CHAPTER : CATALYSIS

CHAPTER 4

CATALYSIS

Introduction

The focus of this chapter is the evaluation of the synthesised magnetic nanocomposites as enzyme supports. This was achieved by quantifying the amount of lipase (Candida Rugosa lipase (CRL) and Pseudomonas Fluorescens Lipase (PFL) immobilised on the nanocomposite support followed by assessing the catalytic activity of the immobilised lipase compared to the free lipase.

The first part of this chapter presents the data and discusses the results on the lipase immobilisation by assessing the surface amine density, followed by Bradford assay to quantify the immobilized enzyme (both physically adsorbed and chemically bonded lipase).

The second part of this chapter aims to examine the catalytic activity of the immobilized lipase using two lipase catalysed reactions. The first reaction used was the widely known model reaction of hydrolysis of p-nitrophenyl palmitate (pNPP) to palmitic acid and p-nitrophenol (pNP). This reaction was repeated in presence of an AC field to evaluate the activity of the immobilized enzyme in the presence of an AC field. The hydrolysis of pNPP has been commonly used as a model reaction since it can be readily monitored using UV-Visible spectrophotometry by measuring the absorbance at λ410nm (Teng and Xu, 2007, Gupta et al., 2002, Leis et al., 2015). The second catalytic application was the hydrolysis of cis-3,5-diacetoxy-1-cyclopentene to produce the chiral optical isomers (1S,4R)-cis-4-acetoxy-2-cyclopenten-1-ol and its enantiomer.

Mesoporous silica coated magnetic core-shell nanoparticles (ME33) were used for enzyme immobilisation through covalent bonding and physical adsorption. The nanoparticles were prepared following the method described in Section 2.4. The nanomaterials were characterized using various techniques such as transmission electron microscopy (TEM), X-ray diffraction (XRD), small angle X-ray scattering, infrared spectroscopy (FT-IR), vibrating sample magnetometer (VSM), scanning column magnetometry (SCM) and Energy-dispersive X-ray analysis (EDAX) as described in Chapter 3.

Colorimetric Assay of Amine Density

The lipase was immobilized on the magnetic nanoparticles covalently, as well as via physical adsorption. To immobilise the lipase on nanoparticles surface via chemical bonding, the magnetic nanoparticles were modified by amine surface functionalization which was then activated by glutaraldehyde as explained in Section 2.8 and 2.10.

Physical and chemical properties of amine functionalised nanoparticles can be determined by the density of the amine groups. The surface amine density was measured following the process explained in Section 2.9. Generally, the aminosilane layer was allowed to react with 4-nitrobenzaldehyde, a UV-sensitive molecule, followed by UV-Visible spectroscopy at λ282nm to confirm the formation of the corresponding imines. Subsequently the imines were hydrolysed to

regenerate the amine groups on the surface of the nanoparticles. The density of the amine groups were quantified by measuring the amount of 4-nitrobenzaldehyde available in the solution and comparing it to the initial concentration. The amount of the 4-nitrobenzaldehyde present in solution was calculated based on the pre-established standard curve.

Standard curves of 4-nitrobenzaldehyde in coupling and hydrolysis solution was prepared by measuring the absorbance of series of dilutions of 4-nitrobenzaldehyde in hydrolysis and coupling solution. Figure 1 and Figure 2 present typical standard curve for dilutions of 4-nitrobenzaldehyde in hydrolysis and coupling solution.

Figure 4-1. Standard curve of 4-nitrobenzaldehyde dilutions in coupling solution (λ282nm).

Figure 4-2. Standard curve of 4-nitrobenzaldehyde dilutions in hydrolysis solution (λ282nm).

As shown in Figure 4-1 and Figure 4-2, a linear trend was observed at the concentration range of 0 to 240 nmol/mL. The initial concentration of 4-nitrobenzaldehyde was 0.7 mg/mL which was not in the range of acceptable UV-visible readings, consequently, all the absorbance including the initial readings were recorded on 20 times diluted samples. Standard curves were reproduced

every six months using fresh samples to maintain the measurements reliability and reproducibility.

Results of both the water method and TPRE method of amine functionalisation are shown in Figure 4-3 and Figure 4-4. The measurements were performed in coupling solution by measuring the amount of unbounded 4-nitrobenzaldehyde left in the solution and comparing that with initial values and it was then confirmed by measuring the amount of 4-nitrobenzaldehyde present in the solution after hydrolysis.

Figure 4-3. Surface amine density of mesoporous silica coated magnetite prepared following water method.

Measuring in coupling solution (a) and measuring in hydrolysis solution (b). Each column represents an independent experiment and error bars present standard deviation between different samples in each experiment

Figure 4-4. Surface amine density of mesoporous silica coated magnetite prepared following TPRE method.

Measuring in coupling solution (a) and measuring in hydrolysis solution (b). Each column represents an independent experiment and error bars present standard deviation between different samples in each experiment

Surface amine density (molecules/nm²) was calculated by integrating the amine values obtained from the colorimetric assay and the surface area of the mesoporous silica coated nanoparticles obtained from nitrogen adsorption-desorption (BET) test. Surface amine density was calculated to be around 0.152 molecule/nm² (0.300 mmol/g) for water method and 0.414 molecule/nm² (0.817 mmol/g) for TPRE method.It is clear from the figures that TPRE method leads to higher surface amine density which is consistence with the literature (Gartmann et al., 2010, Sen and Bruce, 2012a). Higher surface amine density attained by TPRE method could be due to limited water in the system which controls the surface condensation of the aminosilane and leads to uniform amine distribution on the surface. It is well established that water promotes the interaction

of the amino groups with the surface, by proton transfer from the surface silanols to the amino moieties and subsequent electrostatic interaction. The resulting orientation of the amino groups toward the silica surface provides accessible triethoxysilyl moieties for cross-linking, ultimately leading to the formation of clusters. The presence of such clusters can also make pores partially inaccessible for additional APTES molecules and result in a lower amine groups density (Gartmann et al., 2010).