Dextranase Enzyme Production

Dextranase Enzyme ProductionDextranase definition and its engagesDextran is a collective name given to a large class of homopolysaccharides composed of D-glucans with contiguous a-1, 6 glycosidic linkages (95%), with minor subaltern linkages such as a-1, 2, a-1, 3 and a-1, 4 74. It is produced by microorganisms such as Leuconostoc mesenteroides, Streptococcus sp., Acetobacter capsulatus and Acetobacter viscus 44. Dextrans atomic number 18 well soluble in water, have busted toxicity, and relative inertness. These properties make dextrans effective water-soluble carriers for dyes, indicators, and reactive hosts in a wide variety of applications. They be widely utilize in the pharmaceutical and biochemical fields. Dextrans of abject molecular metric weight unit are used as an alternative to blood plasma. They are also used for clinical purposes such as drug deli very 82, and by cross-linking for the product of the chromatographic matrix Sephadex. They are also widely used as both anterograde and retrograde tracers in neurons 94. On the other hand microbial synthesis of dextrans in damaged cane and beets or other products containing sucrose is a serious problem in dirty m 1y and food industry. Dextran is also a structural component of dental plaque which causes the development of dental caries 78, 85.Dextranases are enzymes that cleave the a-1,6 glycosidic linkages of dextran to yield either glucose or isomaltose (exodextranases) or isomalto-oligosaccharides (endodextranases), and are only produced as extracellular enzymes by a small number of bacteria and fungi, including yeasts and perhaps some higher eukaryotes 44.Enzymes in more groups can be classified advertisement as dextranases according to function dextranhydrolases, glucodextranases, exoisomaltohydrolases, exoisomaltotriohydrases, and branched-dextran exo-1,2-alpha glucosidases. In particular the chemical reaction catalyzed is as fol disordereds(1,4-alpha-D-glucosyl)n + (1,4-alpha-D-glucosy l)m (1,4-alpha-D-glucosyl)n-1 + (1,6-alpha-D-glucosyl)m + 1These enzymes belong to the family of glycosyltransferases, specifically the exosyltransferases. The imperious name of this enzyme class is 1,4-alpha-D-glucan 1,6-alphaD-glucan 6 alpha-d-glucosyltransferase. Other commonly used names imply dextrin 6-glucosyltransferase and dextrin dextranase.Many microorganisms are known to produce dextranase, including filamentous fungi be to the genera Penicillium, Aspergillus, Spicaria, Fusarium and Chaetomium, bacteria, e.g. Lactobacillus, Cellvibrio, Flavobacterium etc. The only yeasts report to produce dextranases are members of the family Lipomycetaceae. Only Lipomyces kononenkoae 104 and Lipomyces starkeyi dextranases have been characterized 47.Potential commercial uses of dextranases includeThe synthesis of potentially valuable oligosaccharides 30Potential m let outhwash ingredients since isomaltose may be of significant importance for the prevention of dental caries 40, 41Clear ance of dextran contamination in cane sugar processing 25Dual-stimuli-responsive drug ferment as in biodegradable polymer-structured hydro gels of gelatin and dextran 55. Hydrogels are used for a wide range of biomaterials applications such as contact lenses, drug delivery vehicles and wander adhesives. Dextrans are polymers that mimic biologic sugars found on tissue surfaces. The dextran hydrogel system with tunable mechanical and biochemical properties appears promising for applications in cell assimilation and tissue engine room 58Drug delivery device suitable for delivering drug to the colon 7, 8. Brondsted et al. studied the application glutaraldehyde dextran as a capsule material for colon-specific drug delivery. The dextran capsules were challenged with a dextranase solvent, simulating the stretch of the drug delivery to the colon, so they broke and the drug was released as a dose pump. The out set out high ignitors the dextran capsules as promising candidates for prov iding a colon-specific drug delivery also in site-specific drug delivery systems with the use of antibodies 69The improvement of brewing yeast strain for beer industry. Due to the rising demand for low-calorie beverages, including