• 0 Review(s)

Product Category: Projects

Product Code: 00007192

No of Pages: 74

No of Chapters: 1-5

File Format: Microsoft Word

Price :




The production and characterization of protease and pectinase from bacteria isolated from market solid waste and the bio-scouring of cotton using the enzymes was investigated. Using morphological, cultural and biochemical properties, the isolate was confirmed as Bacillus subtilis. Protease and pectinase were produced by Bacillus subtilis using submerged fermentation in gelatin broth and pectin broth respectively. The enzymes were purified using ammonium sulphate precipitation, dialysis and ion-exchange chromatography. Glucose as a carbon source and peptone as a nitrogen source gave optimum activity for both enzymes. Purified protease exhibited maximum activity of 8.72U/ml at 40oC while pectinase exhibited maximum activity of 8.98U/ml at 50oC. Application of the enzymes on bio-scouring of cotton fabrics was tested. Pectinase was more effective than protease under optimized conditions. Optimum scouring temperature for both enzymes was between 40oC and 50oC with pectinase bio-scoured fabric showing 15.5% weight loss while protease bio-scoured fabric had 14.3% weight loss. Optimum pH for pectinase scouring was pH 9 with 14.8% weight loss in fabric while optimum pH for protease scoured fabric was pH 7 with 12.3% weight loss in fabric. After 120 minutes of bio-scouring, 15.4% weight loss was recorded for pectinase treated fabric while protease treated fabric showed 10.9% weight loss. The application of protease and pectinase for the scouring of cotton fabric revealed that they can be used as bio-scouring agents for the treatment of textile materials.



Title Page                                                                                                                    i

Declaration                                                                                                                  ii

Certification                                                                                                                iii

Dedication                                                                                                                  iv

Acknowledgements                                                                                                    v

Table of Contents                                                                                                       vi

List of Tables                                                                                                              x

List of Figures                                                                                                             xi

Abstract                                                                                                                      xii



1.1       Background of the Study                                                                               1

1.2       Justification                                                                                                     3

1.3       Main Objective                                                                                               3

1.3.1   Specific objectives                                                                                           4


CHAPTER 2: LITERATURE REVIEW                                                              5

2.1       Enzymes                                                                                                          5

2.2       Protease                                                                                                           7

2.3       Pectinase                                                                                                         8

2.4       Bacillus subtilis                                                                                               8

2.5       Fermentation                                                                                                   10

2.5.1    Submerged fermentation                                                                                11

2.5.2    Solid state fermentation                                                                                  12

2.6       Cotton                                                                                                             13

2.7       Scouring and Bio-Scouring                                                                             15

CHAPTER 3: MATERIALS AND METHODS                                                   17

3.1       Sample Collection                                                                                           17

3.2       Isolation of Bacillus subtilis                                                                            17

3.2.1    Gram’s staining                                                                                               17

3.2.2    Endospore staining                                                                                         18

3.2.3    Capsule staining                                                                                              18

3.2.4    Catalase test                                                                                                    18

3.2.5    Oxidase test                                                                                                    19

3.2.6    Indole test                                                                                                       19

3.2.7    Citrate utilization test                                                                                     19

3.2.8    Starch hydrolysis test                                                                                      19

3.2.9    Nitrate reduction test                                                                                      20

3.2.10  Sugar fermentation                                                                                         20

3.2.1        Methyl red test                                                                                                20

3.2.1 2 Voges-Proskauer test                                                                                      21

3.2.13  Urease test                                                                                                      21

3.2.14  Gelatinase test                                                                                                 21

3.2.15  storage of isolate                                                                                             22

3.3       Screening for Enzyme Activity                                                                      22

3.3.1    Screening for protease activity                                                                       22

3.3.2    Screening for pectinase activity                                                                      22

3.4       Protease Production                                                                                        22

3.5       Purification of Crude Protease Extract                                                           23

3.5.1.   Ammonium sulphate precipitation                                                                  23

3.5.2    Dialysis for protease                                                                                       23

3.5.3    Diethylaminoethyl (DEAE) column chromatography for protease                23

3.6       Protease Assay                                                                                                24

