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Module 1: Plant Cell Bioreactors

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Biotransformation in Plant Cultures

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. So,todaywe are going to discuss about Biotransformationand plant cell immobilization and its applications.So, starting with biotransformation; what is biotransformation to you; biotransformationis done using any living organism, you must have already read about it.So, what is biotransformation how is it different from de novo production using bioprocesses.So, majorly what kind of convergence come under biotransformation?De novo synthesis.The question was how it is different from de novo synthesis?What is de novo synthesis?From zero give the . There is organism and .So, from the.Starch kind of thing.From the beginning . So,a will get converted to b, b will get converted to c and this maynot be structurally related.They can be n number of conversions with other intermediates more than 1 which will combinetogether to give you the product moiety.Now, how is bioconversion where is it placed in this synthesis?Close to what you said; so,generally changes in the functional group, addition of certainmoieties in the parent molecule.So, these class of reactions come under biotransformation.Sometimes a low cost substrate can get transformed to give you a high cost product; now for example,glycosylation in the molecules ordemethylation or methylation.So, when we say low cost to a higher cost which mean sometimesthe lead moleculemay besuffering from problems like not soluble in water or toxic.But by biotransformation, the chemical structure is change such that or the functional moietiesare change such that either replacement or displacement or additions such that you getrid of these limitations.So, now when in plant cell technology,the whole cell the plant cell is nothing, butit has rich repository of enzymes.So, generally in biotransformations these reactions are carried out by it is a biotransformation.So, what will be involved in all these reactions?Enzyme . Enzymes.So, either you have a purified enzyme and then carry out these reactions or rather youuse a rich repository of enzymes.If there are more than one steps involved in this complete biotransformation then ratherthan using a package of purified enzymes or a single enzyme you can use the whole cellwhich is nothing, but a rich repository of a number of enzymes dehydrogenesis, hydrolysisthen what else, redox reactions.So, you have so many different kinds of enzymes which can bring about these changes.. So, what is it the one chemical is changeinto another where the precursor is converted into a product using cell suspension or yourbio-catalyst?Now, the bio catalyst can be plant cells growing or in quiescent state which means restingcells, they either can be in the active phase or can be the resting cells.So, why it is linked to plant cell immobilization you will be able to understand when I sayif it is in the quiescent state, how we can exploit this at large scale.So, the bio catalyst can be free in solution it is possible, immobilized or on a solidsupport as a film or entrapped which is immobilization in a matrix.Or even you will say there are different kinds of immobilization we will be talking aboutit, either surface immobilization or embedding it or encapsulating it; so, there are differentways.So, again reiterating; what is biotransformation?Biotransformation is a process through which the functional groups of organic compoundsare modified by the living organisms.Now, why do living organisms do that, they need to do it not everything is de novo synthesizedby the cells.So, what what can be the different reasons?Why the cells are doing this?Defense, what else.So, which means to overcome either toxicity or to convert.So, stress is one form of a any toxic material can get stress there.So, to in order to incorporated to utilize somewhere else; so, in other metabolism.So, that can also be a reason for biotransformation.So, factors such as difficulty of maintaining culture sterility, slow growth rate etceterain plant cell cultures cannot compete with microbial systems for same biotransformationprocess.So, we know the limitations of plant cell cultures, because they are slow growing andwhat else one is slow growing other, but can be a disadvantage.Ok, at large scale imagine heterogeneity mean scale up can be an issue, they generally athigh cell densities they will become non-Newtonian behaviour.So, the scalar becomes limited.So, then how how high cell density can be obtained and in the same process you needto utilize them for biotransformation which can be cell immobilization.You can still have higher cell density, but you can avoid it's in comparison to microbesits shear sensitivity, which can be a problem, when you are using free cells and mass transferlimitations because they tend to aggregate together, the cell to cell contact is highthat can be a problem.Now, what is a key here, whatever process you use for biotransformation?The substrate has to be made available to the cell.So, which means that the substrate has to enter into the cell get bio transformed intothe product and the product should be able to come out in the solution.So, that is a prerequisite.Now the substrate must be therefore because it has to be taken in converted it is notnecessary that all time the substrate will be taken in.In detoxification reactions maybe there can be enzymes hence which can be executed whichare used in bio transforming the toxic metabolite into a non toxic form.So, but generally in case of multiple step biotransformation reactions, the precursormoiety is to be taken in by the cell and then bio transformed into the final product.Now, what is key there it is that the precursor itself should not be toxic to the cells, whenit been taken in by the cells?Sometimes what happens you will see that some of the precursors when they are produced intracellularlythey are not toxic or the same precursors when given exogenously are toxic.So, how is that possible?The cells do not die be because we know biosynthetic pathway that precursor is getting formed,but if you will add that precursor exogenously they become toxic to the cells.So, how is the cell than in synthesizing and managing?Very nice