beer, recombinant strains of Saccharomyces cerevisiae have been produced by combine LSD1 gene of Lipomyces starkeyi 101. S. cerevisiae lacks the ability to produce extracellular depolymerising enzymes that can efficiently liberate fermentable sugar from abundant, polysaccharide rich substratums 75. By introducing the gene mentioned above, adding an exogenous enzyme during beer zymolysis to achieve starch hydrolysis and oligosaccharide reduction can be avoidedCarbohydrase activity produced can also be exploited in sensitive chromogenic cheques for toxicity a mycotoxin bioassay using the intracellular -galactosidase activity of Kluyveromyces marxianus has been developed 20 compartmentalization of dextranase based on amino acidulent sequenceDextranases a re dextran-degrading enzymes that form a diverse group of carbohydrases and transferases. The more new-fangled potpourri divides dextranases into two classes endodextranases (a-1,6-glucan-6-glucnohydrolase also referred to as dextranase) and exodextranases ( glucan-1,6--glycosidase also referred to as dextran glucosidases). The Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUB-MB) provides a system of classification depending on the type of reaction catalyzed and product specificity (Table 1). Contrary to that system, the Carbohydrate Active Enzymes (CAZy) database describes the families on terms of structural and mechanical features of these enzymes enzymes with variant substrate specificities are placed in the same family and those that hydrolyze the same substrate are sometimes placed in different families. In another classification system, Henrissat and Bairoch 33 have divided glucosylhydrolases and glycosyltransferases into five fam ilies on the basis of the similarities in the amino acid sequences (Table 2).Table 1 The IUB-MB classification systemEC3.2.1.11DextranasesEC3.2.1.70Glucan-1,6-glucosidasesEC3.2.1.94Glucan-1,6-isomaltosidasesEC3.2.1.95Dextran-1,6-isomaltotriosidasesEC3.2.1.115Branched-dextran exo-1,2-glucosidasesTable 2 Classification of dextran hydrolysing enzymes, based on amino acid sequences.Dextran-glucosidasesFamilies 13 15IsomaltodextranaseFamily 27IsomaltotriosidaseFamily 49EndodextranasesFamilies 49 66 (no sequence similarities between the two families)Aoki and Sakano (1997) came up with 4 families 2. They isolated and sequenced the isopullunase gene (ipuA) from Aspergillus niger ATCC 9642. The gene shows significant amino acid similarity to the dextranase produced by Penicillium minioluteum (PEMDEX) and Arthrobacter sp. (ARTDEX). Since ASNIPU shows great similarity to PEMDEX and ARTDEX, they can be classified as Family 1. In the same fashion, the researchers compared the amino acid sequen ces of dextranases and dextran-hydrolising enzymes, including ASNIPU.Lipomyces species and Lipomyces starkeyiLipomyces starkeyi and Lipomyces kononenkoae belong to the Lipomycetaceae family and are the only yeasts reported to produce dextranases. The first Lipomyces species was identified by Robert Starkeyi in 1946 during a study of nitrogen-fixing bacteria it was then that he discovered L. starkeyi, a fat-producing, ascosporogenous soil yeast. The family Lipomycetaceae was proposed later, in 1952 by Lodder and Kreger von Rij. Lipomyces species can utilize starch as a restore source of speed of light. Both species contain highly efficient amylolytic systems, permitting produce on starch with very high biomass yields 97.The family Lipomycetaceae is known to utilize certain heterocyclic ring compounds, such as imidazole, pyrimidine, and pyrazine and their derivatives, as sole nitrogen sources 92. Information on the genome organization and molecular genetics of this group of yeasts is very limited.The ascosporogenous soil yeast L. starkeyi has been reported to produce commercially useful extracellular dextranase activity 97, 52, 53, and it can utilize a variety of other compounds, like hexoses, pentoses, alcohols and organic acids, as sole sources of carbon and energy 46. The strains of L. starkeyi currently used are NCYC 1436, IGC 4047, ATCC 12659 and its de-repressed mutant ATCC 20825.L. starkeyi dextranasesCommercial use of dextranase began in 1940s, mainly by producing