3.6.1    Characterization of protease enzyme                                                              24

3.7       Pectinase Production                                                                                      25

3.8       Purification of Pectinase                                                                                 25

3.8.1    Ammonium sulphate precipitation                                                                  25

3.8.2    Dialysis for pectinase                                                                                      25

3.8.3    Diethylaminoethyl (DEAE) column chromatography for pectinase               26

3.9       Pectinase Assay                                                                                              26

3.9.1    Characterization of pectinase                                                                          26

3.10     Scouring                                                                                                          27

3.10.1  Conventional scouring                                                                                    27

3.10.2  Bio-scouring                                                                                                    27

3.10.3  Optimization of different parameters for bio-scouring                                   27

3.10.4  Weight loss determination                                                                              28


CHAPTER 4: RESULTS AND DISCUSSION                                                    29

4.1       Results                                                                                                            29

4.1.1    Screening and identification of protease producing organisms                      29

4.1.2    Screening and identification of pectinase producing organisms                    29

4.1.3    Morphological and biochemical properties of isolate                                     29

4.1.4    Protease activity                                                                                              32    

4.1.5    Pectinase activity                                                                                            35

4.1.6    Optimization of production media                                                                 38

4.1.7    Bio-scouring                                                                                                    43

4.2       Discussion                                                                                                       49


5.1       Conclusion                                                                                                      51

5.2       Recommendations                                                                                          51

References                                                                                                      53

Appendices                                                                                                     53










4.1:      Morphological characteristics of isolate                                                          30

4.2:      Biochemical properties of isolate                                                                    31       



4.1:      Effect of temperature on protease activity                                                     33

4.2:      Effect pH on protease activity                                                                        34

4.3:      Effect of temperature on pectinase activity                                                    36

4.4:      Effect of pH on pectinase activity                                                                  37

4.5:      Protease activity for various carbon sources                                                   39

4.6:      Pectinase activity for various carbon sources                                     40

4.7:      Protease activity for nitrogen sources                                                             41

4.8:      Pectinase activity for nitrogen sources                                                           42

4.9:      Effect of alkali, pectinase and protease on cotton scouring                           45

4.10:    Effect of temperature on pectinase and protease bio-scouring of cotton       46

4.11:    Effect of time on protease and pectinase bio-scouring of cotton                   47

4.12:    Effect of pH on protease and pectinase bio-scouring of cotton                     48       





Enzymes are high molecular weight proteins which catalyse biochemical reactions. The use of microorganisms for the production of extracellular enzymes for industrial use is a preferable option to the  use of plants and animal sources. Enzymes such as lipase, pectinase, amylase and protease can be produced from bacteria and fungi using different fermentation techniques such as Solid State Fermentation and Submerged fermentation under optimized conditions (Machado et al., 2004). Proteases are enzymes which catalyse the hydrolysis of proteins and are of different kinds such as serine, alkaline, acidic, neutral and carboxyl proteases (Ahuja et al., 2004). They are found in all living organisms and are produced extracellularly by bacteria and fungi (Rao et al., 1998). Pectinase is a high molecular weight enzyme that catalyses the breakdown of pectin, a polysaccharide found in plant tissues into smaller molecules. Protease is of industrial importance as it is used in leather processing, food industries, textiles and pharmaceuticals while pectinase is utilized in the food industry and textiles. Bacillus subtilis is one of the widely used microorganisms for the industrial production of both pectinase and protease. They are Gram-positive, mesophilic organisms found commonly in soil and the underneath of vegetation. Culturing and growth of Bacillus subtilis is easy, making it a choice microbe for industrial use (Bandow, 2002). Waste products generated in markets usually consist of both plant and animal material.  Animal waste products from meat, poultry and fish can supply a large amount of protein rich material for bioconversion to recoverable products of importance in various industries (Dalev, 1994). These animal waste consists of portions of an animal that cannot be sold as meat or used in meat-products. Such waste includes tendons, bones, and the contents of the gastro-intestinal tract, blood and organs and these vary with each type of animal (Udandi et al., 2009). Remains of vegetables, cassava, yam and other plant crops make up plant related market waste. Research has been carried out on many other related sources for enzyme production such as slaughter house waste, dairy effluent and sewage waste (Vasantha and Subramanian, 2012). These wastes produced in market places are inexpensive sources for bioconversion processes into products such as enzymes.


