Screening For Ligninolytic Enzymes In Pleurotus HK 37 (Research Proposal Sample)

223431616637000
SCREENING FOR LIGNINOLYTIC ENZYME PRODUCTION
BY PLEUROTUS HK 37 IN SOLID MEDIA
BY
Motatiro Chacha Emmanuel
Reg. No. I411/1305/2013
A project proposal, submitted to the School of Pure and Applied Sciences in partial fulfilment of the requirements for the degree of Bachelor of Science in Biochemistry and Molecular Biology of South Eastern Kenya University.
April, 2017
DECLARATION
I hereby declare that this submission is my own work towards the bachelor’s degree and that, to the best of my knowledge, it contains no materials previously published by another person or material which has been accepted for the award of any other degree except where due acknowledgement has been made.
Motatiro Chacha Emmanuel …………………………… ………………………………
I411/1305/2013 Signature Date
Certified by:
Dr. Mutemi Muthangya ……………………………… …………………………………
(Supervisor) Signature Date
Certified by:
Dr. Kamau ………………………………. ……………………………..
(Chairman of Department Signature Date
ABSTRACT
The mushroom pleurotus HK 37 has a nutritional and medicinal characteristics which relies on the growth substrate. Pleurotus HK 37 is a type of fungi which can grow naturally on dead wood, although it can be artificially cultivated on agricultural wastes such as coffee husks, eucalyptus sawdust, corncorbs and sugarcane bargasse. Degradation of this wastes requires some enzyme complexes which are made up of oxidative laccase, manganese peroxidase lignin peroxidase and hydrolytic enzymes such as cellulases. The operation of this enzymes helps us in understanding ways of improving the productivity of pleurotus HK 37 mushroom cultures and decreases the potential pollution that can be caused by inadequate discharge of agro industrial residues. This work will be essential in assessing the activity of ligninolytic enzymes produced by pleurotus HK 37.
ACKNOWLEDGEMENTS
I express my profound gratitude to the entire SEKU administration and fraternity for allowing me be part of them since I joined them in the year 2014). The conducive environment I received from the institution made me work well and complete my proposal.
I am grateful to the department of Biochemistry and molecular biology for the role the department played in facilitating my proposal. Their constructive criticism acted as a source of inspiration to me.
I appreciate Dr. Kasili for his efforts in taking me through research project unit. He equipped me with much knowledge which made me see a light behind the shadows.
I do owe much thanks to my mentor Dr. Mutemi Muthangya for his supervisory role, patience, humility, hardworking spirit, suggestions and motivation that served as a source of encouragement to me. I am also grateful to him for the great impact he has played in my life, by helping me convert my idea into reality. His good judgments helped shaped this research. May God shower upon him peace, success and above all, strength and endeavors.
I recognize my course mates who shared a continous and mutual support in bringing this work into reality. Thank you all.
DEDICATION
This work is dedicated to my coursemates.
Contents TOC \o "1-3" \h \z \u DECLARATION PAGEREF _Toc479842531 \h iiABSTRACT PAGEREF _Toc479842532 \h iiiACKNOWLEDGEMENTS PAGEREF _Toc479842533 \h ivDEDICATION PAGEREF _Toc479842534 \h vCHAPTER 1 PAGEREF _Toc479842535 \h 11.0 INTRODUCTION PAGEREF _Toc479842536 \h 11.1 PROBLEM STATEMENT PAGEREF _Toc479842537 \h 11.2 RESEARCH JUSTIFICATION PAGEREF _Toc479842538 \h 21.3GOAL AND PURPOSE PAGEREF _Toc479842539 \h 21.4SPECIFIC RESEARCH OBJECTIVES PAGEREF _Toc479842540 \h 21.5 HYPOTHESIS PAGEREF _Toc479842541 \h 2CHAPTER 2 PAGEREF _Toc479842542 \h 32.0 LITERATURE REVIEW PAGEREF _Toc479842543 \h 32.1 On mushrooms PAGEREF _Toc479842544 \h 32.2 Lignocellulose PAGEREF _Toc479842545 \h 42.3 Ligninolytic enzymes PAGEREF _Toc479842546 \h 53.0 MATERIALS AND METHODS PAGEREF _Toc479842547 \h 83.1 pleurotus HK 37 collection and identification. PAGEREF _Toc479842548 \h 83.2 Tissue culture PAGEREF _Toc479842549 \h 83.3 Screening of the isolates for lignolytic activities PAGEREF _Toc479842550 \h 83.4 Enzyme activity assays PAGEREF _Toc479842551 \h 9CHAPTER 4 PAGEREF _Toc479842552 \h 114.0WORK PLAN PAGEREF _Toc479842553 \h 11CHAPTER 5 PAGEREF _Toc479842554 \h 125.0 REFERENCES PAGEREF _Toc479842555 \h 12CHAPTER 6 PAGEREF _Toc479842556 \h 196.0 BUDGET PAGEREF _Toc479842557 \h 19
CHAPTER 1
1.0 INTRODUCTION
Pleurotus HK 37 produces a wide range of extracellular lignolytic enzymes capable of degrading lignin and other woody components (Hatakka, 1994). This species of mushroom contains lignolytic system which is extracellular and nonspecific hence an ability to degrade lignocellulosic wastes. This extracellular multienzymatic system is made up of lignin peroxidase, laccases and manganese dependent peroxidases (Field et al.,1993;Barr and Aust, 1994) expressed in a variety of forms, being the existence of manganese peroxidase and laccase higher than Lignin peroxidase (Wesenberg et al.,2003). The lignolytic machinery in pleurotus HK 37 is highly regulated by nutrients with nitrogen, copper and manganese which are known to have a strong regulatory effect (Bonnarme and Jeffries 1990; Bonnarme et al.,1991).