low-molecular-weight clinical dextran. Therefore, industrially practical mixed culture fermentation of L. starkeyi and Leuconostoc mesenteroides was capable of producing controlled-size dextrans in order to satisfy clinical use, in which dextranase produced by L. starkeyi hydrolyzed the high molecular weight dextran produced by L. mesenteroides to a controlled size 46. The enzyme output signal system of L. starkeyi needs an inducer. Dextran is its normal inducer but it is a relatively expe nsive carbon source for large-scale fermentations. Also, L. starkeyi is reported to have slow growth and difficulty of avoiding contamination from other microorganisms during growth. With that in mind D. W. Koenig and D. F. Day (1989) undertook to establish conditions which would minimize the cost of the inducer for producing an enzyme by using a de-repressed mutant of L. starkeyi ATCC 12659 bad on glucose. Thus the mutant ATCC 20825 is capable of hyperproducing dextranase at low pH to provide biologically contaminant-free supernatant tranquil containing dextranase.Lipomyces starkeyi (IGC 4047), when grown on dextran as a sole carbon source produced a dextranase able to hydrolyse blue dextran and Sephadex G-100. The molecular weight was 23kDa and the isoelectric situation was 5.4 97. The dextranase of L. starkeyi (ATCC 20825) studied by Koening and Day (1988, 1989a, 1989b) was analysed by SDS-PAGE and produced quaternary bands, of molecular weights 65 kDa, 68 kDa, 71 kDa, and 78 kDa. Millson and Evans (2007) have isolated extracellular dextranase of L. starkeyi NCYC 1436 and have found that for their strain the enzyme make its as three molecular weight species and seven isoelectric forms 68.L. starkeyi nutrients (YPDex / YPD)The main ingredient in the chosen media is yeast extract. Yeast extract is a dried autolysate which facilitates rapid and luxuriant growth when used in various media or fermentation broth. It is a good source of amino-nitrogen and vitamins, especially the water-soluble B-complex vitamins. However, yeast extract is reported to enhance glucose metabolism to lipids, but suppress lipolysis 18. The metabolic pathway consists of converting glycerol into pyruvate or glucose and then hydrolysis by a phosphatase gives glycerol again. The disruption of this metabolic pathway, could account for the seemingly truncated numerous bands that SDS gives later on prolonged storage of the yeast. Mycological peptone is incorporated in the media and dis courages bacterial growth because of its acidity.Environment that dextranases favourDextranase activity is affected by temperature, pH, alloy ions and nutrients. According to Lin Chen et al (2007), dextranase activity is optimized between temperatures of 10oC and 60oC at pH of 6.0 12. In the particular study, the effect of pH on enzyme activity was determined by varying the pH between 3.5 and 8.5 under the temperature of 30oC. The pH of 3.4-4.5, 5.0-7.5, and 8.0-8.5 were maintained by sodium acetate modify (20mM), change state and phosphate pilot (20mM) and sodium phosphate buffer (20mM) respectively. The effects of metal ions (AlCl3, CaCl 2, CoCl2, CuSO4, FeCl3, KCl, MgCl2, NaCl, NiSO4, MnCl2 and ZnCl2) and SDS on dextranase activity were assayed by incubation of dextranase with 1mM metal ions or 1 mM SDS at pH 4.5 for 3h at 37oC, and then the enzyme activity of dextranase was determined.Ravi Kiran Purama and Arun Goyal (2008) in a study for optimization of nutritional factors, estimated dextransucrase activity in the cell free extract of Leuconostoc mesenteroides. They analysed the regression coefficients and t-values of six ingredients yeast extract, sucrose, intercept, K2HPO4, beef extract, peptone and Tween 80. Yeast extract, sucrose, beef extract, and K2HPO4 displayed a positive effect for enzyme production whereas, peptone and Tween 80 had a negative effect on enzyme production. The variables with confidence levels greater than 90% were confacered as significant. Sucrose was significant at 99.99% confidence levels for dextransucrase production. K2HPO4 and yeast extract were found significant about 94% level for dextransucrase production. Beef extract was significant 91% for dextransucrase production. Peptone