Immobilization of a-amylase using white lahar as inorganic solid support

Date

2010

Degree

Bachelor of Science in Chemistry

College

College of Arts and Sciences (CAS)

Adviser/Committee Chair

Veronica C. Sabularse

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Abstract

Alpha amylase was immobilized using an inorganic solid support, white lahar, obtained from Botolan Zambales. The lahar sample was treated with concentrated acid and separated into its black and white components. Scanning electron microscopy showed white lahar to be more porous than the black lahar making it ideal for use in immobilization. The treated white lahar was activated using the 3-aminopropyltriethoxysilane (APTS) and glutaraldehyde. The optimum concentration of APTS for activation of lahar was 7.5% (v/v) and that for glutaraldehyde was 2.5% (v/v). The attachment of APTS was confirmed by the nitrous acid and acetyl chloride tests while glutaraldehyde attachment was confirmed by 2,4-DNP test for aldehydes and ketones, and Tollen’s test for aldehydes. Further confirmation of the attachment of the reagents was done by Fourier-Transform Infrared Analysis. The optimum concentration of α-amylase was determined to be 1 mg/mL. Using the determined concentrations, α-amylase was immobilized onto the activated white lahar. The optimum pH, temperature and substrate concentration were also studied for both free and immobilized α-amylase. The optimum pH and temperature for maximal velocity (Vmax) for both enzymes were 6.5 and 65 oC, respectively. The optimum starch solution concentration to serve as substrate was 1% (w/v). The kinetic parameters were also determined. For the free enzyme, Michaelis constant (Km) was 0.53 g starch/100 mL solution and Vmax was 0.07 μmol glucose/mL/min. The Km and Vmax values for the immobilized enzyme were found to be 1.86 g starch/100 mL and 1.46 μmol glucose/mL/min, respectively. The stability of the immobilized enzyme on storage and repeated use was determined. During the 5 weeks of storage, the specific activity decreased for both free and immobilized enzyme. However, at the end of the storage, the immobilized α-amylase retained 58% of its initial activity while the free enzyme retained only 3% of its initial activity. The activity of the immobilized and free enzyme treated with azide were essentially the same as the untreated enzyme. In the course of 5 days of repeated use, the specific activity was found to decrease retaining 33% of its initial activity.

Language

English

Location

UPLB Main Library Special Collections Section (USCS)

Call Number

LG 993.5 2010 C42 D56

Document Type

Thesis

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