1.1 PROBLEM STATEMENT
Lignolytic enzymes from pleurotus HK 37 are useful in a variety of biotechnological fields and environmental fields such as effluent detoxification and production of important industrial products such as bioethanol. However the enzymes in pleurotus are difficult to produce and hence this marks as the greatest limitation (Merwe, 2012) to its large scale application. In natural environment, Pleurotus HK 37 and other mushroom species grows on substrate different from those used in many laboratories in Kenya and different parts of the world hence resulting to low lignolytic activity in liquid cultures (Zhao et al.,1996).Hence high lignolytic activity can be achieved through improvement of culture media through which they can exhibit themselves clearly, this will be done in a way which mimics the natural environment under which pleurotus species does well, hence my selection of the solid media. Properties of ligninolytic enzymes have been reported elsewhere in the world, where it seems to vary from species to species with more than one isoenzymes being expressed within different species under different cultures (Baldrian, 2005).
1.2 RESEARCH JUSTIFICATION
Currently, there is a problem with treatment of waste and industrial effluents, to an extent that many industries dump their wastes in rivers, a state which seems to threaten marine life among other lives on earth. Given that lignolytic enzymes can help solve this problem of waste treatment, it is important for efforts to be made in the vast production of this enzymes so that they can replace the cruel means of waste disposal. Above that, many people from different parts of the world sees mushroom as a one-purpose fungi, apart from being edible, it should be known that mushroom has a variety of enzymes which can not only treat waste, but can also be used in production of products such as bioethanol (Avit and Mberek, 1998: Envirocare, 2000).
1.3GOAL AND PURPOSE
The overall goal of this study is screening for ligninolytic enzyme production by Pleurotus HK 37 in solid media.
1.4SPECIFIC RESEARCH OBJECTIVES
1 To screen for the presence of ligninolytic enzymes in Pleurotus HK 37 in solid media.
2.To optimize culture conditions for pleurotus HK 37 in solid media for the enhanced production of ligninolytic enzymes.
1.5 HYPOTHESIS
Ligninolytic enzymes are available in pleurotus HK 37 and can be used in degradation of industrial waste.
CHAPTER 2
2.0 LITERATURE REVIEW
2.1 On mushrooms: They are special classes of fungi used in the scientific classification. Mushrooms do not belong in a class of both animals and plants but since they perform all the life processes, they are still considered as organisms. The word “mushroom” has many meanings and different explanations in different spheres of the world. Many researchers have tried to explain it but still many definitions still comes out. According to Ganopedia (2011), it is described as a fungus which is in possession of a stem, a cap and gills or pores on the underside of the cap. (Cho and Kang2004) described mushroom as a macro fungus having distinctive fruiting body which can be either epigeous (growth is close to the ground) or hypogenous (growth is underground). The life cycle of a mushroom begins from a spore which under favorable conditions germinates to a mass of branched hyphae of mycelia which colonize a substrate (Dike et al. 2011). Mushrooms goes through two phases of the reproductive phase and the vegetative phase. When the hypha fully colonizes its substrate the vegetative phase is terminated while the reproductive phase begins after development of primordial by the hyphae (Dike et al. 2011). There are three groups of mushrooms, this includes; edible mushrooms, toxic mushrooms and medicinal mushrooms (Ganopedia, 2011). Edible mushrooms such as oyster mushrooms are mushrooms...