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Amylase extraction from soil, Schemes and Mind Maps of Research Methodology

Basically a thesis in extraction of amylase bacteria from soil

Typology: Schemes and Mind Maps

2024/2025

Uploaded on 07/04/2025

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Biotechnological Production of Microbial Amylase for Industrial
Applications
A Project Report
Submitted by
Payasvani Raina(22BBT10084)
in partial fulfillment of the award of the degree of
BACHELOR OF SCIENCE
IN
BIOTECHNOLOGY
Chandigarh University
April 2025
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Biotechnological Production of Microbial Amylase for Industrial

Applications

A Project Report

Submitted by

Payasvani Raina(22BBT10084)

in partial fulfillment of the award of the degree of

BACHELOR OF SCIENCE

IN

BIOTECHNOLOGY

Chandigarh University

April 2025

BONAFIDE CERTIFICATE

Certified that this project report “Biotechnological Production of Microbial

Amylase for Industrial Applications” is the bonafide work of “PAYASVANI

RAINA” who carried out the project work under our supervision.

SIGNATURE SIGNATURE

Dr. Nancy George Dr. Gunjan Mukherjee

HEAD OF THE DEPARTMENT SUPERVISOR (DIRECTOR)

UIBT UIBT

Submitted for the project viva-voice examination held on

INTERNAL EXAMINER EXTERNAL EXAMINER

TABLE OF CONTENTS

List of Figures……………………………………………………………..….…i

List of Tables..……………………………………………………………….…ii

List of Abbrevations…………………………………………………………..iii

Abstract…………………………………………………………………………iv

Graphical Abstract………………………………………………………………v

Chapter 1.………………………………………………………….…………vi-x

Chapter 2 .……………………….…………………………….…………..xi-xxv

Chapter 3 ………..…………………………………………….………..xxvi-xxxi

Chapter 4. …………………………………………………….………..xxxii-xlix

Chapter 5. ………………………………………………………………….…….l

References...……………………………………………………………….……li

i

List of Figures

Figure 2 .1 : A comparative analysis of solid substrate fermentation (SSF) and

submerged fermentation (SmF)………………………………………...……...xx

Figure 2 .2: Observing α-amylase activity with DNS and dextrinizing

techniques…………………………………………………………….………xxii

Figure 4.1 : Rice and Sugarcane field from where the soil sample was

collected…………………………………………………………………..…xxxii

Figure 4.2 : Enrichment media with soil sample and other salts to promote

growth of microorganisms……………………………………………….…xxxiii

Figure 4.3 : Serially diluted Rice and Sugarcane samples with pH 7 ………xxxiv

Figure 4.4 : 10 ³ Starch agar plates showing bacterial colonies…………..…xxxv

Figure 4.5 : Pure cultures obtained after streaking thrice…………………..xxxv

Figure 4.6 : Clear zones of hydrolysis around bacterial isolate after iodine

screening……………………………………………………………,,,,,,,…xxxvi

Figure 4.7 : Biochemical test results of the two bacterial strains (Rice and

Sugarcane samples)……………………………………………………….xxxviii

Figure 4.8 : Before ssdndadding ssdndsamples to the mnsjdnf biochemical

kit…………………………………………….……………………………xxxviii

Figure 4.9 : Gram staining result of rice samples and sugarcane sample under

microscope under 40X……………………………………………………..…...xl

Figure 4.10: Showcasting at 100 X microscope …………………..…………..xl

Figure 4.1 1 : After adding DNSA – degree of conversion of starch into

maltose…………………………………………………………………….....xliii

iii

List of Abbrevations

DNSA.............................. Dinitrosalicylic acid

NA.................................... Nutrient agar

NB...................................... Nutrient broth

SmF...................................... Submerged Fermentation

Ph......................................... Potential of hydrogen

OD....................................... Optical Density

NaCl..................................... Sodium Chloride

K 2 HPO 4 ................................ Dipotassium hydrogen phosphate

Fecl3..................................... Ferric chloride

(NH 4 ) 2 SO 4 ........................... Ammonium sulphate

CaCl 2 .................................... Calcium chloride

iv

Abstract

Amylase is an essential enzyme in the brewing and sugar production, starch

processing, textile, and detergent manufacturing sectors. It facilitates the

conversion of starch into its simpler sugar components. The current study aims

to isolate the bacterial species that produce amylase from rice and sugarcane

fields, characterize the isolated species, conduct various biochemical analyses,

and assess the enzymatic activity of the amylase that these species create. The

isolated bacteria were first screened, and then their morphological and

biochemical characteristics were further characterized. The isolated bacterial

cultures' amylase enzyme activity was measured.Our results depict higher

enzyme activity of amylase producing bacterial species isolated from rice field

in compared to that isolated from sugarcane field.

