Industrial Biotechnology Flashcards
Define sustainability
Creating and maintaining the conditions under which humans and nature can exist harmoniously that permits the fullfilling of social, economic and other requirements of the present and the future.
Rationalise the argument of oil vs. bio-feedstock
- thinking about what the supply chain of the future will look like - will it be bio or chemical based
- important to consider competition of land for food vs. land for chemical feedstocks and fuels
- most likely both i.e a mixed oil/bio economy
- biotechnology requires new industrial manufacturing methods and
biocatalyts - use of food based feedstock for e.g. bioplastics increases the pressure on food prices but release pressure on oil prices
What are some current sustainability concerns?
- mounting waste
- scarcity of quality water
- difficulty associated with extracting oil and coal reserves
- diminishing metal / material resources
What are some examples of industrial biotechnology all products from everyday life?
- insulin
- biological washing powders
- stone wash jeans
- cheese
Describe the production of insulin
- originally made from pigs, whose insulin is similar to human insulin
- the gene that makes insulin was found and copies of it made and inserted into E.coli
- this allowed them to group colonies/ batches of E.coli that produce human insulin
What is the role of insulin in the body?
- a hormone that helps take up sugar from the blood
- diabetics cannot make insulin and so have to have regular injections of insulin
Describe the industrial biotechnological process of making stonewashed jeans
- biological washing powders contain a variety of enzymes that break down and remove stains
- enzymes can be effective at temperatures
List 3 enzymes commonly found in detergents and what they down
- proteases - break down proteins e.g. Blood, egg, gravy
- lipases - break down fats and grease
- amylases - break down starches
Describe the industrial biotechnological process of stonewashed jeans and the associated advantages
- produced by washing jeans with the enzyme cellulase produced naturally by a bacteria and fungi
- jeans need to be rinsed few times using less water energy
- jeans are also not damaged as much thereby increasing their durability
What is the original process of stonewashed jeans and the disadvantages associated with it?
- using pumice stone
- stones can damage material making it less durable
- stones can also damage washing machine
- labour intensive because the stones need to be removed from the pockets of the jeans by hand
- jeans need to be rinsed several times which uses more water and adds cost
Describe the biotechnology case study of cheese
- traditionally made by taking milk then adding bacteria and enzymes from the stomached of calves (rennet)
- for vegetarian cheese cheese-makers sometimes use enzymes from plants i.e. fig trees, nettles, and thistles
- the enzymes do not work as well and those from the calf and the cheese doesn’t taste as good
- so the calf’s enzyme gene was inserted into a microorganism to produce recombinant enzyme
What are the three basic facts of natural selection?
- organisms produce more offspring than can survive
- individual characteristics vary
- many characteristics are inherited by offspring
Describe the process of sugar cane to tyres.
- the manufacturing of isoprene from sugar cane, corn, corn cobs, switch grass or other biomass that involve:
- microbial strain development
- large scale fermentation
- recovery and purification
- using bioisoprene reduces the seven gallons of petroleum feedstock per tyre required to close to zero
Describe the IB of rubbish to bio ethanol
- the conversion of cheap biomass and wastes to clean fuel and energy
- delivers a step change in green house gas emissions
- can be achieved safely, reliably, in a cost effective and sustainable way
- process: feedstock –> gasification –> renewable power –> fermentation –> bio ethanol which can be used as fuel
What are some examples of biocatalyts?
- isolated enzymes
- library of protein variants
- microbial culture collection
What are the screening methods associated with developing an active enzyme?
1- whole cells -genetic selection, colorimetric tests
2- microlitre plates -enzyme coupled assays, labelled substrates
3- analytical instruments -GC, HPLC, TLC, NMR, IR, MS
Briefly describe enzymes.
- proteins whose MW is 20-300KD
- usually unstable and low activity outside pH 5-9 therefore often need to be used in buffers
- commercially typically supplied as lyophilised powders and maybe crude preparations
- activity defined in units (U) per mg
Define the unit used to describe enzyme activity
U/mg - 1 Unit is the amount of enzyme that catalyses the conversion of 1μmol of substrate per min understand standard defined conditions of temperature and pH
Describe the process of the production of whole cells
- a sample of the desired microbial strain is obtained- either as an ampoule from a culture collection or glycerol stock stored in a -80 degree freezer
- microbial strain is cultivated on solid phase in a static incubator
- the microorganisms are maintained on agar plates or on slopes
- the microbial strain is grown in liquid phase in a shaking incubator
- enzymes of interest are produced within microorganism
- the microorganism cell containing the enzyme is harvest and centrifuged
- whole cells can be used for biotrnasformations
- cells can be lysed and the enzyme isolated
What is recombinant DNA technology?
- allows a foreign gene to be put into a host organism
- simplest host organisms are bacteria e.g. E.coli
- the organism can be programmed into producing the corresponding protein
- plasmids are used to shuttle DNA into host organism
What are the steps of gene cloning that lead to recombinant protein expression?
- organism is selected
- DNA is isolated and the gene identified
- the gene cloned
- plasmid vector containing the gene is inserted to the microorganism
- bacteria is cultivated into colonies on agar plates
- the protein is then purified from the bacteria cultures
What are plasmids and describe their role.
- circular double stained DNA that contain antibiotic genes
- they contain restriction sites that can be used for the insertion of foreign DNA
- easier to manipulate than chromosomal DNA
- many different plasmids are commercial available
What is a case of antibiotic resistance?
