Professional Documents
Culture Documents
Johnry S. Maloles
MCB 260 Advanced Industrial Microbiology November 28, 2012
Although enzymes are formed within living cells, they can continue to function in vitro
PLANTS
Papain Ficin
ANIMALS
MICROBES
Amylase Protease Cellulase Xylanase
Rennet
ENZYME
PRODUCTION
Microbial Fermentations
Extracellular enzymes
Plant Latex
Intracellular enzymes
liquids
WASTE
Purification
Concentration Finishing
PRODUCT
Source: Aehle, 2008 Enzymes in Industry
Extremozymes Enzymes that function at high temperatures or extreme environmental (cold, very high salt, very acid or very alkaline pH) - Used as biocatalysts - e.g. Taq polymerase, thermostable proteases, amylases,and cellulases
Immobilized Enzyme In industrial processes, it is desirable to attach enzymes to a solid surface Isolated enzymes can be immobilised so that they do not contaminate the end product and can be used again and again
Source: Madigan et al, 2011 Biology of Microorganisms
Fungi
Produced various individual enzymes vary in amounts Commercial quantities can be obtained by isolating microbial strains
Bacteria
>Saccharomyces cerevisiae, Aspergillus sp., and Bacillus sp.
(http://etc.usf.edu/clipart/15400/15447/grwyeastcell_15447.htm) (http://www.bioquicknews.com/archive/story/2009?page=6)
PROPERTIES
Fungi
Bacteria
>Saccharomyces cerevisiae, Aspergillus sp., and Bacillus sp.
(http://etc.usf.edu/clipart/15400/15447/grwyeastcell_15447.htm) (http://www.bioquicknews.com/archive/story/2009?page=6)
1. Capable of producing the enzyme of interest 2. Grows fast and produces enzyme in large scale production 3. Compatible with substrates 4. Easily manipulated genetically 5. Genetically stable 6. Safe
NO
NO
YES
Downstream processing cost minimized? Purity stable? YES
success NO
Improve downstream processing lower cost and waste, higher purity success NO
NO
Induced
The rate of mutation can be increased by various factors and agents called mutagens. ionizing radiations X-rays, gamma rays non-ionizing radiations ultraviolet radiations various chemicals mustard gas, benzene, ethidium bromide, Nitrosoguanidine-NTG
Site-Directed
Change in the base sequence of DNA
(http://users.wmin.ac.uk/~redwayk/lectures/sdm.htm)
- efficient way to induce heterokaryon formation and recombination with high frequency
- the organism selected for fusion should be genetically related - Types: Intraspecific hybridization Interspecific hybtidization Intergeneric hybridization
Other Types
- Specific Goals:
(1) to get multiple copies of specific gene
Gene encoding for alkaline protease is amplified by PCR - template DNA - F primer (5- CATATGTTTGGGTACTCTATGG-3) - R primer (5-GGATCCTTATTGGCCGGGAACGGAA-3) - taq polymerase, dNTPs - optimal thermocycling profile
Product is analyzed using gel electrophoresis and digestion with restriction enzymes
FERMENTATION PROCESS
Most enzyme production is carried out in deep submerged fermentation; a few are best produced in semi-solid media.
FERMENTATION PROCESS
(http://www.anilbioplus.com/infrastructure/manufacturing.htm)
FERMENTATION PROCESS
Most industrial enzymes are products of batch processes; few are currently produced via continuous fermentation Fermenters for bulk enzyme production are up to 100m3 capacity, but fine enzymes may be produced on smaller scales of a few hundred litres or less The medium must be chosen to stimulate the microbe into synthesizing the correct enzyme What type of medium would you use to stimulate a microbe to synthesize a specific enzyme?
