Abstract

Maize is an agricultural product widely distributed in Cameroon and amylase is one of the main enzymes in germinating maize and catalyses the hydrolysis of starch molecules into simpler sugars.

The objective of this study is to extract amylase from germinating maize. In this study, amylase enzyme was obtained from germinating maize. The seeds were grown in the laboratory and the amylase was extracted from the germinating seeds after a series of processes like washing, crushing, decanting, centrifugation and gel filtration.

The study was successful and the amylase was extracted and proven to be the main enzyme because out of the five protein samples amylase was found in two of those samples.

Amylase is applicable in many industries such as the textile, food, detergent, in medicine, in pharmacology and alcohol/fuel industries.

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.1 INRTODUCTION

1.1.1 BACKGROUND

Maize is an agricultural product widely distributed in Cameroon with minimal processing to minimize cost. Obtaining amylase from germinating maize can increase the value of maize thus increasing the price of maize culture which in turn will improve the standards of living of maize farmers in Cameroon.

Amylase is one of the main enzymes found in germinatingmaize and it catalyses the hydrolysis of starch molecules into polymers composed of glucose subunits. This is done by the hydrolysis of alpha D 1,4 glucosidic linkage in starch components.

Amylases have several applications in industrial processes such as the food industry, the pharmaceutical industry, textiles, fuel alcohol production, detergent production.

There are three types of amylases, alpha, beta and gamma amylase; the former is an extracellular enzyme which is widespread between animals, higher plants and microbes. It has an isoelectric point of 5.4, excellent pH and and temperature of enzymatic activity at 4.7 and 55°C respectively, its molecular weight depends in its source. Beta amylase is also an extracellular enzyme found in plants and microbes and hydrolyses amylopectin and glycogen, has an optimum pH between 4.5 and 6.5, an isoelectric point of 4.8, an excellent temperature of enzyme activity at 55°C but is inactivated at temperaturesabove 60°C, its molecular weight is between 20-50 kDa. Gamma amylase is found in animals and microbes and cleaves the last alpha 1,4 glycosidic bond, they work at an optimum pH of 3.0 but is not used in brewing like the other two.

1.1.2 Rationale

Despite the availability of maize in Cameroon, thus the availability of amylase, the enzyme is still purchased from foreign countries thus increasing the cost of production of products from the industries involved such as the food industry, the textile industry, pharmaceutical industries, fuel/alcohol industries and detergent industries.

Production of amylase by the industries themselves will reduce cost of importation of already made amylase thus reducing the cost of production of goods making it available and affordable for consumers.

1.1.3 Objectives

General Objective

The objective of this study is to extract amylase from germinating maize.

Specific Objectives

1. Planting and germination of maize.
2. Extraction of proteins from germinated seeds.
3. Identification of amylase enzyme from protein mixture.

1.1.4 Hypothesis

Germinating maize contains a variety of proteins with amylase being one of the main proteins which can be identified and extracted for use both locally and industrially.

1.2 LITERATURE REVIEW

1.2.1 MAIZE

CLASSIFICATION

Kingdom: Plantae

Order: Poales

Family: Poaceae

Subfamily: Panicoideae

Tribe: Andropogoneae

Genus: Zea

Species: Z. mays

Scientific name: Zea mays

Maize is grown in large scale in Cameroon and is mostly consumed as corn beer locally called “sha” or ground to powder and consumed as corn fufu or consumed as corn pap by both adults and infants. It is hardly or  never processed and mostly sold unprocessed.

Extracting amylase which is useful to many industries from germinating maize will increase the standards of living of maize farmers and reduce the cost of production of the various industries that require amylase in their production process because they won’t have to import amylase and as a consequence contributing to the improvement of the living standards of Cameroonians as a whole.

Maize (Zeamays L) is one of the most versatile emerging crops having wider adaptability under varied agro-climatic conditions. Globally, maize is known as queen of cereals because it has the highest genetic yield potential among the cereals. It is cultivated on nearly 150 m ha in about 160 countries having wider diversity of soil, climate, biodiversity and management practices that contributes 36 % (782 m t) in the global grain production.

Maize is one of the oldest human-cultivated crops. The center of origin is believed to be the Mesoamerica region, at least 7000 years ago when it was grown as a wild grass called teosinte in the Mexican highlands (FAO, 2006).

Maize spread around the globe after European discovery of the Americas in the 15thcentury (OGTR, 2008). Maize has tremendous variability in kernel color, texture, composition and appearance. Botanically, maize belongs to the grass family gramineae (Poaceae); it is an annual plant with an extensive fibrous root system. It is a diploid species, with a chromosome number of 2n = 2x = 20 (Cai, 2006).

The importance of cereal grains in human nutrition is widely recognized, as they provide substantial amounts of energy and protein to millions people, especially in developing countries (FAO, 2011).

According to Walcott, (2003) Seed-borne pathogens are a serious threat to seedling establishment. Amongst many assays used for detection of seed-borne pathogens visual examination based on characteristic symptoms including discoloration, shriveled and reduced seedsize is the most common, for separating healthy and diseased seeds.

For the amylase to be extracted the maize must be germinated to ensure that seed dormancy is broken and the production of proteins has begun. During the maize seed germination process, the embryo produces and secretes natural gibberellins to the endosperm. These hormones induce the development of hydrolytic enzymes in the aleurone layer such as alpha and beta amylase enzymes, which are responsible for the degradation of the reserves that are found in the endosperm.

1.2.2 Amylase

1.2.2.1 Definition

Amylase is an enzyme that hydrolyses starch into glucose polymers. This enzyme is extracted from germinating maize after it has been crushed to make available the protein mixture from which the enzyme with be extracted. It is essential to work in a sterilised medium to avoid any contamination.

All amylases are glycoside hydrolyses and act on alpha 1,4 glycosidic bonds. Alpha amylases can actanywhere on the substrate and is responsible for 90% of amylolytic activity in maize seeds, beta amylases act on the second alpha 1,4 glycosidic bond from the non reducing end while gamma amylases act on the last. Both alpha and beta amylases are present in seeds in the inactive form prior to germination and alpha amylase appears once germination has begun.

Amylases are starch degrading enzymes. They are widely distributed in microbial, plant and animal kingdoms (Banks et al, 1975). They degrade starch and related polymers to yield products characteristic of individual amylolytic enzymes. Initially the term amylase was used originally to designate enzymes capable of hydrolysing α-1, 4- glycosidic bonds of amylose, amylopectin, glycogen and their degradation products (Damien et al, 2010). They act by hydrolysing bonds between adjacent glucose units, yielding products characteristic of the particules enzyme involved (Dhanya et al, 2009).