PRODUCTION, AND ACTIVITY ASSAY OF KERATINASE ENZYME PRODUCED BY STREPTOMYCES ISOLATED FROM KARU YAM FARM SOIL

PRODUCTION, AND ACTIVITY ASSAY OF KERATINASE ENZYME PRODUCED BY STREPTOMYCES ISOLATED FROM KARU YAM FARM SOIL.



TABLE OF CONTENTS


TITLE PAGE i

DEDICATION ii

CERTIFICATION iii

ACKNOWLEDGEMENT iv

TABLE OF CONTENTS v-vii

LIST OF FIGURES viii

ABSTRACT ix

CHAPTER ONE 1-5

1.0 INTRODUCTION AND LITERATURE REVIEW 1

1.1 INTRODUCTION 1

1.2 AIM AND OBJECTIVES 2

1.2.1 Aim 2

1.2.2 Objectives 2

1.2.3 Justification 2-3

1.3 LITERATURE REVIEW 3-5

1.3.1 Keratinase 3

1.3.2 Sources of Keratinase 3-4

1.3.3 Streptomyces 4-5

CHAPTER TWO 6-8

2.0 MATERIALS AND METHOD 6

2.1 MATERIALS 6

2.1.1 Chemical and reagents 6

2.1.2 Apparatus/equipment 6

2.2 METHODS 6

2.2.1 Collection and preparation of sample 6-8

2.2.2 Preparation of potato dextrose agar 6-7

2.2.3. Isolation of Streptomyces 7

2.2.4. Sub-culture 7

2.2.5. Enzyme production 7-8

2.2.6. Enzyme assay 8

CHAPTER THREE 9-10

3.0 RESULTS 9-10

3.1 Isolation and identification of Streptomyces and production of Keratinase 9-10

3.2 Keratinase Activity Assay 10

CHAPTER FOUR 11-12

4.0 DISCUSSION, CONCLUSION AND RECOMMENDATION 11-12

4.1 DISCUSSION 11

4.2 CONCLUSION 11-12

4.3 RECOMMENDATION 12

REFERENCES 13-19


 

LIST OF FIGURES 


Figure 1A. PDA Isolation Plates showing colonies of Streptomyces………………….……....9

Figure 1B. Subculture plate showing pure isolates of Streptomyces.…………………..……..9

Figure 2A. Submerged fermentation culture for enzyme production………………..…….…10

Figure 2B. Cell-free supernatant.…………………...……………………………………..…10


 



ABSTRACT

The study was conducted to select the best promising keratinolytic bacterial strain. Microbial keratinases have become biotechnologically important since they target the hydrolysis of highly rigid, strongly cross-linked structural polypeptide “keratin” recalcitrant to the commonly known proteolytic enzymes. Soil samples collected from a yam farm in Karu LGA, Abuja was enriched for keratinase producers on Whole feather agar containing whole feathers as a sole Carbon and Nitrogen source. Among 8 bacterial isolates, 4 isolates showed keratinase activity. The best keratinase producing bacterium was selected and identified as Streptomyces strain, based on morphological, cultural, biochemical characteristics and 16S rRNA sequence analysis. The isolate exhibited maximum keratinase production in a optimized feather meal medium containing feather meal powder (5), NH4NO3 (1), NaCl (2), NaH2PO4 (2.6), FeSo4 (1.0), MgSo4·7H2O (0.5) with pH 7.5, and incubated at 37°C on rotary shaker (250 rpm) overnight. The optimum enzyme activity was observed to be (0.665U/ml). Therefore, Streptomyces might be used for large scale production of keratinase for industrial purposes in less time. 

 

CHAPTER ONE

1.0 INTRODUCTION AND LITERATURE REVIEW 

1.1 INTRODUCTION

Keratins are insoluble fibrous proteins that make up the external protective surfaces in vertebrates and are the structural components of wool, hoof, horns, hair, nail and feathers (Sharma and Gupta, 2016; Gunes et al.,2018). Keratins are known for their complex degradation and high stability caused by the firm stabilization of their polypeptide chains, tightly packed with hydrogen bonds and hydrophobic interactions (Sahni et al., 2015). The disulfide bonds crosslinking the chains contribute to their stability and tolerance to the degrading effect of the usual proteases such as pepsin, trypsin and papain (Brandelli et al., 2010; Vidmar and Vodovnik, 2018).