FERMENTATION PROCESS
Factors to consider prior to fermentation:
1) use of GRAS-listed (generally regarded as safe) organisms - incubation period 2) knowledge of the properties of the enzyme of interest - optimum pH and heat resistance
FERMENTATION PROCESS
Semi-solid Medium
aka Koji or moldy bran method of solid state fermn
medium consists of moist sterile wheat/rice bran acidified with HCl; mineral salts including trace minerals are added; inducer is usually added organism used are fungi amenable to high enzyme production condition
FERMENTATION PROCESS
Submerged Production
generally greater ease of controlling temperature, pH and other environmental factors in a fermentor
medium must also contain all the requirements for growth (C, N, various metals, trace elements)
*medium adequate for growth may not be satisfactory for enzyme production
inducers are also added that act as substrates rennet prodn soy bean proteins are added to induce protease prodn by fungi
FERMENTATION PROCESS
Submerged Production
inducer is not always the substrate for enzyme production but sometimes, the end product is the substrate (cellobiose stimulate cellulose prodn) medium components may repress enzyme production by catabolite repression
*glucose -amylase in B. licheniformis and B. subtilis *fructose enzymes in B. stearothermophilus *isoleucine and proline protease in B. megaterium *sulfur-containing aa protease in Aspergillus niger
temperature, pH, and oxygen requirements should be optimized (most mcgs used are aerobic)
FERMENTATION PROCESS
Production of Protease
proteases of all species of Bacillus are produced in a submerged culture fermentation system carbon source: starch, ground barley, or lactose ...nitrogen source: soybean meal or casein 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] and free amino acids ... inducers: peptides and proteins
FERMENTATION PROCESS
Production of Protease
Materials - culture medium casamino acids (variable), glucose (variable), 1 g/L KH2PO4, 3 g/L K2HPO4, 2 g/L Na2SO4 and 0.1 g/L MgSO47H2O - microorganism - orbital shaker - cooling centrifuge - agroindustrial residues (substrate) - salt solution - incubation chamber in Aspergillus oryzae - 4 proteases are formed in submerged fermentation - 2 proteases are formed in semi-solid medium and are less heat resistant
PRODUCT RECOVERY
- Whether enzyme is intracellular or extracellular - How pure the final product needs to be
PRODUCT RECOVERY
Generally account more than 50% of total enzyme production costs Dependent on degree of purity
PRODUCT RECOVERY
Stabilizers may be added: calcium salts proteins sugars starch hydrolysates Destabilizing metals are removed using EDTA Antimicrobials (optional) benzoates sorbate
Source: Waites et al, 2009 Industrial Microbiology
PRODUCT RECOVERY
Intracellular Enzyme
cells are needed to be disrupted mechanically or enzymatically
MECHANICAL
ball mills use of small abrasive particles ultrasonic disruption use of high sound frequency blenders blades rotate at speeds of 6,000 - 50,000 rpm freeze fracturing water crystals as abrasives
NON-MECHANICAL
chemical permeabilization (e.g., organic solvents, surfactants) enzymatic permeabilization (e.g., glycanases, proteases) osmotic shock (e.g., high sucrose medium)
PRODUCT RECOVERY
Intracellular and Extracellular Enzyme
separation of cellular debris from medium
A. Centrifugation
tubular bowl scroll
http://www.sgconsulting.co.za/flottweg-sedicanter.php
multichamber disc-stack
http://www.jvcgyroscreen.com/tab_cent.html
http://www.sciencedirect.com/scienc e/article/pii/S0015188205705875
PRODUCT RECOVERY
Intracellular and Extracellular Enzyme
separation of cellular debris from medium
B. Microporous Membrane Filtration
microfiltration (0.1 to 1 m) suspended solids, cells ultrafiltration (0.01 to 0.1 m) protein fractionation nanofiltration (0.001 to 0.01 m) low-molecular weight organics, color removal, desalination reverse osmosis (< 0.001m) demineralization
(http://www.winesecrets.com/wine-ultra-filtration/wine-ultra-filtration.asp)
PRODUCT RECOVERY
Further Purification by CHROMATOGRAPHY impurities (e.g., proteins, DNA and others) further purification when safety (e.g., recombinant DNA, viruses) or functional reasons (impurities disturbing catalytic function) basic knowledge of protein property necesary molecular weight (MW) isoelectric point (pI) cofactors pH range greater than 10% of enzyme is lost due to multiple-step manipulation
PRODUCT RECOVERY
Further Purification by CHROMATOGRAPHY
Enzyme Properties Size-exclusion chromatography Anion-exchange chromatography Cation-exchange chromatography Hydrophobic chromatography Metal-Affinity chromatography Bifunctional/Multifunctional chromatography Biospecific chromatography Molecular weight Surface charge (at pH > pI) Surface charge (at pH < pI) Hydrophobicity of enzyme surface Histidines at the surface or His-tags Surface charge and hydrophobicity
Specific hydrophobic and electrostatic binding interaction with inhibitors, antibody or biospecific tags
PRODUCT RECOVERY
Final treatment of Enzymes enzymes distributed as solutions, powders or immobilized Solution preservation and stabilization agents sterilization by filtration (e.g., therapeutical enzymes) Powder freeze-dried (small particles can be inhaled) spray-dried and coating/cover with protecting layer (particle size can not be inhaled)
