Ezymes present in various microoragisms are indispensable part of industries because of their ability to act as metabolic catalyst. This enzymes are extensively utilized for manufacturing of approximately 500 commercial products showing ubiquitious importance of enzymes for commercial application. since long back microbes are one of the most dependable source of obtaining industrially important enzymes. for obtaining industrially useful enzymes, variety of microorganisms like yeast algae fungi bacteria are studied worldwide. Microbial enzymes are booming in various fields like food and beverages as well as one of the most integral part of pharmaceutical industries.
Utilization of enzymes provides various advantages like higher reaction rate, greater reaction specificity and along with enzymes can also under milder reaction condition in comparision to chemical catalyst. Specific enzymes are utilized on the basis of commercial interest and specific properties like thermostable, withstand elevated temperature, pH and pressure.
However there are several drawbacks like poor catalytic efficiency, one of the most encountered problem is when need arises for production of stereospecific chiral compounds. In order to overcome all this problems enzymes are the most reliable source which can be utilize for production of stereospecific chiral compoundas well as catalytic efficiency can be improved by inculcating various approaches of genetic engineering”,protein engineering, metagenomics and bioanalytical techniques .
Moreover Purity of an enzyme is an aspect which is most emphasized because of economical, biopharmaceutical, and medicinal importance. Commercially important enzymes are purified by using various methods like affinity chromatography, size exclusion chromatography, ion exchange chromatography, e.t.c”,
1.1.1 Tartaric acid utilizing microorganisms:
Modes of tartrate utilization organisms References
Aerobic L-(+)-tartrate utilization Rhodopseudomonas sphaeroides
Pseudomonas putida Friedrich giffhorn and Anita Kuhn(1983)
Setsuo Furuyoshi , Hidehiko Tanaka
And Kenji Soda(1987)
Anaerobic L-(+)- tartrate utilization E.coli k-12 Ok Bin Kim And Gottfried unden(2006)
Anaerobic D-(-)-tartrate utilization E.coli k-12 Ok Bin Kim”,Sebastian Lux , Gottfried unden(2007)
Anaerobic Meso-tartrate and L-(+)-tartrate utilization Pseudomonas spp. Shilo And Stanier 1957; Rode And Giffhorn (1982)
IMPORTANCE OF TARTRATE DEHYDROGENASE.
-Tartrate dehydrogenase is an unique NAD-dependent enzyme which displays numbers of catalytic reactions at single active site.one of the paramount reason behind this spectacular feature is ability of this enzymeto catalyze a reaction pathway in which variety of substrate undergoes similar initial catalytic steps but following intermediate dissociates from enzyme during different stage of catalysis which results in formation of variety of products.
Reaction pathway for TDH of pseudomonas putida.
*Oxidation of L-(+)-tartrate:
-Oxidation of L-(+) tartrate leads to formation of oxaloglycolate”,which rearranges nonenzymatically to form dihydroxyfumerate.
Hydroxypyruvate+Tartronic semialdehyde +co2
Oxidation of D-malate:
-TDH catalyses oxidation of D-(+)-malate thereby forming oxaloacetate and reduction to NADH.
-Decaroxylation of oxaloacetate leads to formation of pyruvate and carbondioxide.
Decarboxlyation of mesotartrate
-TDH catalyses decarboxylation of mesotartrate to yielding hydroxypyruvate”,which remains bound to enzyme and reduces to D-glycerate “,then releases from enyme.
Substrate Intermediate Product
IMPORTANCE OF DIFFERENT PRODUCTS:
-It is utilized for production of biodiesel
-It is useful as chiral synthon in synthetic organic chemistry.
-it useful as corrosion inhibitor and antisealing agent
-it is useful as plating agent and surface treating agent.
– It is utilized as anticaking agent in gypsum processing.
-It is useful as flavouring agent in food industry
Catalytic mechanism of tartrate dehydrogenase in pseudomonas putida.
-Enzyme tartrate dehydrogenase follows general acid base mechanism for metabolizing substartes.when malate is utilized as substrate by tartrate dehydrogenase, during early catalytic step proton abstraction is mediated by Lys192 from C2 hydroxyl atom of substrate.Thus Lys192 acts as base.This process results in transferring of hydride to NAD, thereby forming oxaloacetate intermediate. monovalent metal ion (cation) like NH3+ plays amazing role in stabilizing the processs of hydride transfer to NAD. Decarboxylation of oxaloacetate leads to generation of CO2 and pyruvate which in turn dissociates from enzymes. This whole reaction step is supported by general acid Tyr141.when reduced cofactor i.e.NADH is released and proton transferred from Lys192 to Tyr141″,the enzyme is regenerated.
Kinetic mechanism involving oxidation of L-(+)-tartrate by tartrate dehydrogenase in pseudomonas putida.
