Effect of carbohydrates and amino acids on fermentative production of alpha amylase

Abstract

Extracellular alpha amylase was produced from Aspergillus oryzae under solid state fermentation. House hold agro-wastes were used as medium which were considered as one of the major pollutants due to unfavorable gas production via natural fermentation beside creating disposal problem. Investigations were carried out to evaluate the effect of various carbohydrate sources e.g. glucose, maltose, lactose, sucrose and soluble starch with different concentrations on the production of fungal alpha amylase utilizing agricultural wastes as the fermentation medium. Studies were also carried out to evaluate the effect of various heat stable amino acids e.g. glycine, histidine, proline, leucine and isoleucine with different concentrations on the production of fungal alpha amylase utilizing agricultural wastes as the fermentation medium. The results indicated that maximum activity of alpha amylase (6639.85 U/gds) was obtained at 0.5% concentration of sucrose solution with a r2 of 0.9692 when compared to control (3319.95 U/gds) and other carbohydrates used. Maximum activity of alpha amylase (10407.80 U/gds) was also obtained at 0.75% concentration of proline solution with an r2 of 0.6816 when compared to control (3035.60 U/gds) and other amino acids used in our present study.

Key words: alpha amylase, solid state fermentation, Aspergillus oryzae, agricultural wastes, carbohydrates, amino acids.

Introduction

Alpha Amylases (E.C. 3.2.1.1.) are starch-degrading enzymes that catalyze the hydrolysis of internal a-1,4- O -glycosidic bonds in polysaccharides with the retention of a-anomeric configuration in the products. Most of the a-amylases are metalloenzymes, which require calcium ions (Ca2+) for their activity, structural integrity and stability. They belong to family 13 (GH-13) of the glycoside hydrolase group of enzymes1. Amylases are one of the most important industrial enzymes that have a wide variety of applications ranging from conversion of starch to sugar syrups, to the production of cyclodextrins for the pharmaceutical industry. These enzymes account for about 30 % of the world’s enzyme production2. The a-amylase family can roughly be divided into two groups: the starch hydrolyzing enzymes and the starch modifying, or transglycosylating enzymes. The enzymatic hydrolysis is preferred to acid hydrolysis in starch processing industry due to a number of advantages such as specificity of the reaction, stability of the generated products, lower energy requirements and elimination of neutralization steps3.Alpha amylase is produced by a variety of plants, animals, and microorganisms4. Some α-amylase producing fungi are from the genera Aspergillus, Penicillium, Cephalosporium, Mucor, Candida, Neurospora, and Rhizopus 5.

Agricultural waste is composed of organic wastes (animal excreta in the form of slurries and farmyard manures, spent mushroom compost, soiled water and silage effluent) and waste such as plastic, scrap machinery, fencing, pesticides, waste oils and veterinary medicines. There are a number of methods used to treat agricultural waste. These include spreading the waste on land under strict conditions, anaerobic digestion and composting. Residues from fruit and vegetable processing units are also considered as agricultural wastes. These wastes are one of the major causes for environmental pollution. In general most of this “wastes” may be used as cattle feed or converted to biogas or compost. But, greater environmental and economic benefits could result from the conversion of these by-products of higher value. This can be achieved either by using such materials as multifunctional food ingredient or in order to other processes within the concept of low-residue food production. Thus bio-conversion of these wastes not only reduces disposal problem but also environmental pollution along with production of value added products6. Fourteen different agro-residues were screened for alpha amylase production using Bacillus amyloliquefaciens ATCC 23842, where wheat bran (WB) and groundnut oil cake (GOC) in mass ratio of 1:1 was proved as the best substrate source7.

Solid-state fermentation (SSF) is an alternative to the submerged fermentation (Smf). A closer evaluation of these two processes in recent years in several research centers throughout the world has revealed the enormous economical and practical advantages of SSF over SmF. These include non-aseptic conditions, use of raw materials as substrates, use of a wide variety of matrices, low capital cost, low energy expenditure, less expensive downstream processing, less water usage, lower wastewater output, potential higher volumetric productivity, higher concentration of the products, high reproducibility, lesser fermentation space, easier control of contamination, and generally simpler fermentation media8,9,10,11. This, however, does not mean that SSF can be taken as a foolproof technology to replace SmF. SSF too has some disadvantages, such as: difficulty in agitation of the substrate bed, resulting in heterogeneously distributed physiological, physical and chemical environment in the substrate bed; difficulties in fermentation control, mainly in heat buildup; control of moisture level of the substrate and control of aeration; difficulty in rapid determination of microbial growth and other fermentative parameters; limited types of microorganisms that can grow at low moisture levels8,9,12. Wheat bran (WB) showed the highest enzyme production under optimum conditions of solid-state fermentation (SSF) carried out using corncob leaf (CL), rye straw (RS),wheat straw (WS) and wheat bran (WB) as substrates by a fungal culture of Penicillium chrysogenum 13.

Carbohydrates such as maltose, starch, cellobiose, lactose, glucose, fructose and galactose favours induction of amyloglucosidase(glucoamylase)14. Experimental studies had revealed that, different carbon sources such as glucose, sucrose, maltose, lactose and starch at 1% (w/w) concentration when mixed with substrate, shows an increased production of the alpha amylase enzyme, especially in cases with lactose and maltose, even with different strains of Aspergillus oryzae 15. Starch and fructose may also serve as potential activators in case of amylase synthesis16. Amylase production was found to be stimulated due to fructose, lactose and maltose in all five species of Fusarium (F. dimerum, F. moniliforme, F. oxysporum, F. roseum and F.semitectum) while, Sucrose inhibited F. oxysporum 17. Alpha amylase was also produced under solid-state fermentation by Bacillus cereus MTCC 1305 using wheat bran and rice flake manufacturing waste as substrates where glucose showed enhanced enzyme production, whereas supplementation of different nitrogen sources showed decline in enzyme production18.

Amino acids such alanine, arginine, glycine, leucine, phenylalanine, proline, and cystine can act as stimulator for the production of alpha amylase from microorganisms19 . Whereas, glycine, lysine, isoleucine and histidine, had proved to be vital for glucoamylase synthesis from Aspergillus sp. using rice bran as fermentation medium14.

Considering the above factors, studies were carried out to evaluate the effect of various carbohydrates e.g. glucose, maltose, lactose, sucrose and soluble starch and heat sterilizable amino acids e.g. glycine, histidine, proline, leucine and isoleucine with different concentrations on the production of fungal alpha amylase utilizing agricultural wastes as the fermentation medium.

Materials & Methods

Microorganism

Aspergillus oryzae (NCIM No. 645) collected from National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratory, Pune(India) was maintained on Czapek Dox agar medium and stored under refrigerated condition at 4oC. A suspension of the mold i.e. one loopfull in 5ml of sterile water blank was used as the inoculum for each Roux bottle in our present study. A constant ratio of 4:1 (w/v) of waste to inoculum was maintained through out the study.

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