White rot fungi will be grown on different lignocellulosic substrates in SSF to select the best strain, inducer substrate for the production of liP, MnP and laccase enzymes. The fermentation parameters including incubation time, moisture level, pH, temperature, inoculum size, additional carbon & nitrogen sources will be optimized through Response Surface Methodology (RSM) in a split plot experimental design. The effect of surfactants like tween-80 and sodium dodecyl-sulphate (SDS) will also be investigated. The enzymes produced on pilot scale (10L fermentor) will be purified through ion exchange, affinity and gel filtration chromatographic and characterized through kinetic studies.The effect of immobilization of enzymes (LiP, MnP, laccase) in hydrophobic gels on activity and stability of the enzymes will also be investigated. The native and chemically modified enzymes will also be compared in terms of kinetic, thermostability and thermodynamic characteristics to investigate their suitability for industrial applications
b. Problem statement:
Ligninolytic enzymes of white rot fungi have extensive applications in the pulp and paper industry, in biopulping and bibleaching of kraft lignin, for denim washing on commercial scale and bioremediation of industrial effluents. However, slow growth rate of WRF giving low enzyme yields in fermentation media and low activity and thermostabilty properties of enzymes limit their use in industrial processes. An aspect of great concern for industrial applications of ligninases for delignification of lignocellulosic biomass is their activity and thermostability. These lignolytic enzymes can be used after hyperactivation through enzyme engineering. The immobilization often causes activation and enhanced thermostability of enzymes. The immobilized enzymes also have the advantage of their reusability, convenient handling of enzyme preparations, controlled product formation and separation of enzymes in most of the cases examined Immobilization of LiP has been found to enhance its pH and temperature optima as well as thermostability and catalytic properties making it suitable for applications in paper and pulp industry and bioethanol production Immobilized enzymes can be more robust, stable, and capable of functioning in a wide range of conditions.
- Enhanced production of ligninase enzymes by selected WRF cultures through optimization of production process.
- Development of Immobilized of ligninases for their hyperactivation and and thermostabilization.
- Characterization of the chemically engineered LiP, MnP and laccase for industrial applications.
- Application of native and selected hyperactive and thermostable modified enzymes for delignification of lignocellulosic residues and decolorization of textile dyes.
1. Fungal Cultures
The newly isolated indigenous cultures of Pleurotus ostreatus IBL-02, Phanerochaete chrysosporium IBL-03, Coriolus versicolor IBL-04, Ganoderma lucidum IBL-05 and Schyzophyllum commune IBL-06 will be obtained from industrial Biotechnology Laboratory, University of Agriculture, Faisalabad. The fungi will be maintained on PDA slants at 40C in refrigerator and periodically activated by transferring on fresh slants.
2. Collection and Preparation of Lignocellulosic substrates for Fermentation
Abundantly available lignocellulosic substrates including wheat straw, rice straw, corncobs, corn stover, sugarcane bagasse, banana stalk and banana pseudostem will be used as substrates for solid state fermentation (SSF). The substrates will be sun dried, oven dried and ground to 40mm mesh, and stored in airtight plastic jars.
3. Screening of WRF Cultures on Lignocellulosic substrates
All the WRF cultures will be cultured on all substrates in SSF using Kirk,s basal nutrient medium (Tien and Kirk 1988) as moistening agent to select the best cultures on basis of maximum LiP, MnP and Laccase production. It will form the basis for use of selected cultures on selected substrates for the development of production process for individual enzymes.
4. Optimization of fermentation Process for Production of ligninases through studying the:
- Effect of pH and temperature on enzyme production
- Effect of Moisture content and inoculum size
- Effect of additional carbon and nitrogen sources
- Effect of surfactants like Tween-80, Tween-20, Triton X100 and SDS
- Effect of low molecular mass mediators like varatryl alcohol, manganese sulphate, ABTS, Oxalate and H2O2.
- Effect of inorganic and organic activators and inhibitors
5. Production of LiP. MnP and Laccase in 10 L bioreactor under optimum conditions
6. Purification of enzymes: Through standard purification techniques including ammonium sulphate
precipitation, gel filtration and Fast Protein Liquid Chromatography (FPLC).
7. Characterization of purified LiP, MnP and Laccase: Through kinetic studies by studying the effect of follwing on enzyme activity:
i. Effect of pH ii. Effect of temperature
iii. Effect of substrate concentration: Determination of Vmax and KM values
iv. Molecular mass determination:Through native &SDS-Polyacrylamide gel electrophoresis (SDS-PAGE).
8. Immobilization: Purified enzymes will be immobilized in gels of different hydrophobicities
9. Investigation of the effect of immobilization: Effect of immobilization on activities and thermostabilities of LiP, MnP and Laccase will be monitored through kinetic studies