vi

CHAPTER-I

INTRODUCTION

Certain biochemical reactions are regulated by biological catalysts called enzymes. Extracellular enzymatic activity in a range of bacteria has drawn attention in recent years due to the possibility of using microorganisms as biotechnological sources of industrially relevant enzymes. Proteases and amylases are regarded as the most well-known industrially significant enzymes due to their extensive use in the food, detergent, and brewing industries [Doss, A. & Anand, SP. 2012].The enzyme amylase can hydrolyze starch molecules into polymers that include glucose units. Amylase is used by a number of industries, including the food, fermentation, and pharmaceutical sectors, for its possible use in a variety of industrial processes. Living things, such as plants, animals, and microbes, can provide amylases (Souza, 2010). Amylase is a vital digestive enzyme that plays a central role in breaking down carbohydrates in the body. Produced primarily by the pancreas and salivary glands, it initiates the chemical process of digestion by converting complex starches into simpler sugars. Amylase functions as an endoglycosidase enzyme that specifically targets α-1,4-glycosidic bonds within carbohydrate molecules. TYPES OF AMYLASE Amylases can be broadly classified into three categories (Castro et al., 2018). α- Amylase Alpha-amylase is the most widely distributed form, found in humans, animals, plants, and microbes, functioning as a calcium metalloenzyme that breaks random α-1,4 glycosidic bonds in starch molecules .[ Henrissat, B & Bairoch, A. 1993.] β-Amylase Beta-amylase, primarily occurring in plants and microbes, works differently by cleaving the second α-1,4 glycosidic bond, releasing two maltose molecules at a time.0A wide range of microorganisms contain β-amylases and glucoamylases.[ Pandey, A; Nigam, P; Soccol, CR; Soccol, VT; Singh, D & Mohan, R. 2000.] γ-Amylase γ-amylase breaks down α 1-6 glycosidic bonds, including those at the non-reducing end of

vii amylose and amylopectin, resulting in the production of glucose. γ-amylase, unlike other amylases, performs best in acidic settings with an optimal pH of 3. Table 1.1: Types of Amylase and their properties MARKET POTENTIAL The global enzymes market is poised for substantial growth, projected to reach USD 20. billion by 2029, with a compound annual growth rate (CAGR) of 7.8% from 2024. This expansion is driven by diverse applications across multiple industries and a rising demand for sustainable solutions. The market is categorized into industrial enzymes—used in sectors such as food and beverage processing, bioethanol production, textiles and leather, detergents, and paper and pulp—and specialty enzymes, which serve the pharmaceutical, research and biotechnology, diagnostics, and biocatalyst industries. Within this industry, the amylase market exhibits strong growth potential. Amylase, particularly sourced from barley and wheat, plays a vital role in the brewing sector by breaking down starches into fermentable sugars, enhancing production efficiency and quality. Moreover, amylase enzymes are increasingly recognized as eco-friendly alternatives to conventional synthetic chemicals, as they function under mild conditions and contribute to lower energy consumption. SOURCES OF AMYLASE Plant sources Type Source Function Optimum pH Products α-amylase Animals, plants, and microbes Random cleavage of starch 6.7-7.0 Maltose, dextrins β-amylase Plants, microbes End-wise cleavage 5.4-5.5 Maltose γ-amylase Animals, microbes Complete hydrolysis 4.0-4.5 Glucose

