Colorado State University-Pueblo Isolation of cellulase enzymes from Penicillium spinulosum Higgins, Nicole R. April 24, 2017 Senior Honors Thesis Abstract Cellulases are enzymes produced by select microorganisms and fungi that possess the ability to degrade cellulose, a particularly stable biopolymer found in plant cell walls. These enzymes are important to the organisms that produce them because they provide a carbon source that is inaccessible to most other organisms. They are also of particular interest in industry due to their potential to convert cellulose to biofuels that may serve as an alternative for non-renewable petroleum based fuels. Therefore, the isolation of cellulases with traits that make the production of biofuels on an industrial scale economically sustainable is of particular interest. This study aims to isolate cellulase genes from the fungus Penicillium spinulosum. Ultimately, the goal is to obtain the most efficient cocktail of enzymes for biofuel production whose gene sequences can be inserted into a genetically engineered bacterial vector for easy use in industrial applications. Therefore, this study also aims to determine the optimal conditions for the isolation of cellulases at all steps using techniques such as gradient PCR. In addition, a brief bioinformatics analysis of the primers developed for isolation of cellulases from P. spinulosum was conducted using NCBI BLAST to determine if any similarity with other cellulases or fungal organisms existed. BLAST search results indicated that the target cellulases are relatively novel. Introduction The cell walls in plants are primarily composed of cellulose, a biopolymer containing β-1,4- glycosidic linkages1. Structurally, cellulose is a homo-polysaccharide that arranges into unbranched chains of glucose molecules called microfibrils1. These microfibrils are difficult to degrade due to the β- 1,4-glycosidic linkages between each glucose molecule as well as the presence of hydrogen bonding between the cellulose fibers2. As a result, enzymes specialized for hydrolyzing the β-1,4-glycosidic linkages in cellulose have developed in many bacterial and fungal species3. These enzymes, termed cellulases, are produced by select microorganisms and fungi and provide such organisms with a carbon source that is unattainable by the majority of organisms. The general term cellulase encapsulates three distinct groups of hydrolytic enzymes capable of degrading cellulose: endoglucanases, exoglucanases, and β-glucosidases (BGL)3. Cellulolytic enzymes are classified into one of these three groups based on the method by which they cleave β-1,4-linkages as well as their specificity for particular substrates3. The first class of cellulases, endoglucanases, acts randomly on the cellulose chain to produce smaller oligosaccharides3. Exoglucanases, on the other hand, act more specifically by cleaving off units of cellobiose from the ends of cellulose chains3. Cellobiose is the smallest cleavable unit of cellulose that can be converted to glucose3. BGL, also known as cellobiase, is then responsible for hydrolyzing cellobiose to glucose2,3. Ultimately, these three groups of enzymes work synergistically in order to hydrolyze cellulose to glucose for use in metabolic processes2,3. It is important to note that the final conversion of cellobiose to glucose monomers by BGL is frequently the limiting step of the process3. This is due to the fact that BGL exists in a smaller ratio compared to the other two classes of cellulases3. In addition, BGL is inhibited by both its end product, glucose, as well as its substrate cellobiose 3. Apart from their importance in the metabolic processes of many microorganisms, cellulases have a number of important industrial roles including their potential to convert cellulose and its derived sugars to renewable biofuels that may provide a viable substitute for non-renewable petroleum based fuels. Biofuels produced via this mechanism (shown in Figure 1) are commonly referred to as lignocellulosic ethanol, named after dry plant matter termed lignocellulose2. The use of lignocellulose in the production of biofuels is appealing for several reasons. First of all, a large portion of this raw plant material is inedible and therefore its use for biofuel production would not negatively impact food production4. In addition, biomass has a low economic cost and has been reported as one of the largest available energy resources in the world with India alone possessing an annual availability of about 500 million metric tons2,5. It has been suggested that in the United States alone, approximately 30% of the current petroleum usage could be offset by switching to biofuels2. Finally, the use of fuels derived from lignocellulose has the potential to substantially reduce many of the negative effects of climate change due to the fact that their combustion releases lower levels of greenhouse gases in comparison to fossil fuel combustion4. Figure 1: General schematic of the biofuel