MCB 260: MICROBIAL ENZYMES Prepared by: Johnry Maloles

2nd Sem: AY 2012 - 2013

Enzyme Production: Isolation/Development of Organism Fermentation Process Enzyme Recovery Purification of Enzyme Product Enzymes - protein molecules that are being produced by living organisms that act as catalysts, increasing the rate of different biochemical reactions - their activity is very specific (stereospecific), they are fully biodegradable, and works under mild conditions (pH and temperature) - activity is retained even enzymes are outside the living cells (functions in vitro) - can be obtained from plants, animals, and microbes - usually needs a cofactor or a coenzyme for the enzyme to be active Endoenzymes (Intracellular) - group of enzymes that are produced in small amounts inside the cell that are responsible for catalysing different metabolic pathways required for proper cell metabolism Exoenzyme (Extracellular) - enzymes that are produced inside the cell in large amounts that are being excreted into the environment - has a role on the digestion of insoluble polymers such as cellulose, proteins, lipids, and starch - Advantages/Importance in Commercial Production: 1) already outside the cell making it easier for isolation; 2) limited number of types of secreted enzyme, so easier to purify; and, 3) more robust compared to intracellular enzymes. Extremoenzyme - enzymes that are being produced by extremophiles that can function under extreme environmental conditions such as very high or low pH, temperature, salinity or other factors - cannot be easily denatured by typical enzymes making these enzymes of interest to a variety of biotechnical applications - examples: Taq polymerase, Pfu polymerase, thermostable proteases/amylases/cellulases Immobilized Enzymes - enzymes attached to an insoluble solid surface such as calcium alginate that provides increase resistance to changes in conditions such as pH or temperature - Reasons for Immobilizing Enzymes: 1) Convenient miniscule amounts of protein dissolved after each reaction, so workup is much easier 2) Economical immobilized enzymes can be easily removed in a reaction making it easy to recycle the biocatalyst 3) Stable have greater thermal and operational stability than the soluble forms of enzymes; denaturation process is retarded - Techniques for Enzyme Immobilization: 1) Carrier-bounding enzyme bonded to a carrier by adsorption, ionic or covalent bonding 2) Entrapment enclosing enzymes in microcapsules, gels, or fibrous polymers 3) Cross-linking use of cross-linking agents such as glutaraldehyde Page | 1

A. STRAIN SELECTION AND DEVELOPMENT Properties of Desirable Microorganism: 1) capable of producing the enzyme of interest; 2) grows fast and produces enzyme in large scale process (cost effective); 3) compatible with substrates; 4) can be easily manipulated genetically; 5) genetically stable; and, 6) safe *Microorganisms can be isolated from the environment by means of biodiscovery and screening processes, and can also be obtained from different existing culture collections. *Bacillus sp. (bacteria), Saccharomyces sp. (yeast), and Aspergillus sp. (fungi) are the microorganisms that are mostly used for the production of microbial enzymes, which are generally regarded as safe (GRAS) organisms.

Development of Microbial Strains for Enzyme Production - involves microbiology, biochemistry, and genetics - usually done when yield in wild type organism is low - also done when desired enzyme is produced by a nonGRAS-listed microorganism Strategies for Strain Development: 1) Mutation any change in the base sequence of the DNA (deletion, insertion, inversion, substitution) a) Spontaneous occurs spontaneously at a very low frequency that can be caused by tautomerism, depurination, deamination, or slipped strand mispairing b) Induced mutation is brought and hastened using various mutagenic agents c) Site-Directed creating specific point mutations with specific change in the base sequence of the desired mutants Approaches for Site-Directed Mutagenesis: - Cassette Mutagenesis - PCR Site-Directed Mutagenesis - Whole Plasmid Mutagenesis - In vivo Site-Directed Mutagenesis 2) Recombination combining of genes or characters from two parental strains of microorganisms a) Transformation acquisition of DNA molecule of a bacterium from the environment b) Transduction DNA is transferred between bacteria via bacteriophages or viral vectors c) Conjugation transfer of DNA segment from one bacterium to another bacterium d) Protoplast Fusion two protoplasts are fused to form hybrid cells that can grow into mature hybrid organisms 3) rDNA Technology construction of recombinant DNA molecule from two or more different DNA molecules

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B. FERMENTATION PROCESS Factors to consider prior to fermentation: 1) Use of GRAS-listed organisms 2) Knowledge of the properties of the enzyme to be produced (optimum pH and temperature) 3) Source of inexpensive carbon and nitrogen feedstock 4) Knowledge of enzyme stability for downstream processing

What type of medium to use to stimulate a microbe to synthesize a specific enzyme?