Quality Control:
PRODUCT RECOVERY
Recovery and Purification of Protease
first step is separation of cell biomass and insoluble nutrient ingredients from the supernatant purification is by precipitation and chromatographic procedures recovery of protease: A. Ammonium Sulfate Fractionation - protein fractions are precipitated by successively increasing the concentration of salt (sulfate/sulfite) - proteins tend to aggregate and precipitate out of the solution B. Gel Filtration Chromatography (1) Purity Quality - separates protease using proper filtration matrix (2) Activity Control: (3) Identity of enzyme produced
PRODUCT RECOVERY
Recovery and Purification of Protease
Materials - 1% NaCl - 80% (NH4)2SO4 - 0.2M citrate phosphate buffer, pH 6.5 - magnetic stir plate and stir bar - centrifuge - dialysis tubing - chromatography equipment - appropriate gel matrix Quality check: measurement of protease activity - a colorimetric assay using azocasein (chemically modified (1) Purity protein) as a substrate can be used to determined the Quality (2) protease activity inActivity crude and purified samples
Control:
APPLICATIONS
Detergents Starch processing and related carbohydrases Cheese and plant juice production Textile and leather manufacturing Treatment of wood pulps Catalysts in organic synthesis
APPLICATIONS
Detergents Lower Temperature & No Phosphate Clothes Washing
- introduction of proteases that are active at alkaline pH - a well-known example is subtilisin, a bacterial alkaline serine protease from Bacillus licheniformis and B. subtilis, which is used extensively in laundry detergents - enzyme has now been engineered to improve pH and temperature characteristics, and reduce sensitivity to peroxide - protease (degrades protein stains) cutinase (degrades mixutre of fatty acids) amylases and lipases
APPLICATIONS
Starch processing and related carbohydrases Sugar syrups from Starch
- manufacturing of sugar syrups containing specific mixtures of sugars that could not be produced by conventional acid hydrolysis of starch
- -amylase generates glucose, maltose, and maltotriose industrially employed in brewing and baking thermostable types from Bacillus species - amyloglucosidase breaks down starch and dextrin into glucose from Aspergillus niger and Rhizopus species - glucose isomerase converts glucose into fructose (sweetener) from Bacillus and Streptomyces species - invertase converts sucrose into glucose and fructose (syrup and confectionery) from A. niger and A. oryzae
APPLICATIONS
Dairy Applications Cheeses
- rennet from animals were replaced with proteases from Rhizomucor miehei and R. pusillus (vegetarian cheese)
- calf chymosin gene has been introduced to several microorganisms like E. coli and Aspergillus species
Cheese Flavors
- microbial lipases for the hydrolysis of fatty acid esters to accelerate flavour development (wide variety of high quality cheeses)
APPLICATIONS
Textile Manufacturing
- removing the adhesive components (may be composed of starch, starch derivatives, vegetable gum, and water-soluble cellulose derivatives) before dyeing, bleaching, and printing
- use of amylases and cellulases as they are non-corrosive and produce no harmful effluent wastes
- cellulases are used for biopolishing cotton and other cellulose fibers *for smoother and glossier appearance - cellulases for stonewashing *accelerates abrasion and aid the loosening of the indigo dye
APPLICATIONS
Plant Juice Production Leather Processing Treatment of Wood Pulps
APPLICATIONS
(Waites, 2001)
(http://www.anilbioplus.com/aboutus/industrial-enzymes.htm)
THANK YOU
References:
Aehle W. 2008. In Enzymes in Industry.Hoboken, NJ: Wiley-VCH. Barredo, J. 2005. Microbial enzymes and biotransformations. Totowa, N.J.: Humana Press. Lehninger. The Foundations of Biochemistry Madigan M, J Martinko, D Stahl and D Clark. 2011. Brock: Biology of Microorganisms. 13th edition. San Francisco, CA. Benjamin Cummings Okafor, Nduka. 2007. Modern Industrial Microbiology And Biotechnology. Enfield, (NH): Science Publishers. Polaina, J and MacCabe, AP. 2007. Industrial enzymes: Structure, function and applications. Dordrecht: Springer. Sadeghi HM, Rabbani M, Naghitorabi M. 2009. Cloning of alkaline protease gene from Bacillus subtilis 168. Research in Pharmaceutical Sciences: Vol 4(1): p. 43-46. Vermelho AB, Supuran CT, and Guisan JM. 2012. Microbial Enzyme: Applications in Industry and in Bioremediation, Enzyme Research, vol. 2012, Article ID 980681, 2 pages, 2012. doi:10.1155/2012/980681 Waites MJ, Morgan NL, Rockey JS, and Higton G. 2009. Industrial Microbiology: An Introduction. In Industrial Microbiology pp19). Hoboken, NJ: Wiley-Blackwell.
http://www.iogen.ca/company/enzymes_technology/index.html http://www.bioquicknews.com/archive/story/2009?page=6 http://etc.usf.edu/clipart/15400/15447/grwyeastcell_15447.htm http://www.accessscience.com/search.aspx?rootID=792645 http://users.wmin.ac.uk/~redwayk/lectures/sdm.htm http://www.anilbioplus.com/infrastructure/manufacturing.htm http://www.sgconsulting.co.za/flottweg-sedicanter.php http://www.jvcgyroscreen.com/tab_cent.html http://www.winesecrets.com/wine-ultra-filtration/wine-ultra-filtration.asp http://www.sciencedirect.com/science/article/pii/S0015188205705875 http://loschmidt.chemi.muni.cz/peg/lecture/biocat_lecture08.pdf