Mn+2 NAD+ L-(+)-tartrate oxaloglycolate NADH Mn+2
Above mention Cleland diagram indicates that NAD+”,Mn+2 and L-(+)-tartrate are added to enzyme to enzyme in sequential manner showing ordered bi bi mechanism.Mn+2 is added first, followed by NAD
and lastly L-(+)-tartrate is added. oxaloglycolate is released as first product “,followed by NADH and Mn+2.
It has been noted in strain of pseudomonas sp.group ve-2″,L-tartrate decarboxylase mediates conversion of L-(+)-tartrate into D-glycerate in single step with almost 100%selectivity and its optical purity is about More than 92%e.e.
Comparision of tartrate dehydrogenase from pseudomonas putida and Rhodopseudomonas sphaeroids Y.
*Similarity between L-(+)-tartrate dehydrogenase D-(+)-malate dehydrogenase of R.sphaeroides and pseudomonas putida:
-Enzyme is tetrameric in nature.
-molecular weight for each of subunit is 36″,800Da.
-enzyme from both organism requires NAD as cofactor”,Mn+2and NH4+ cation for activity.
Organisms P.putida R.sphaeroides
Substrates KM values KM values
Mesotartrate 8.3×10-4M –
L-tartrate 1×10-3M 2.3×10-3M
Malate 5×10-5M 1.7×10-4M
Above data (Fredrich Giffhorn* and Anita Kuhn) indicates that in comparasion to L-tartrate, mesotartrate is better substrate for tartrate dehydrogenase in P.putida”,while in R.sphaeroides, mesotartrate acts competitive inhibitor for tartrate dehydrogenase.
From several laboratory experiment, it is observed that for tartrate dehydrogenase of bacillus subtilis (crude lyaste) KM values of dihydroxyfumerate as substrate is 0.2×10-3M and for L-(+)-tartrate as substrate is 50×10-3M.
For P.putida tartarte dehydrogenase “,the KM value of L-(+)-tartrate is quite lower i.e”,1×10-3M than KM value of L-(+)-tartrate( 50X10-3M) for Bacillus subtilis tartrate dehydrogenase (crude lysate).In contradictory to this for P.putida tartrate dehydrogenase KM value of dihydroxyfumerate is 11×10-3M which quite higher than KM value of dihydroxyfumerate( 0.2×10-3) for bacillus subtilis tartrate dehydrogenase (crude lyaste).
Infact for R.sphaeriodes Y. dihydroxyfumerate acts as noncompetitive inhibitor so it may be inferred that Bacillus subtilis tartrate dehydrogenase is a type of unusual enzyme that favours reversible reaction to greater extent than forward reaction.
L-(+)-tartrate is a cheap by- product that is obtained from wine industry but it is mostly present in form of salts especially bitartrate.The procedure for isolating tartaric acid from waste (pulp residue) that is generated during grape juice production is quite tedious and labour intensive .Additionally this procedure provides very low yield of tartaric acid acid.Moreover optical purity can be suspected.
So ability of tartrate dehydrogenase of bacillus subitis to calatyse reversible reaction with better efficiency than forward reaction can be exploited for production of stereospecific L-(+)-tartrate.
APPLICATION OF L-(+)-TARTRATE:
*L-(+)-tartrate is useful for production of chiral compounds:
-It is utilized to synthesize an intermediate to make an enantioselective epoxidation catalyst.
-It is useful as chiral resolving agent for resolution of 2″,2’-bispyrrolidine.
-It acts as precursor material for preparation of 1″,4 di-o-benzyl L- threitol.
-L-(+)-tartrate can be utilize for kinetic analysis of recemic allylic alcohol and alpha-furfuryl amides.
*THERAPEUTIC APPLICATION OF L-(+)-TARTRATE:
-It is exentsively used as chiral agent for synthesis of isoquinoline alkaloids and arundic acid which produces therapeutic effect in acute ischemic stroke therapy.
-It is useful for treating COPD
-It is also useful for determining prostatic serum acid phosphatase.
-Tartaric acid derivative have imparted their role as therapeutic agent for treating HIV.
-It has wide range of pharmaceutical importance as for example:intermediate in antibiotic”,effervescent antacid.
*GENERAL APPLICATION OF L-(+)-tartrate:
-L-(+)-tartrate is utilize for baking purpose in order to improve the flavor of bread. L-(+) –tartrate is mainly used in wine industry for balancing the naturally occurring tartaric acids and mallic acid in wine. Common commercial esters are diethyl-dibutyl derivative used for lacquers and in textile printing.
-It is commomly used in soft drink industry, confectionery products”,gelatin desserts”,bakery products.
-Many other fields revolves around utilization of tartaric acid like tea processing, textile, fertilizer, tannery and building materials like gypsum and cement.