*Most industrial enzymes are products of batch culture fermentation processes because enzymes are usually produced at the end phase of exponential phase of the growth curve until the early stationary phase. *Most enzyme production are carried out in deep submerged fermentation; only a few are best produced in semi-solid media *Fine and expensive enzymes may be produced on small scale fermentation processes (<100 L)

Semi-solid Medium VS Submerged Fermentation Semi-solid medium - also known as Koji or moldy bran method of solid state fermentation - medium consist of sterile wheat or rice bran acidified with HCl; mineral salts including trace minerals are added - inducer is usually added (e.g. 10% starch for amylase, gelatin and pectin for protein and pectinase production, respectively - fungi are the used organisms, which appear amenable to high enzyme production because of the low moisture condition and high degree of aeration of the semi-soluble medium - production period is usually 30-40 hrs, but could be as long as seven days Submerged Production - method for most of industrially produced enzymes - less labor intensive and cheaper compared to semi-solid fermentation - controlling of temperature, pH and other environmental factors can be done easier - medium must contain all the requirements for growth, including adequate sources of carbon, nitrogen, various metals, trace elements, growth substances, etc. - production of commercial enzyme lasts from one to seven days Submerged fermentation is really much more advantageous compared to semi-solid medium fermentation because of the higher yield output, but it requires constant aeration all through out. Page | 3

C. PRODUCT RECOVERY - generally accounts for 50% 60% of total enzyme production cost - involves separation, purification, stabilization, and preservation Things to consider: 1) Is the enzyme produced intracellular or extracellular? 2) How pure the final product needs to be? *In order to limit contamination and degradation of enzyme, medium should be cooled to about 20 C as soon as the fermentation is over. *Stabilizers can be added such as calcium salts, proteins, sugar, and starch hydrolysates. Destabilizing metals may be removed using EDTA. *Anti-microbials can also be used like benzoates and sorbates. *Medicinal enzymes must be extremely pure. The purer the enzyme, the more complex the downstream processing, and the more expensive it is. *Sometimes enzymes may be precipitated using a variety of chemicals such as methanol, and be further purified by dialysis or chromatography before being dried in a drum drier. Intracellular enzymes need to be freed from cells before the extraction process can be done Mechanical cell disruption: use of ball mills, ultrasonic sound, blenders, and freeze fracturing Non-Mechanical cell disruption: chemical and enzymatic permeabilization, and osmotic shock Cellular debris must then be separated from the medium that contains the enzymes produced Centrifugation use of tubular bowl centrifuge / scroll centrifuge / multi-chamber disc-stack Microporous Membrane Filtration can be microfiltration, ultrafiltration, or nanofiltration Enzymes can be further purified by Chromatograpy - impurities can be proteins, DNA or other cellular debris - possible if the properties of the enzyme is known such as molecular weight, isoelectric point, cofactors required, and optimum pH and temperature range - greater than 10% of the total enzyme is lost due to multiple-step manipulation Final Treatments: enzymes can be in solution form or powder form - enzymes are packaged preferably in liquid form, but when solids/powders are used, the enzyme is usually mixed with a filler, Quality check: Toxicity Testing and Standardization - should be tested by animal feeding to show that it is not toxic - potency of enzyme preparation, based on tests carried out with the substrate should be determined - shelf life and conditions of storage for optimal activity should also be determined Page | 4

D. APPLICATIONS Microbial Enzymes

Proteases

Source
Aspergillus, Bacillus, Rhizomucor, and Kluyveromyces spp.

Applications
- biological detergents - baking (dough modification) - beer brewing - leather baiting and tendering - cheese manufacture - biological detergents - leather processing fat removal - flavour production in dairy

Lipases

Bacillus, Aspergillus, Rhizopus, and Rhodotorula spp.