Keratin is an insoluble fibrous protein macromolecule with very high stability and low degradation rate. On the basis of secondary structural conformation, keratins have been grouped into α (α-helix of hair and wool) and β (β-sheets of feather) (Voet and Voet, 1995; Akhtar and Edwards, 1997). Keratins are also classified as Type I (acidic) keratin and Type II (basic) keratin and have molecular weights ranging from 30 KDa to 70 KDa (Steinert, 1993; Selvam and Vishnupriya, 2012). There are approximately 30 different types of keratins which are generally grouped into epithelial keratins (in epithelia cells) and trichocytic keratins which make up hair, nails, horns and reptilian scale. Keratins are also classified as soft keratins and hard keratins (Zhou et al., 1988; Chou et al., 2015). The most distinctive feature of keratin is, they are mechanically robust and chemically unreactive (high resistance to proteolytic degradation) due to presence of higher degree of cross-linking by disulphide bridges, salt bridges, hydrogen bonds and hydrophobic interactions (Bockel et al.,1995; Balaji et al.,2008; Jones et al.,1997).


1.2 AIM AND OBJECTIVES

1.2.1 Aim

The aim of this research was to conduct the production and activity assay of keratinase by streptomyces isolated from soil sample collected from Abuja.

1.2.2Objectives

The specific objectives are:

To isolate Streptomyces from soil.

To produce the enzyme keratinase from the isolated Streptomyces.

To assay the activity of keratinase on chicken feather substrate.



1.2.3 Justification

Poultry feathers contain more than 90% of crude protein in keratin form, found as wastes or by products at poultry processing plants (Howie et al., 1996). Increasing quantities of feathers could have effect to the environmental pollution (Rajput and Gupta, 2013). The crude protein content in feather wastes could have a great potential nutrient value and may have some advantage as a protein sources for substitute from more expensive dietary ingredients for animal feed such as poultry and ruminant animal (Xie et al., 2010). Worldwide, commercial poultry processing generates 5 millions of tons of feathers per year, which are currently converted to feather meal through steam pressure and chemical treatment (Freeman et al., 2009). Including in Nigeria, poultry industries are growing faster comparing to the other livestock industry due to the high demand of poultry meat as cheap and high quality protein sources for human consumption. Therefore, there is need to produce keratinase by streptomyces isolated from the soil sample collected in Nigeria.


1.3 LITERATURE REVIEW

1.3.1 Keratinase

Keratinase is a protease that can specifically degrade keratin. Keratinases belong to group of proteolytic enzymes which have ability to hydrolyze insoluble protein keratin more efficiently than other proteases (Onifade et al., 1998). Due to the strength and stability of keratin, very few microorganisms are able to degrade keratin and utilize it as carbon, nitrogen and sulphur source (Marchisio, 2000). Total degradation of keratinous material by specialized microorganisms can generate a chain of events involving breaking of disulfide bridges (sulfitolysis) and proteolysis (Błyskal, 2009; Ramnani et al., 2005). It is due to the cooperative action of keratinolytic protease and disulfide reductase enzyme which may be produced by same or different microorganisms (Gupta et al., 2015). Microbial keratinases are mostly extracellular enzymes which are inducible in nature but some are membrane linked (cell bound) and intracellular. Keratinases are by and large serine or metalloproteases which are capable of degrading the structural keratinous protein (Gupta and Ramnani, 2006). Keratinase are also classified into two major groups “endopeptidases and exoproteases” based on their site of cleavage action. These enzymes are produced by bacteria, actinomycetes and fungi which can hydrolyze a large number of keratin substrates (Saibabu et al., 2013; Tork et al., 2010).

1.3.2 Sources of Keratinase

Keratinase is an inducible enzyme that is synthesized only when an inducer (keratin) appears in the environment. Keratinase can be produced by many kinds of microorganisms, and has broad application prospects in the fields of feed, leather, medicine, food and other industrial, as well as environmental governance. As early as the beginning of the 19th century, people discovered that some organisms could degrade keratin, and the separation of such strains has been ongoing since then. At present, there are more than 30 kinds of microorganisms that can degrade keratin, including bacteria, actinomycetes and fungi, such as Dermatophyte and Cnadina albicans in fungi, Streptomyces in actinomycetes, Bacillus licheniformis and Bacillus subtilis in bacteria, etc. The keratinase produced by different microorganisms has a certain difference, mainly in terms of the existence of the enzyme, as well as the structure, composition, stability, optimum reaction temperature, and pH value of the enzyme. For example, some forms of keratinase exist mainly in the cell, and some of them are mainly secreted to the outside. The keratinase of the fungus is found both intracellularly and extracellularly.

1.3.3 Streptomyces

Streptomyces is the largest genus of Actinobacteria and the type genus of the family Streptomycetaceae. Over 500 species of Streptomyces bacteria have been described (Kämpfer and Peter, 2006). As with the other Actinobacteria, Streptomycetes are gram-positive, and have genomes with high GC content (Euzéby JP, 2008). Found predominantly in soil and decaying vegetation, most streptomycetes produce spores, and are noted for their distinct "earthy" odor that results from production of a volatile metabolite, geosmin. (Madigan and Martinko, 2005).