so, there are enzymes; so, this is also class of biotransformation to preventitself from the toxic effect of that metabolite.So, it is formed and either it is bio transform to make it to detoxify it or it is and thenit is stored at a suitable position with the enzyme nearby.So, that when the need arises the enzyme can convert it into the required metabolite.What other prerequisite, the rate product formation must significantly exceed the rateof further metabolism then only it is advantages.If you want a desired biotransformation reaction to happen then the rate of formation has tobe more than the rate of its subsequent metabolism, if the cell does not needed it has to be metabolizethen or it has to be leased out.So, the rate of transfer of the product outside then becomes more limiting than the rate ofbiotransformation for the success of such process or its further metabolism rate.Now, what are the advantages of advantages of biotransformations over chemical transformation;what are the advantages?This is; this you must have already read when we use enzymes, how are enzymatic becausethese are enzymatic reactions.So, how are enzymatic reactions in what way they are better than chemical reactions.So, high specificity means what?There is specificity what else there is regio specificity, I hope you understand.So, enzymes give you the leverage, enzymes provide you stereo specificity and regio specificityand at the same time milder conditions, lesser toxic byproducts.So, they are more environmental friendly processes.Wide range of biotransformation reactions can be carried out using plant cells as repositoriesor plant cell enzymes are as bio-transformants, but here we are talking about plant cell asit is as repository of enzymes.So, what kind of reactions for example, glycosylation, glucosyl esterification, hydroxylation, oxidoreduction between alcohols and carbonyl compounds, then reduction carbon double bonds saturation-unsaturationthen hydrolysis isomerizations sometimes for example, I must have also talked about vitamine alpha tocopherol during my earlier discussions.So, tocopherols in nature they exist in four different isomeric forms; so, these are alpha,beta, delta, gamma.So, there are four different forms, but the most bioactive form which is preferentiallyabsorbed by humans and is used in your vitamin e capsules are in cosmetics where the pricerises the minute it is added.So, that is alpha tocopherol.Now, therefore, when you use plant cells versus chemical synthesis then plant cells can producethe natural form of alpha tocopherol, where the chemical synthesis leads into a recemicmixture; even methylation, demethyation, dehydrogenation all these reactions also come under biotransformations.Now, glucose conjugations of exogenously added substances are considered to be detoxificationreactions generally.So, ifglycosylation is needed.So, then glucose, moiety, addition is needed then plant cells can be used as bio-transformermants.The terpenoids and cholesterols which are hydrophobic may disturb the membranes of thecells and organelles when the molecules are taken in the cell directly.So, they may cause generation of reactive oxygen species in the cell .So, therefore, when these kind of products or substrates are given then the cell wouldglycosylated or add the glucose moiety to detoxify.If the substrates are non-toxic to the cells, the cells may not respond to them.So, what factors which will affect your bio transformation efficiency; improvement ofcell viability.So, you need to see at what stage the cell is whether the resting cell is needed or whetheryou need the cells in the active phase.So, many substances may be harmful to the cell.So, it is necessary to decrease the toxicity in order to increase the yield of the product.For example sugar could increase the cell viability via glycosylation of phenolic compounds.Now, selection of plant species the capacity for biotransformation is very diverse in plantspecies where now culture age is also an important factor; what age of cells, what phase of cellsyou are using similarly which means whether active cells or resting cells.Now, immobilization of plant cells it has advantages; what all advantages, reuse itmakes the process continuous, reuse of the biological catalyst is possible, but whatis the prerequisite here, the product must be excreted out what kind of immobilizationmaterials are known PUF; PUF stands for any guess?Poly Urethane Foam.Very nice Poly Urethane Foam, it is an immobilization material inert material which has been successfullyused to immobilize plant cells.But we need to take care while selecting the material and depending on the species on whichwe are working.So, we will see in details when we go on to plant cell immobilization.Now, what are the factorsaffecting biotransformation for example, root cultures they have beenshown to have higher glycosylation activity than cell cultures.So, sometimes it also depends what kind of cultures you are using.So, organ cultures like root cultures may have better biotransformation ability thanthe free cells in suspension.Now, for substrates which are hydrophobic and organic solvents like ethanol detergentslike tween eighty are often used to solubilize the substrates for biotransformation.One of the, I said it is very important for the substrate to be readily available to thecell for uptake.Now, some of these substrates cannot be taken as it is by the cells maybe because of thetoxicity.So, in order to facilitate its transport inside the cell one of the molecules which is calledas cyclodextrin.It is very commonly used to encapsulate the substrate and such that it can be it formsa bicycle like structure which can be easily then taken in by the cell.So, cyclodextrin acts like a solubilizer for poorly water soluble substrates, glucosylationof podophyllotoxin was facilitated after complexation with cyclodextrin as it is it cannot be takenin it may be toxic, but when you complex it with cyclodextrin then it was able to getglycosylated.Now, pH of the medium can also play a role affecting the activity of the bio transformingenzymes.So, what are the class of compounds, which can be involved in biotransformation monoterpenoids,diterpenoids these are some example class of compounds secondary metabolites which gounder biotransformation reactions in plant cells .