ABSTRACT
Enzymes are considered environmentally friendly (green) chemicals that have the potential to completely replace or reduce the use of hazardous chemicals in industrial processes, thereby promising sustainable production and manufacturing. Proteases derived from microbes have almost all of the properties required for biotechnological applications. This study was conducted with the aim of screening for potent protease-producing bacteria from Bacillus subtilis, determining optimal production conditions and partially characterizing the stability of the protease with regards to some physicochemical parameters. Hence, identification of Bacillus subtilis at a molecular level and purification as well as detailed characterization of the types of the proteases are recommended for effective utilization in different area of applications as the purification of the extract from the study yielded 71.3%-fold with high specific activity of 2787.1U/g. Based on optimization of physicochemical parameters and nutritional parameters for the optimum production of protease, the optimum temperature of protease production was 40°C with protease concentration of 32.9 ±1.60U/g. pH 9 was the optimum for production of protease with protease concentration of (14.1±1.40U/g and 22.7±2.1U/g) for both phosphate and carbonate buffers respectively. Among the metallic ions, media containing Mn2+ performed better than Cu2+, Mg2+, Fe2+, and Zn2+ with a protease concentration of 24.6±2.45U/g and 23.3±1.31U/g at 5mM and 1mM respectively. Studies on the effect of concentration of divalent ions revealed that both the activity and stability of protease was better in 5mM than in 1mM concentration. Furthermore, evaluation of some agro-industrial wastes as potential substrates for protease production indicated that wheat bran was better for carbon agro-residues (3.9±0.30U/g), whereas soybean meal was better for nitrogen agro-residues (4.0±0.23U/g). Since protease was produced from readily available complex substrates and agro-residues, Bacillus subtilis appear to have substantial potential for application in various proteolytic processes. Also, from the study, it was observed that immobilized alkaline protease from Bacillus subtilis was most compatible with the laboratory-based detergent Tween 20 and consequently showed highest protease activity of 6.3±0.62U/g, and also the enzyme showed highest compatibility with the commercial detergent omo and ariel and consequently showed highest protease activity of 5.8±0.55U/g and 5.0±0.10U/g respectively. Finally, in this study, stain removal was very efficient and haemolysis of blood occurred within a minute of enzyme action on the blood stain. An addition of purified alkaline protease from Bacillus subtilis effectively removed the stain. It could therefore be used as an additive in laundry detergents.
TABLE
OF CONTENTS
Page
Title
Page i
Declaration
ii
Dedication iii
Certification iv
Acknowledgments
v
Table
of Contents vi
List
of Tables x
List
of Figures xi
Abstract
xii
CHAPTER 1: INTRODUCTION 1
1.1
Background of the Study 1
1.2
Statement of the Problem 2
1.3
Justification of the Study 3
1.4
Aim and Objectives of the Study 5
CHAPTER
2: LITERATURE REVIEW 6
2.1 An Overview of Enzymes 6
2.2 Protease Enzymes 7
2.3 Sources of Protease 9
2.3.1 Animal proteases 9
2.3.2 Plant proteases 10
2.3.3 Microbial proteases 10
2.4 Classification of Microbial Proteases 13
2.4.1 Glutamic acid proteases 15
2.4.
2 Serine proteases 15
2.4.3 Cysteine proteases 15
2.4.4. Aspartase proteases 16
2.4.5 Threonine proteases 16
2.4.6 Metalloproteases 16
2.5 Bacillus
Subtilis 17
2.6 Utilization of African Oil Bean (Ugba)
as Substrate for
Enzyme Production 19
2.7 Protease Production 19
2.8 Process Optimization 21
2.8.1 Optimization of protease parameters 21
2.8.2 Optimization of physical parameters 22
2.9 Purification
and Characterization of Protease 23
2.10 Commercial Application of Protease Enzymes 24
2.10.1 Detergent additives 26
2.10.2 Proteases in leather industry 28
2.10.3
Processing of keratin wastes 29
2.10.4 Fibrinolytic proteases as thrombolytic agents 30
2.10.5 Proteases for biofilm removal 31
2.10.6 Proteases for silk degumming 33
2.10.7
Proteases in photographic industry 35
2.10.8 Proteases in food industry 37
2.10.9 Proteases for contact lens cleansing 38
2.10.10 Proteases for bio polishing of wool 38
CHAPTER
3: MATERIALS AND METHODS 40
3.1 Materials 40
3.1.1 Collection of materials 40
3.1.