Cotton is the most valuable natural source of cellulose and its use is constantly increasing. It is highly hydrophobic and slightly coloured (Boylston and Herbert, 1995). To prepare cotton fibres for further treatment and use, pre-treatment processes are needed and these include scouring and bleaching.  In these treatments large amounts of auxiliary agents are added. Due to the high working temperatures, a lot of energy is consumed. Large amounts of water are used to rinse and deactivate the alkaline-scoured and bleached fabrics. Conventional scouring of cotton entails the use of alkali (soda ash and caustic ash) and detergents for the removal of waxes, fats, pectin, mineral salts and other impurities from raw cotton fabrics (Mosjov, 2012). This process effectively removes impurities but has a high demand for energy, water and auxiliary agents. The effluent generated is also ecologically undesirable. These chemicals also attack cellulose leading to heavy strength loss and weight loss in the fabric. These setbacks has led to alternative processes such as bio-scouring being considered. Bio-scouring is a novel process based on the use of specific enzymes for the removal of impurities from textile materials (Mangesh and Tesfaye, 2016).


Consequently, the textile industry is considered to be one of the biggest water, energy, and chemical consumers. To comply with the increasingly more rigorous environmental regulations and to save water and energy, biotechnology based alternatives such as the application of enzymes have been introduced in the the textile sector. Bio-scouring with enzymes such as pectinase and protease is an alternative to alkali scouring in the removal of non-cellulose substances from the cotton-fibre surface.

The process has the capacity to occur at moderate temperatures in a slightly acidic or alkaline medium which is dependent on the type and nature of enzyme used. The use of enzymes in the scouring of textile materials such as cotton is gaining global recognition due to their non-toxic nature. Enzymes have been tested in every step of wet textile treatment starting from desizing to scouring. Microorganisms are the best sources of industrial enzymes as their production is economically viable with low resource consumption and low emissions (Adivarekar et al., 2007).  Enzymatic treatment of textiles is of great interest as it is effective under mild treatment conditions (Deng et al, 2010).




Conventional scouring of cotton fibre is essential for the removal of impurities such as wax and fats. However, it has a high energy demand and its effluent is non-biodegradable hence making it toxic ecologically. It also leads to reduction in strength and loss of fabric weight. Therefore, the use of enzymes such as protease and pectinase presents an ecologically responsive option as they are easily biodegradable. It also leaves the cellulose structure of the cotton fabric almost intact, preventing excessive weight and strength loss.




The main objective is to produce pectinase and protease and compare their effects on bio-scouring of cotton fibres.



1.3.1   Specific objectives

v  Isolation of Bacillus subtilis from  market solid waste.

v  To produce and purify protease enzyme.

v  To perform the assay of protease enzyme.

v  To characterize the protease enzyme produced.

v  To produce and purify pectinase enzyme.

v  To perform the assay of pectinase.

v  To characterize the pectinase enzyme produced.

v  To compare conventional scouring of raw cotton fabric and bio-scouring using enzymes.

v  To determine and compare the scouring potentials of pectinase and protease application on cotton.



Click “DOWNLOAD NOW” below to get the complete Projects


+(234) 0814 780 1594

Buyers has the right to create dispute within seven (7) days of purchase for 100% refund request when you experience issue with the file received. 

Dispute can only be created when you receive a corrupt file, a wrong file or irregularities in the table of contents and content of the file you received. shall either provide the appropriate file within 48hrs or send refund excluding your bank transaction charges. Term and Conditions are applied.

Buyers are expected to confirm that the material you are paying for is available on our website and you have selected the right material, you have also gone through the preliminary pages and it interests you before payment. DO NOT MAKE BANK PAYMENT IF YOUR TOPIC IS NOT ON THE WEBSITE.

In case of payment for a material not available on, the management of has the right to keep your money until you send a topic that is available on our website within 48 hours.

You cannot change topic after receiving material of the topic you ordered and paid for.

Ratings & Reviews


No Review Found.

To Review

To Comment