Streptomycetes are characterised by a complex secondary metabolism. They produce over two-thirds of the clinically useful antibiotics of natural origin (e.g., neomycin, cypemycin, grisemycin, bottromycins and chloramphenicol) (Madigan and Martinko, 2005). The now uncommonly used streptomycin takes its name directly from Streptomyces. Streptomycetes are infrequent pathogens, though infections in humans, such as mycetoma, can be caused by S. somaliensis and S. sudanensis, and in plants can be caused by S. caviscabies, S. acidiscabies, S. turgidiscabies and S. scabies (Kieser et al., 2000). In general Streptomycetaceae family can be distinguished by physiological and morphological characteristics, chemical composition of cell walls, type of peptidoglycan, phospholipids, fatty acids chains, percentage of GC content , 16 SrRNA analysis and DNA-DNA hybridization (Korn-Wendisch & Kutzner, 1992). In terms of number and variety of identified species, Streptomyces represents one of the largest taxonomic items of recognized Actinomycetes (Bhattacharyya, pal & Sen, 1998). They are distinguished as gram-positive bacteria, aerobic, non-Acid- Fast and with a high GC content more than 70% (Dehnad, Parsa Yeganeh, Bakhshi & Mokhtarzadeh, 2010). Streptomycetes can grow in different environments (Maleki, Dehnad, Hanifian & Khani, 2013). They produce layer of aerial hyphae that can differentiate into a chain spores (Korn-Wendisch & Kutzner, 1992).

Scientific classification (Waksman & Henrici, 1943):

Kingdom: Baceria

Phylum: Actinobacteria

Class: Actinomycetes

Order: Actinomycetales

Family: Actinomycetaceae

Genus: Streptomyces

 

CHAPTER TWO

2.0 MATERIALS AND METHOD


2.1 MATERIALS

2.1.1 Chemical and reagents

Soil sample, Mgso4.7H2O, Phosphate buffer, distilled water, NaH2Pp4, Nacl, NH4So4, Potato dextrose Agar (PDA), Chicken feather powder, 75% ethanol.

2.1.2 Apparatus/equipment

Water bath (DH – 426), beakers (Pyrex, England), conical flask (Pyrex, England), measuring cylinder (Pyrex, England), test tubes (Pyrex, England), micropipette (Perfect instrument, London), dry air oven (Haraeus, thermo electron corporation), petri dishes, centrifuge machine (Beckman couler, U.S.A), shaking machine, spectrophotometer (Spectrum lab 23A, Gulfex Medical and scientific, England), autoclave, weighing balance (Satorius, TE 2145, Germany), test tube rack, hot plate (Barnstead international, HPA 1910M, Kerper Boule vard, U.S.A), pH meter (Metrohm 800), spatula, incubator (Boekel scientific CCC 5 – od).


2.2 METHODS

2.2.1 Collection and preparation of sample

The soil sample was collected from a yam farm in Karu LGA, Abuja. The soil sample was transported to the laboratory in a sterile polythene bag. 

2.2.2 Preparation of potato dextrose agar

Thirty-nine gram (39g) of potato dextrose was dissolved into 1000 mls of distilled water. The solution was heated to ensure subsequent dissolution of agar on a hot plate and it was They are characterized by their tough, leathery, frequently pigmented colonies and their filamentous growth, when first discovered, these organisms were thought to be fungi, but closer examination revealed a lack of a nuclear membrane and the presence of peptidoglycan, demonstrating their prokaryotic autoclaved at a temperature of 121°C for 30 minute and the media was allowed to cool for 45°C and dissolved into sterilized petri dishes. And was finally allowed to solidify.

2.2.3. Isolation of Streptomyces

The soil sample collected was pre-treated to eliminate the commonly found microbes using physico-chemical methods. Soil was dried overnight in foil trays under sterile cheesecloth. The soil was placed in a drying oven at 60°C for 90 minutes. The soil was allowed to cool. One gram of soil sample was suspended in 9ml of normal saline and swirled vigorously and allowed to stand for 30 minute, thereafter soil sample was serially diluted up to 10-4. An  aliquots of 100 μL of each dilution was inoculated by spread plate method onto the isolation media plates using aseptic technique. The plates were incubated at 37 °C for 72 hours.

2.2.4. Sub-culture

Streptomyces colonies showing distinct morphological characteristics were selected from the isolation plates and further sub-cultured to obtain pure culture. About 50ml of the prepared agar was poured into a 3 fresh petri dishes aseptically and allowed to solidify. The plates were divided into four portions with a marker, one colony each was picked from the isolation plate using a sterile toothpick and streaked on each portion and incubated at 28°C for 24 hours.

