobilization was achieved in nanoclay (Cloisite®30B) (58.47%), followed by kenaf core fibre (53.39%) and Na-feldspar (12.07%). In the same way, nanoclay (Cloisite®30B) contained the highest protein loaded of 45.30 mg protein/g support, followed by kenaf core fibre and Na-feldspar, 36.71 mg protein/g support and 7.55 mg protein/g support, respectively. In adsorption isotherms analyses, nanoclay (Cloisite®30B) showed a sharp and steep slope at the lower concentration range, and a flat ‘plateau’ at the high range which was best described by the Langmuir isotherm. Kenaf core fibre and Na-feldspar systems however, showed typical characteristics of Freundlich isotherm with a ‘step’ or two plateaux over the Ce range of 60 protein mg/ml solution. Characterization of the immobilized lipases using SEM showed substantial, thick and rough external surface due to existence of lipase. Meanwhile, black spots of the lipases confined on the surface of supports were observed using TEM. The fingerprint elements of CRL and the supports were identified in EDX analysis. Successful immobilization of CRL onto supports was observed through dissimilarities in basal spacings shown by the XRD patterns and the significant differences in the values of surface area, pore diameter and pore volume between the supports and immobilized lipases obtained through the physisorption of nitrogen analysis. In screening of immobilized enzymes with high activities in the synthesis of butyl oleate, Nanoclay-CRL and Kenaf-CRL showed higher catalytic activity with 6.32 x 10-4 and 4.99 x 10-4 mmol/min/mg of protein, respectively. Optimal butyl oleate production (>80%) was achieved using Nanoclay-CRL and Kenaf-CRL after 12 hours of reactions. Nanoclay-CRL and Kenaf-CRL also showed high thermal stabilities with more than 85% retained relative activities after incubation at 70 oC. Nanoclay-CRL and Kenaf-CRL were also able to retain high activities and took over 16 to 19 cycles, to reach 50% of ester conversion. In the kinetic study of native and immobilized CRL, the lower Km(But) value suggested that the Nanoclay-CRL displayed higher affinity towards butanol (But) than towards oleic acid (Ol), Km(Ol) > Km(But). However, Native-CRL and Kenaf-CRL exhibited higher affinity towards oleic acid than towards butanol, Km(Ol) < Km(But). Despite of that, the Vmax(But) value showed that the initial rate catalyzed by Native-CRL in reactions containing varying butanol concentrations was higher than those catalyzed by Nanoclay-CRL and Kenaf-CRL. The immobilized lipases also demonstrated considerably higher Vmax(Ol) compared to Native-CRL in reactions containing varying oleic acid concentrations. The experimental findings of one substrate (Nanoclay-CRL and Kenaf-CRL) and two substrates (Native-CRL) competitive inhibition were best explained by the Ping-Pong Bi-Bi mechanism.