ix natural availability of starch. Soil samples from these sites regularly show higher enzyme activity than other agricultural soils. Amylase Production Amylase can be produced through submerged fermentation (SmF) or solid-state fermentation (SSF), depending on physicochemical factors. SmF is preferred for commercial enzyme production due to its controlled regulation of temperature, pH, aeration, and moisture (Gangadharan et al., 2008). SSF, which mimics natural microbial growth conditions, is favored for value-added products due to its high yield, specificity, and low moisture content, reducing bacterial contamination (Souza, 2010). While SSF is generally suited for fungal amylase production, studies suggest that bacterial cultures can also be adapted for this method (Pandey, 2003). Table 1.2: Optimal conditions for different microorganisms Characteristics/properties of Amylase Physical and chemical factors play a crucial role in microbial growth for amylase production, with bacterial and fungal requirements extensively studied (Gupta et al., 2003; Shalini, 2014). These parameters are essential for optimizing fermentation processes, impacting both economic feasibility and practicality (Saini et al., 2017). Key factors influencing amylase production include pH, temperature, metal ions, carbon and nitrogen sources, surfactants, phosphate levels, and agitation. Applications of α- Amylase Microorganism pH Optimal/Range Temperature Optimal/Range Bacillus subtilis 7.0 37 °C Bacillus amyloliquefaciens 7.0 33 °C Bacillus sp. 4.5 70 °C Aspergillus niger 5.5 70 °C

x Starch is a primary storage component in many key crops like wheat, rice, maize, tapioca, and potatoes, supporting a growing starch processing industry. Over time, starch hydrolysis has shifted from acid-based methods to enzyme-driven processes, with starch-converting enzymes now accounting for 30% of global enzyme production. These enzymes are widely used in producing maltodextrin, modified starches, and glucose/fructose syrup, as well as in industrial applications like detergents and baking. In the food industry, amylases are essential for bread making, brewing, and sweetener production by converting starches into fermentable sugars. In textiles, they remove excess starch from fabrics, while in detergents, they help dissolve starch-based stains. The paper industry uses amylases to enhance sizing and reduce starch consumption. In pharmaceuticals, amylases aid in drug formulation, pancreatic enzyme therapy, and diagnostic testing. They are also expanding into biofuel production, breaking down starch into fermentable sugars for ethanol conversion. OBJECTIVES OF THIS PROJECT

  1. Isolation of the amylase producing microbes
  2. Screening of the microbes for amylase production
  3. Purification of the amylase producing microbial strain
  4. Selection of potent amylase producing microbes based on qualitative and quantitative assay.
  5. Characterization of amylase producing strain
  6. Optimisation of the enzyme production for the maximum amylase activity

xii

FLOW CHART OF HISTORY

1811 - Kirchhoff discovers starch-degrading enzyme in wheat 1833 - Anselme Payen isolates and names "diastase" (early amylase) 1894 - First industrial fungal amylase used for digestion treatment 1914 - Takamine pioneers fungal amylase production via Solid-State Fermentation (SSF) 1917 - Boidin & Effront commercialize bacterial α-amylase production (Bacillus species) 1925 - Kuhn names "α-amylase" based on hydrolysis product configuration 1930 - Ohlsson identifies and names "β-amylase" 1990s - 3D structures of amylase enzymes analysed and compared Present - Amylases widely used in industrial biotechnology and therapeutics Types of Amylases Amylases, including endoamylases and exoamylases, are enzymes capable of hydrolyzing starch. They are classified based on how they attack glycosidic bonds. Starch-degrading enzymes belong to several glycoside hydrolase families, with the most notable being the GH13 family.[ Coutinho, P & Henrissat, B. 1999.][. Henrissat, B & Bairoch, A. 1993]. Amylase enzymes exist in three main categories, each with distinct characteristics and functions. α-Amylases (EC 3.2.1.1) Endoamylases break α-1,4 glycosidic linkages found in amylose or amylopectin chains. A well-known example is α-amylase, found in a wide spectrum of microbes, including Archaea and bacteria.[Pandey, A; Nigam, P; Soccol, CR; Soccol, VT; Singh, D, & Mohan, R. 2000]. The enzyme generates branched oligosaccharides with an α-configuration and α-limit dextrins. α-Amylases are classified according on their level of substrate hydrolysis:  Saccharifying α-amylases break down 50-60% of glycosidic bonds.  Liquefying α-amylases break down 30-40% of the connections. α-amylases are metalloenzymes that require calcium for action. They degrade long-chain carbohydrates at random sites throughout the starch molecule, producing maltotriose and

xiii maltose from amylose and maltose, glucose, and limited dextrin from amylopectin. Because of their random activity, α-amylases act faster than β-amylases.[Fukumoto, J, and Okada, S. 1963.] Salivary and pancreatic amylases are part of the α-amylase family. Plants, fungi, and bacteria (including Bacillus species) all contain the enzyme. Exoamylases, the second type of amylase, work by cleaving starch molecules from the outer glucose residues. They are further grouped according to their specificity.