production process using cellulase enzymes. However, isolation and production of cellulase enzymes remains a costly process3–5. In addition, the enzymatic efficiency of many commercially available enzymes is still too slow to maintain biofuel production on an industrial scale3–5. Therefore, developing more efficient methods to isolate cellulases, as well as identifying cellulases with traits that eliminate economic constraints, have become widespread areas of research. Ultimately, a successful transition to the use of biofuels on a commercial level will depend on the development of a “cellulase cocktail” that completely and efficiently hydrolyzes the intended biomass substrate in a cost-effective manner2,3. As previously stated, cellulases are produced by a number of microorganisms including fungi and both aerobic and anaerobic bacteria, however fungi are the most studied of these microorganisms3. The reasons for this are twofold, the first being that they tend to have a higher enzyme yield2,3. Furthermore, fungi secrete cellulase enzymes extracellularly making them more accessible than the majority of their bacterial counterparts2,3. In particular filamentous fungi, including those from the genera Penicillium, tend to produce higher levels of cellulase enzymes3. More importantly, such fungi produce all three types of cellulases giving them the ability to completely hydrolyze cellulose polymers3. More specifically, the fungus P. spinulosum, is a fast growing, non-pathogenic fungal species, making it an attractive choice for the study of cellulolytic enzymes. Ultimately, this study aims to isolate the gene sequence for cellulase enzymes from the genome of P. spinulosum as well as determine the optimal conditions under which this isolation is most efficient. Optimal conditions for the isolation the enzymes will be carefully considered at all steps in order to obtain the most efficient cocktail of enzymes for biofuel production. Methods Standard Growth Media Preparation Two 500 mL batches of standard growth media were prepared, one with glucose and the other with sucrose as a carbon source. In both cases, the sugar solutions were prepared separately from the salt solutions. Sugar solutions were prepared by adding 28.84 g glucose and 28.82 g sucrose to their respective 500 mL flasks and dissolving in 250 mL of distilled, deionized water. Salt solutions were prepared together in a 1 L flask by combining the remaining macronutrients and micronutrients in a 500 mL of distilled, deionized water according to the concentrations listed in Table 1. All of the micronutrients, except iron sulfate, were in solution. Each micronutrient was pipetted into the solution of macronutrients and iron sulfate at a final volume of 250 μL/L. After preparation, the salt solution was divided into two 250 mL portions and placed in 1 L flasks. All of the salt and sugar solutions were autoclaved and allowed to cool. Upon cooling, the sugar solutions were each aseptically combined with a salt solution by adding the sugar solution to the salt solution in a biosafety cabinet (Fig. 2). Table 1: Standard Growth Media Preparation Nutrients Constituents Concentrations Macronutrients mM g/L Carbon Glucose/Sucrose 320 57.6 Nitrogen NH4Cl 80 4.3 Phosphorus Na2HPO4 20 2.8 Sulfur Na2SO4 2 0.28 Buffer/Chelator Sodium Citrate 12.5 3.0 Citric Acid 12.5 2.2 Potassium K2CO3 3 0.42 Magnesium MgCO3 4 0.34 Sodium NaCl 20 1.2 Micronutrients Element, ppm Salt, mg/L . Iron FeSO4 7H2O 10 50 Zinc ZnCl2 5 10.4 . Manganese MnCl2 4H2O 5 18.0 . Molybdenum (NH4)6Mo7O24 4H2O 2 3.7 . Calcium CaCl2 5H2O 0.5 1.8 . Copper CuSO4 5H2O 0.4 1.6 Vanadium NH4VO3 0.2 0.46 Boron H3BO3 0.1 0.57 . Chromium Cr2(SO4)3 12H2O 0.1 0.93 . Nickel NiCl2 6H2O 0.1 0.40 . Cobalt CoCl2 6H2O 0.1 0.40 Sucrose (28.82 g) in Glucose (28.84 g) in 250 mL H O in 500 mL 250 mL H2O in 500 mL 2 flask (calc. for 500 flask (calc. for 500 mL) mL) 500 mL Sucrose Std. 500 mL Glucose Std. Growth Growth Salts in 250 mL H O in Salts in 250 mL H2O in 2 1 L flask (calc. for 1 L) 1 L flask (calc. for 1 L) Figure 2: Schematic Diagram of Standard Growth Media for P. spinulosum cultures. Spore Suspension A spore suspension to be used for current and future inoculations was prepared by adding sterile water to three Penicillium spinulosum cultures in petri dishes and scraping them with a sterile glass spreader. The spores were resuspended in an aqueous 0.12% (w/v) Tween 20 solution with 0.9% (w/v) NaCl to increase the density of the solution and suspend the spores. The Tween 20 is hydrophobic and therefore coats the spores to prevent them from germinating. Media Inoculation Each liquid media preparation was inoculated with 0.20 mL of the Penicillium spinulosum from the spore suspension using a sterile micropipette. The inoculated media flasks were then placed in the shaker and allowed to grow for approximately 1 week. Fungal DNA Extraction In order to extract DNA from the prepared cultures, the MasterPure Yeast DNA Purification kit was used6.