- plasmids contain origin of replication
- amp gene - ampicillin antibiotic resistance gene
- naturally found on plasmids to make bacteria antibiotic resistant
- can be used to select from bacteria with the desired plasmid
What enzyme carries out DNA joining reactions
DNA ligase
What is the restriction enzyme action of EcoRI?
- it cuts both DNA strands at the same site
- DNA fragments then join at the sticky ends by action of DNA ligase
What is gene cloning
- invokes the selection and amplification of a particular gene from a mixed population
- use N-terminal proteins to design primers for PCR application
- codon degeneracy mains it is necessary to synthesise all possible primers as a mixture of synthetic oligonucletides
What is the process of gene cloning?
- the chromosomal DNA fragment to be cloned is inserted into the vector DNA
- the cDNA is joined to the vector DNA by DNA ligase
- the recombinant DNA molecule is introduced into the bacterium
- the bacterium is grown in a rich medium
- ## protein expression is analysed by SDS-PAGE
What are some of the problems associated with insulin from pigs?
- small structural differences which can cause side effects such as allergies, resistance, insulin lipodystrophy
Describe the production of human insulin
- the gene was isolated and cloned and litigated into plasmids
- this was mixed and oxidise which resulted into production of insulin
What is PCR used for
- amplifying and modifying DNA
- cloning genes
What is plasmid DNA
- extra-chromosomal circular DNA that is easily manipulated and can carry foreign genes
When is it possible to use gene synthesis
- if the gene sequence is known
What are the uses of restriction enzymes/DNA ligase
- to cut DNA at specific sites and stick it back together
What is the purpose of transformation/ electroporation
- to reintroduce plasmids and genes back into microbes
What is the purpose of electrophoresis
- to separate and analyse protein and DNA mixtures
What is preload rule concerning which face of ketone is reduced
- reduction or ketone occurs from top face
Define the term generic API
All active pharmaceutical ingredients that can be obtained from multiple sources or that are manufactured and supplied to multiple customers
-APIs on the other hand are sold only by the originator company
What type of molecule is NADH and KRED?
- cofactors reducing agents that donate electrons
What kind of drug is Sitagliptin
An oral anti-diabetic
Describe the case study of Sitagliptin
- 1st generation API process involves use of NH4OAc, and a Rh complex
- carbon treatment for the removal of Rh
- 2nd generation API process uses transaminase as the biocatalyst
In terms of the Sitagliptin case study in C-N bond formation using transaminase what are the advantages associated with using (R)-selective transaminase
- substrate walking is used to evolve large binding pocket, this results in:
- increased yield
- higher e.e.
- higher productivity
- reduced waste
In the C-N bond formation using transaminase associated with the second generation API synthesis of Sitagliptin
What is the cofactors used
- pyridoxial phosphate also called PLP
What is montekulast used in the treatment of
- it is a leukotriene receptor agonist
- used in the maintenance treatment of asthma and for the relief of seasonal allergies
- usually orally administered
What are the cofactors used in the enzymatic production of montekulast
- NADH and KRED
In the chemical process of montekulast synthesis what is the main reagent used and what is the temperature that the reaction occurs at
- (S)-DIP-Cl
- -20 degrees
What are the conditions of biocatalytic synthesis of montekulast
- temp 45 degrees
- product isolation by direct filtration
- e.e. >99.9%
- solvents used: IPA, Water, Toluene
- waste generation: biodegradable enzyme, cofactors
In the (S)-DIP-Cl synthesis of montekulast what are the conditions associated with the reaction
- reaction is stoichiometric
- temperature -25 degrees
- production isolation occurs by extraction with high dilution
- e.e. 99% after recrystallisation
- solvents used: DLM, THF
- waster generation: non-biodegrade borane, pinene
What are the applications our erucamide
- an anti-sticking lubricant
- release and anti-static agent of polyethylene and polypropylene
- used as an anti slip agent in plastic bags
- used in manufacturing of printer inks
What is the chemical process associated with the synthesis erucamide
- reagent: ammonia
- temperature: 180 degrees
- 125-150 psi
- temperatures >200 degrees result in the formation of nitrile
- amidation Is continued with periodic removal of water
Describe the enzymatic process associated with the synthesis of erucamide
- solvent amidation at 90 degrees
- catalysed by immobilised biocatalyst novozyme
- ammonium carbamate (or urea) used as ammonia source
- 95% conversion obtained in 12 hrs
What are the applications of isopropyl myristate
- it is an emollient
- used in cosmetics as a natural oil substitute- because it easily absorbs into the skin and has excellent spreading properties
- can be used as an emulsifier
- used as an antibacterial in the treatment of headlice
What is the enzymatic process associated with the synthesis of isopropyl myristate
- enzyme/biocatalyst : novozyme (a lipase)
- temperature : 60 degrees
- yield >99%
- water is removed by vacuum
- myristic acid reacts with propan-2-ol
What are the advantage associated with the practice of often immobilising enzyme on a resin
- improved stability
- ## aids separation and recovery of product
What are the major advantages associated with IB
- biocatalysts catalyse reactions when high rates and selectivity and reduced side reactions
- generally operate at room temperature and neutral pH under partially aqueous conditions
- reduced cost of goods
- avoidance of metals - cost of contamination
- improved safety and environmental impact
- used increasingly 2nd generation manufacturing processes
- can lead to sustainable manufacturing
What are the four types of biotechnology?
- medical - use of stem cells to regenerate damaged tissue
- industrial- includes the production of new chemicals or new fuels
- agricultural - principally developing pest resistant grains or the accelerated development of disease resistant animals
- marine - concerns itself with processes in marine and aquatic environments