2.2.5. Enzyme production

The keratinase enzyme production was carried out in the basal medium containing in g/L: feather meal powder (5), NH4NO3 (1), NaCl (2), NaH2PO4 (2.6), FeSo4 (1.0), MgSo4·7H2O (0.5) with pH 7.5. The media was autoclaved at 121°C for 30 minutes and allowed to cool. A 50ml of the production media was transferred into a 100 ml conical flask and using a sterile tooth pick the pure isolate from the sub-culture plate was collected and agitated into the production media. The flask was placed on a rotary shaker overnight with shaking at 250 rpm. The next day, the fermented broth was centrifuged at 5000 rpm for 20 minutes at 40°C. The cell free supernatant was collected and used for keratinase activity assay.

2.2.6. Enzyme assay

The enzyme activity was determined by keratin digestion method using 1% keratin in 0.05 M phosphate buffer (pH 8.0) as substrate according to. The reaction mixtures contained 3.5 ml chicken feather meal substrate solution and 0.5 ml crude enzyme solution. The reaction was incubated at 40 °C in water bath for 10 min and reaction was terminated by heating in water bath at 100°C for 30 minutes. The control also was made by incubating the enzyme solution with phosphate buffer without addition of chicken feather. The mixture was then centrifuged at 3825 rpm for 30 min and absorbance was measured at 320 nm by spectrophotometer against the control. a rotary shaker overnight with shaking at 250 rpm. 

 

CHAPTER THREE


3.0 RESULTS

3.1 Isolation and identification of Streptomyces and production of Keratinase

In the process of isolation and identification, colonies with different morphology such as large white colonies with fluffy spores, small white sporulation powdery colonies producing light brown pigment which was viewed from the base of the plate, yellow colonies pigment diffused through the media and large white colonies with greyish centre and fluffy spores were observed on PDA isolation agar plates (Fig.1A). Pure isolates of Streptomyces were obtained by subculture (Fig. 1B).

A B

         

Fig. 1. (A) PDA isolation plate showing colonies of Streptomyces with different morphological characteristics. (B) Subculture plate showing pure isolates of Streptomyces.


The keratinase enzyme was produced from submerged fermentation (Fig 2A). To obtain a cell-free enzyme the fermented broth was centrifuged and the supernatant was used as keratinase solution (Fig. 2B). Fig. 2. (A) Submerged fermentation culture for enzyme production. (B) Cell-free supernatant as enzyme solution.

    A B

      


3.2 Keratinase Activity Assay

The feather-hydrolysing activity of this enzyme at pH 8 was found to be 0.665 U/ml. Alkaline pH possibly favours keratin degradation as higher pH modifies cystine residues to lathionine, making it more susceptible to keratinase attack (Friedrich and Antranikian 1996; Gupta and Ramnani 2006). These results suggest that keratinase is an efficient keratinolytic protease for β-keratin (chicken feathers) under high-temperature and alkaline conditions.

 

CHAPTER FOUR

4.0 DISCUSSION, CONCLUSION AND RECOMMENDATION 

4.1 DISCUSSION

In the present study, bacteria were isolated from collected soil samples and screened for keratinase producing capability on the basis of clear zone formation. The higher clear zone forming isolate on both media was considered as better keratinase producer. The bacterial isolate was gram positive and confirmed as Streptomyces. In most cases, keratin degradation is executed by gram positive bacteria (Gupta and Ramnani, 2006). It was also found in previous study that keratinases are produced by coccus Arthrobacter sp. (Pereira et al.2014). Arthrobacter creatinolyticus KP015744 has been reported as better keratinase producer (Kate and Pethe2014). Thus Streptomyces appears to be a potential candidate for keratinase production. Microbial keratinase is an inducible enzyme (Malviya et al.1992). The isolated Streptomyces produced maximum keratinase when keratinous protein elements were present in the media. Similar result was found for Arthrobactercreatinolyticus KP015744 that gave highest keratinase production in presence 1% feather powder (Kate and Pethe, 2014). (Kainoor and Naik, 2010) also achieved the maximum keratinase production in presence of 1% feather meal with Bacillus sp. JB99. The production of keratinase depends on the presence of keratin and its concentration. Enzyme production might be declined in the presence of higher concentration of feather meal indicating catabolic suppression (Saibabu et al.,2013).

4.2 CONCLUSION

In conclusion, this study shows that the properties of this enzyme increase the probability in industrial application at high temperature. Streptomyces might be a good candidate for keratinase production. 


4.3 RECOMMENDATION

Further studies should be carried out to purify, optimize and fully characterize the enzyme and to determine the sequence of this keratinase gene for future improvement for industrial application through genetic engineering approaches.

 

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