Protease enzyme production in tray bioreactor using solid state fermentation process

Dissertation for receiving a master's degree

in the field of chemical engineering, biotechnology orientation

Abstract

Microbial proteases are among the most important hydrolyzing enzymes, which account for about 60% of the global industrial enzyme market. have given In this research, synthesis of protease enzyme in solid state fermentation process using Bacillus bacteria. licheniformis was investigated. Various agricultural wastes and products including wheat bran, rice bran, bagasse, corn husk, corn flour and barley flour were used as substrates. Protease produced from wheat husk has shown more activity than other substrates. The effect of different extraction parameters, including the type and amount of extractant solution and residence time in the shaker, was evaluated. The highest amount of protease recovery was obtained using 50 ml Tris buffer after 1 hour in the shaker. In addition, the effect of operating parameters such as time, temperature, pH, initial humidity, cabin humidity, particle size and amount of inoculum on enzyme production was investigated. The results showed that the highest amount of protease enzyme activity was obtained after 48 hours of fermentation at a temperature of 35°C, initial pH of 9, cabin humidity of 90%, particle size in the range of 1-2 cm, and initial humidity of 200% for the upper tray and 150% for the middle tray. Also, the effect of different carbon and nitrogen supplements on protease production was investigated. The results showed that by enriching the substrate with rice bran (1% w/w) and corn flour (2% w/w), the highest activity of protease enzyme was obtained. The maximum protease activity after 48 hours, under all optimal conditions, the activity level of 1281/1281 U/gds and 1048/7 U/gds was obtained for the upper and middle tray, respectively. The effect of temperature and pH on enzyme activity and stability was investigated. The results showed that the produced protease had an alkaline nature and had a very significant stability in the range of temperature changes in the range of 30-85 C? and pH values ??between 7-13, and the highest activity was obtained at a pH value of 8 and a temperature of 65 C?. Also, the use of produced enzyme in leather processing and hydrolysis of gelatin layer from photographic films and also as an additive to detergent was investigated. The results showed that the alkaline protease obtained from B. licheniformis showed a very good ability to remove various stains from fabric, remove hair from cow skin and digest the gelatin layer of photographic films. Also, the production of protease enzyme using solid state fermentation process in bioreactor and flask was investigated and the two systems were compared in terms of enzyme production rate.

Key words: protease, wheat bran, solid state fermentation, tray bioreactor, Bacillus. licheniformis

1-1. Introduction

In this chapter, general explanations about enzymes, their structure and characteristics are discussed. Then the enzyme groups and related reactions are expressed. Brief information about proteases as one of the most important enzyme groups is mentioned below. Then the solid state fermentation process is introduced as an efficient fermentation system. At the end, the needs and goals of this project are discussed.

1-2. Enzyme definition

Enzymes are the most important group of proteins that are responsible for carrying out biochemical reactions and speeding them up and can increase the speed of the reaction up to 107 times. Enzymes are produced by living organisms, plants and microorganisms. Carrying out all reactions in a living cell requires a special enzyme. As shown in figure (1-1), enzyme like an inorganic catalyst is not consumed in the reaction but provides a lower energy pathway for the reaction to take place [1, 2]. Catalysts remain unchanged in reactions, but enzymes, like other proteins, do not remain stable under different conditions. Some enzymes are mainly active in a limited range of pH, temperature and ionic strength, and at temperatures and pHs higher than the optimum, the enzyme is destroyed and loses its activity [3, 4]. Due to the special conditions of many of these enzymes, they are not recommended for industrial use, so trying to find new species that meet the needs of the industry is a continuous process [5].

1-3.Enzyme history

The catalytic activity of enzymes has been used for thousands of years in various processes such as making cheese, wine and bakery [6]. By the 19th century, it was known that processes such as the souring of milk and the fermentation of sugar into alcohol could only occur through the action of a living organism. In 1833, the sugar activating agent was relatively purified and named diastase [1], which is now called amylase [2]. A few years later, they isolated a substance from the gastric juice of a person who was on a protein diet and named it pepsin [3]. These compounds are called under the general name of yeast. Liebig [4] stated at that time that these yeasts could be non-living materials derived from living cells, while Pasteur [5] and others still believed that yeasts must contain living materials. Despite these disagreements, Cohen [6] called these molecules enzymes in 1878. Buchners[7] showed in 1897 that when yeast extract is added to sugar, sugar fermentation takes place. In 1926, Sumner [8] purified urease enzyme [9] from bean extract and crystallized it. Since then, they were able to crystallize a large number of enzymes [7, 8]. Simultaneously with the emergence of the knowledge of enzyme purification, their applications multiplied and, of course, with the use of engineered enzymes, the number of choices for industrial processes increased [6]

Abstract

Microbial protease is one of the most important enzymes and represents 60% of total industrial enzyme sales. In this study, protease synthesis in solid state fermentation using B. licheniformis was investigated. Several agricultural residues such as rice bran, sugarcane bagasse, wheat bran, barely bran, corn meal and corn husk were used as substrates. Protease from the wheat bran showed higher activities than other substrates. Effect of extraction parameters such as type and volume of extracted medium and mixing time in shaker were investigated. Maximum recovery of protease obtained with 50 ml of Tris-HCl buffer after 1 hour incubation in the incubator-shaker. Furthermore, the influence of operational parameters such as fermentation time, temperature, pH, initial moisture, cabin humidity, particle size and inoculum level were studied. Results showed that maximum activity of protease was obtained after 48h of incubation, cabin temperature of 35 °C, initial pH of 9, cabin humidity of 90%, particle size in the range of 1-2 cm and initial moisture of 200% for the top tray and 150% for the middle tray. Moreover, the effect of different carbon and nitrogen supplementary sources for protease production was investigated. Results showed that, maximum protease activity was obtained with supplementation of substrate with rice bran (1% w/w) and corn meal (2% w/w). Maximum protease activities achieved under all of the desired conditions were 1281.1 and 1048.7 U/gds for the top and middle trays respectively. Effect of temperature and pH for protease activity and thermal stability was investigated. The results showed that, the enzyme found to be a typical alkaline protease which was stable in broad temperature range (30-85 ?C) and pH values ??(7-13), with maximum activity was defined at 65 ?C and pH value of 8. Moreover, the application of produced enzyme in leather processing, hydrolysis of gelatin layers of photographic films and also an additive in detergents were investigated. Results showed that, alkaline protease from B. licheniformis, have variety of application in detergent formulation for the removal of stained from cotton cloths, dehairing of cow hide and hydrolysis of gelatin layers of photographic films. Also, protease production in two solid state fermentation systems including flask and bioreactor were evaluated. Finally, the protease production in two systems was compared.

Key words: protease, wheat bran, solid state fermentation, tray bioreactor, Bacillus.