  • β-Amylase only breaks α-1,4 glycosidic linkages.
  • Amyloglucosidase (E.C. 3.2.1.3) and α-glucosidase (E.C. 3.2.1.20) may break down α-1, and α-1,6 bonds. These enzymes generate various end products: Glucoamylase and α-glucosidase produce glucose, while β-amylase produces maltose and β- limit dextrin. β-amylase and glucoamylase change the anomeric structure of maltose from α to β. Glucoamylase breaks down long-chain polysaccharides more efficiently than α-glucosidase, which preferentially hydrolyzes short maltooligosaccharides to produce α-configuration glucose.Enzymes, especially β-amylases and glucoamylases, are present in a range of microorganisms. γ-Amylase γ-amylase breaks down α 1-6 glycosidic bonds, including those at the non-reducing end of amylose and amylopectin, resulting in the production of glucose. γ-amylase, unlike other amylases, performs best in acidic settings with an optimal pH of 3. Type Primary Locations Notable Features α-Amylase • Salivary glands
    • Pancreas
    • Human tissues Present in humans, animals, plants, and microbes β-Amylase • Plant seeds
    • Fruits
    • Microorganisms Primarily found in plants and microbes

xv Substrate Characteristics and Production Patterns

  1. Starch  Highest production levels (3.64 IU/ml/minutes maximum)  Most consistent performance across experiments  Primary substrate for industrial applications  Standard reference for production studies
  2. Rice Powder  Second-highest production (2.93 IU/ml/minutes maximum)  Reliable alternative to pure starch  Economically viable option  Suitable for large-scale production
  3. Wheat Powder  Moderate production levels (2.67 IU/ml/minutes maximum)  Stable production patterns  Good substrate for bacterial growth  Consistent enzyme activity
  4. Ragi Powder  Lower production levels (2.36 IU/ml/minutes maximum)  Contains 72% carbohydrates  Rich in fiber and protein  Alternative substrate option SOURCES OF AMYLASE Enzymes can be produced by plants and microorganisms, with microbial sources being widely recognized for meeting industrial demands (Pandey et al.). Research on bacterial,

xvi fungal, and viral amylases has gained prominence due to their ease of large-scale production and low downstream processing costs, as they are extracellular in nature (Ashis et al., 2009). Amylases, namely α-amylases, have been identified from fungi, yeasts, bacteria, and actinomycetes. However, enzymes of fungal and bacterial origin are more commonly utilized in industrial applications and dominate the market. Notably, Aspergillus and Rhizopus species generate amylase with high efficiency (Pandey et al., McVey, 2002). The consistency, cost-effectiveness, reduced time and space requirements for production, and ease of process optimization and modification make bacterial and fungal amylases the preferred choice for industrial applications (Ellaiah et al., 2002). Plant Sources of Amylase Amylases are found in many different organisms, including mammals, fungi, plants, unicellular eukaryotes, bacteria, and archaea. While plants and animals produce amylases, microbial enzymes are most commonly used in commercial applications because to their high productivity, thermostability, and simplicity of cultivation (Reddy, R; Reddy, G & Seenayya, G. 1999). A key advantage of enzymatic production is its high selectivity, efficiency, and specificity, ensuring high yields of the desired product (Kim, S & Dale, BE. 2004.). Bacillus species are the primary bacterial sources used in commercial enzyme production (Olafimihan, C & Akinyanju, J. 1999), while other bacteria such as Escherichia, Pseudomonas, Proteus, Serratia, and Rhizobium also produce significant amounts of amylase (Oliviera, A; Oliviera, L; Andrade, J & Chagas, A. 2007). Fungal species such as Aspergillus, Rhizopus, Mucor, Neurospora, Penicillium, and Candida are also known to produce commercially significant extracellular amylases (Gupta, R; Gigras, P; Mohapatra, H; Goswami, VK & Chauhan, B. 2003). Despite their presence, plant-derived amylases have not been widely explored as enzyme sources (Azad, MAK; Bae, JH; Kim, JS; Lim, JK; Song, KS; Shin, BS & Kim, HR. 2009). However, agricultural waste materials offer a sustainable alternative by reducing pollution while serving as substrates for solid and liquid fermentation, lowering production costs. These carbon- and nitrogen-rich agricultural byproducts include pearl millet starch, orange