2 Instrumentation and apparatus 40
3.1.3 Chemicals and reagents 40
3.2 Methods 41
3.2.1 Sterilization 41
3.2.2 Media preparation 41
3.2.3 Sample preparation and inoculation 41
3.2.4 Purification and storage of cultures 42
3.2.5 Identification and characterization of
proteolytic bacteria 42
3.2.6 Screening for alkaline protease enzyme from
bacterial isolates 48
3.2.7 Molecular characterization and
identification of isolates 49
3.2.8 Quantitative estimation of protease 51
3.2.9 Enzyme purification 51
3.2.10 Ninhydrin test to detect hydrolysis of casein
by enzyme
alkaline protease 53
3.2.11 Estimation of alkaline protease activity 54
3.2.12 Quantitative protein assay 56
3.2.13 Enzyme specific activity 58
3.2.14 Optimization of growth and physical parameters
for
enzyme production 58
3.2.15 Enzyme immobilization 60
3.2.16 Detergent compatibility and alkaline protease activity
using immobilized enzyme 60
3.2.17 Efficacy of alkaline protease enzyme in stain
removal 61
3.2.18 Statistical analysis 61
CHAPTER 4: RESULTS AND
DISCUSSION 63
4.1 Results 63
4.1.1 Morphological and biochemical properties of
isolates 63
4.1.2 Screening and identification of protease
producing organisms 65
4.1.3 Genotypic characterization of isolate 68
4.1.4 Crude enzyme activity and purification 71
4.1.5 Optimization of physiological and
nutritional parameters
on protease production 73
4.1.6 Compatibility of alkaline protease and
detergents with their
protease activity 83
4.1.7 Efficacy of enzyme on biological stain
removal 85
4.2 Discussion 87
CHAPTER
5: CONCLUSION AND RECOMMENDATIONS 92
5.1 Conclusion 92
5.2 Recommendations 92
References 93
Appendices 104
LIST
OF TABLES
Page
2.1. Microorganisms having protease activity 12
2.2. Protease general classification with
enzyme code and specific
mechanism of action for each sub group 14
4.1. Phenotypic characterization of isolates 64
4.2. Purification steps of protease enzyme from
Bacillus subtilis 72
4.3. Protease
production at different pH values 76
4.4. Effect
of divalent metal ions on protease production 78
LIST
OF FIGURES
Page
2.1 A representation of general industrial
application of protease enzyme 25
2.2 Steps involved in biofilm formation 32
2.3 Structure of silk fiber and degumming of
silk. 34
2.4 Gelatin hydrolysis for release of silver
for x-ray film 36
3.1 Tyrosine standard curve 57
4.1 Representation of clearance zone for
gelatin hydrolysis of the isolate 66
4.2 Representation of the zone of casein
hydrolysis shown by test organism 67
4.3 Phylogenetic tree showing the evolutionary
distance of the
bacterial
isolates 69
4.4 Agarose
gel electrophoresis of the 16SrRNA gene selected
bacterial
isolates 70
4.5 Temperature effect on protease production 74
4.6 Effect of carbon agro-residues on
protease production 80
4.7 Effect of nitrogen agro-residues on
protease production 82
4.8 Detergent and alkaline protease
compatibility and their protease activity 84
4.9 Wash test and efficacy of alkaline
protease on stain removal 86
CHAPTER
1
INTRODUCTION
1.1 BACKGROUND
OF THE STUDY
Advances in industries, agriculture, and
biotechnological domains have recently driven the quest for microorganisms with
innovative features that can be used to boost scientific and industrial output.
Due to a number of factors, such as substrate selectivity and high catalytic
activity, metabolic products such as enzymes generated by these microorganisms
have a significant advantage over conventional chemical catalysts (Rai and
Mukherjee, 2011). Proteases, lipases, and amylases, among other enzymes
produced by these microbes, are widely used in medical, molecular,
environmental biotechnology, and agricultural applications (Reddy et al., 2008).
Microbial proteases, which have a wide range of
applications in bio-industries such as food, pharmaceuticals, textiles,
photography, leather, and detergents, currently dominate the global enzyme
industry (Singh and Bajaj, 2017). However, for successful industrial use,
proteases must be robust enough to withstand the adverse process conditions.
Proteases used in biotechnological processes must be resilient and capable of
kinetic and structural adjustments in harsh industrial microenvironments, such
as high temperatures, low pH, and the presence of inhibitors (Singh et al., 2014).
Bacillus
spp. proteases have a specific significance among microbial proteases since
they are renowned for their ability to create strong enzymes that may be
suitable for industrial process conditions. Enzyme-based solutions promise
effective raw material use, minimum or no waste formation, and avoid the use of
harmful chemicals (Singh and Bajaj, 2017b). Enzymes are the basic molecules
that govern a variety of metabolic processes in living systems, and they are
essential for life to exist.
Proteases are one of the most important classes of
industrial enzymes that have received a lot of attention in recent years (Sarouk
et al., 2012). Proteases (EC 3.4) are
a type of enzyme found in all living things that perform a wide range of
complex physiological and metabolic tasks (Theron and Divol, 2014). These
proteases could come from microbial sources, particularly extremophiles
(microbes that live in severe biological niches) (Ali et al., 2016).