Carbohydrases -Amylase

Aspergillus and Bacillus spp.

- starch processing liquefaction - baking partial starch degradation - brewing during wort preparation - textile manufacturing desizing - maltose syrup production - brewing - increasing wort fermentability - whey syrup production hydrolysis of lactose - milk and dairy processing lactose reduction - prevention of sandy texture in ice-cream - manufacturing of high-fructose syrups hydrolysis of glucose to lactose - sweets and confectionery production liquefaction of sucrose - invert sugar production sucrose conversion to glucose and fructose - plant juice and oil extraction - fruit juice and wine clarification - coffee bean fermentation - malting speeds modification of grains - textile processing biopolishing of cellulose fibres - wood pulp processing - animal feedstuffs - neutraceutics - wood pulp processing - bleaching textiles - cheese manufacture - addition to feeds for monogastric animals in Order to release phosphate from phytic acid - wine production - ceramics manufacturing to enhance the manufacture and quality of modern ceramics which are of importance for the computer and aeronautics industries or for orthopaedics - for antibiotic conversion for making the base molecules for the synthesis of semisynthetic penicillins and cephalosporins - resolution of L-amino acids from acylated racemic mixtures of D,L-amino acid mixtures

-Amylase

Bacillus, Streptomyces, and Rhizopus spp.

-galactosidase

Bacillus, Kluyveromyces, and Candida spp.

Glucose Isomerase

Actinoplanes, Arthrobacter, and Streptomyces spp. Saccharomyces and Kluyveromyces spp.

Invertase

Pectinases

Aspergillus niger and Penicillium spp.

-Glucanases

Trichoderma reesei, Penicillium funiculosum, Aureobasidium pullulans, and Bacillus spp.

Hemicellulases

Cryptococcus and Trichosporon spp.

Catalases Phytases Ureases

Aspergillus niger, Corynebacterium glutamicum, and Micrococcus lysodeikticus Recombinants of Pichia spp. Lactobacillus fermentum

Penicillin and cephalosporin acylases L-amino acid acylases

Bacillus megaterium, Escherichia coli, and Pseudomonas spp. Aspergillus spp.

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COMMERCIAL PRODUCTION OF THE PROTEASE ENZYME Proteases - proteolytic enzymes that accounts for 60% of the total worldwide sale of enzymes - play a critical role in many physiological and pathological processes giving them extensive applications in food industry, laundry detergents, leather treatment, bioremediation processes, and the pharmaceutical industry - they also play a major role in nutrition due to their depolymerising activity - can be an exopeptidase or endopepdidase depending on its mode of action Isolation of Proteolytic Microorganisms - can be obtained from protein-rich soil samples - screening of pure cultures for proteolytic activity using nutrient agar medium containing 1% casein zone of clearing around the growth indicates positive result Development of Microorganism (Sadeghi et al., 2009) - cloning of gene coding for alkaline protease - Steps for the cloning of the gene: 1) Genomic DNA of Bacillus subtilis with gene coding for alkaline protease is extracted 2) Gene encoding for alkaline protease is amplified by PCR 3) Product is analyzed using gel electrophoresis and digestion with restriction enzymes 4) Amplified gene is inserted to a cloning vector 5) E. coli competent cells are prepared and transformation is executed 6) Screening of transformed colonies on LB agar-ampicillin culture medium Fermentation Process - proteases of all species of Bacillus are produced via submerged culture fermentation system - carbohydrate is added continuously in small amounts to keep concentration low - usually supplemented with 10-15% dry substance and high protein content - repressors: high carbohydrate concentration and free amino acids inducers: peptides and proteins - in Aspergillus oryzae, 4 kinds of proteases can be formed in submerged fermentation, and 2 kinds of proteases in semi-solid medium Recovery and Purification of Protease - first step is separation of cell biomass and insoluble nutrients from the supernatant - purification is by precipitation or chromatographic procedures - recovery of protease can be done by: a) Ammonium Sulphate Fractionation salting out method b) Gel Filtration Chromatography use of proper filtration matrix Quality Check - a colorimetric assay using azocasein (chemically modified protein) as a substrate can be used to determine the protease activity in crude and purified samples

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