Bacillus
spp. strains are used to make industrial enzymes and have grown in importance,
accounting for 35% of total microbial enzyme sales (Jayakumar et al., 2012). This is due to Bacillus spp capacity to create enzymes
that are well suited to industrial process settings (Rehman et al., 2017). Bacillus spp. enzymes are considered poly-extremotolerance, meaning
they can function in a wide range of process conditions, including extremes in
temperature, pH, the presence of solvents, detergents, and other potential
enzyme inhibitors (Joshi and
Satyanarayana, 2013).
For biotechnological operations involving protein
production, numerous species of Bacillus,
viz. Bacillus subtilis, Bacillus
amyloliquefaciens and Bacillus
licheniformis and others, have become the most popular due to their
superior fermentation capabilities, high product yields and the complete lack
of harmful by-products (Guleria et al.,
2016).
1.2 STATEMENT OF THE PROBLEM
Enzymes are obtained
commercially from any biological source, including animals, plants, and
microorganisms. These naturally occurring enzymes are frequently not easily
available in sufficient numbers for commercial application, but the number of
proteins generated via recombinant methods is rising at an exponential rate. The
cost of manufacture, purification, yield, and stability to particular
environmental conditions, which has notably influenced high quantity yield of
proteases, are the key impediments to proteolytic application for industrial
enzymes. In particular, there is an urgent need for the development of
microbial strains from various sources capable of producing more protease
enzymes at a more thermostable and moderate pH activity in industrial
production. Given the wide range of applications for proteases, discovering
incipient sources for protease production has piqued the interest of numerous
scientific communities. Identifying substrates and optimizing their nutritional
content for the generation of more proteolytic enzyme-producing microorganisms
with a broad spectrum of activity has thus been a major issue in the
manufacturing industry. The choice of substrate is a critical aspect in the
production process. The most crucial factor to consider is that the substrate
should be inexpensive and stimulating in nature, allowing the enzyme production
to increase and the cost to drop. Solid state fermentation (SSF) is preferred
over submerged fermentation because it offers various benefits, including
highly concentrated products, the use of low-cost substrates, semi-sterile
conditions, simplified downstream processing, and less pollution. The effect of
agro-residue particle size and moisture content (humidity) on alkaline protease
synthesis is significant. As a result, there has been a need to broaden the
search for microorganisms with the potential and needed characteristics for
protease synthesis.
1.3 JUSTIFICATION OF THE STUDY
The use of protease
enzyme in the manufacturing industry has skyrocketed in recent years. On
account of their technological and economic contributions to many industrial
production domains, these enzymes are currently considered important. These
advantages and contributions can be realized by maximizing diverse substrates
for production, such as low-cost agricultural residues, which are considered
waste products. Ugba is a readily available substrate and a successful
usage of enzyme from ugba fermentation isolates will create a new
channel for ugba's utility value to be boosted in this regard. The cost
of production in relation to the value of the end product is the most important
issue when isolating enzymes on an industrial scale for commercial use.
Alkaline proteases are commonly used in commercial applications as crude
preparations. Nonetheless, alkaline protease purification is critical for gaining
a better knowledge of the enzyme's action. Environmentally friendly approaches
to the synthesis of fine chemicals have gotten a lot of attention in recent
years, therefore the usage of agricultural waste for the production of various
enzymes has skyrocketed. Various industries have concentrated on using
agro-residues as the only source of carbon and nitrogen in large-scale
fermentation medium. In terms of medium ingredients, these leftovers are
readily available in big quantities at low cost and with minimal capital
expenditure. Enzyme production is based on the waste to energy principle,
thus, the use of agro-waste for the manufacture of commercially essential
compounds does not disrupt the food chain and reduces reliance on non-renewable
resources. Screening methods
are being used to quickly discover enzymes with potential industrial
applications. Protease
enzymes are intended to fill the gap between chemical and biological processes
as a result of their peculiar features, and they have been widely used in
industrial operations. The ability to recognize and optimize the spatial and
temporal variation of microorganisms expressing different enzymes, as well as
the factors militating their activities, is critical. This has necessitated the
use of Bacillus subtilis as potential
microorganism in production of protease despite the fact that it is pathogenic
nature yet it has found its potential use in industrial production.
1.4 AIM AND OBJECTIVES OF THE STUDY
The aim of the
research is to screen, characterize and optimize protease enzyme isolated from Bacillus subtilis. Specific objectives
of the study include;
i.
Isolation, screening and identification of proteolytic Bacillus subtilis from traditionally
fermented ugba for synthesis of protease enzyme.
ii.
Purification and characterization of alkaline protease.
iii.
Optimization of physicochemical and nutritional parameters for maximum
production of protease.
iv.
Detergent compatibility and efficiency of alkaline protease in
protenious stain removal: an empirical study.
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