and Tween 80 were found unnoticeable with negative coeffficients for enzyme activities.Methods used for enzyme activity measurementEnzymatic activity is metric with the help of laboratory systems called enzyme assays. All enzyme assays measur e either the consumption or production of product over time. Enzyme assays can be split into two groups according to their sampling manner continuous assays, where the assay gives a continuous reading of activity, and discontinuous assays, where samples are taken, the reaction stopped and then the concent balancen of substrates/products determined 11, 20.Continuous assaysSpectrophotometry in which you follow the course of the reaction by measuring a transmute in how much light the assay consequence absorbsFluorimetric assay in which we make use of the difference in the fluorescence of substrate from product to measure enzyme reaction. These assays are in general much more sensitive than spectrophotometric assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to lightCalorimetric assay in which the heat released or absorbed by chemical reactions is measuredChemiluminescence in which the light emitted by some enzyme reactions is measured so as to detect product formation. The detection of horseradish peroxidase by ECL is a common method of detecting antibodies in western blottingDiscontinuous assaysRadiometry in which the internalisation of radioactivity in substrates is measuredChromatographic assays measuring product formation by separating the reaction mixture into its components. This is usually done by high-performance quiet chromatography (HPLC), but thin layer chromatography can also be used. Although this approach needs a lot of consumables its sensitivity can be increased by labelling the substrates/products with a radioactive or fluorescent tagMethods and assays for dextranase activity measurementThe large variability of available substrates makes it difficult to estimate the enzyme activity, because the reaction product is very much an undefined mixture of sugar polymers. The existing assays try to compromise convenience, speed and accuracy 44Viscosimetric analysis was amon g the first to be used 31, 35, 36. This method measured the amount of enzyme which reduced the specific viscosity of the dextran solution by half in 10min. and it is more suitable when dextranase hydrolyses the dextran molecule at random, producing long oligosaccharides.Reducing-sugar assay or saccharogenic methods measure the rate of increase in reducing sugar as measured with the Somogyi assay, the 3,5-dinitrosalicylicacid method (DNS) 102, thiourea borax-modified O-toluidine colour reagent (35) and alkaline potassium ferricyanide solution (225). These methods test the social movement of free carbonyl group (C=O). It is a simple method commonly used to analyze for reducing sugars produced from enzymatic hydrolysis of substrates such as starch and sucrose 67.The approximately common substrates applied are Dextran T2000,47 T-260,3 and T110 54, 72. A number of substances have been reported as interfering with DNS colour development and citrate is one of them. acetate rayon and cit rate are reported to enhance colour development and the true antagonist in this reaction is the proton (H+) 96. This method is based on the release of shortsighted coloured products from polymeric blue dextran and their selective colorimetric detection at 610-650nm after hurriedness of the polymer. DNS colorimetric assays reported in literature are oft modifications of the method of Webb and Spender-Martins (1983). E. F. Khalikova and N. G. Usanov (2001) developed a dextranase assay using an isoluble substrate, namely, Sephadex G-200 with Remazol Brilliant Blue dye 45. The action pattern of dextranase was then, studied by means of exclusion chromatography. Overall, this assay was reported as convenient for quantitative dextranase detection, relatively independent of the enzyme source, and is proposed as an inexpensive alternative to the known procedures utilizing coloured substrates.The dextranase substrates can be either dye-releasing or fluorogenic. The assay procedures based on these substrates are accurate, fast and can be recommended for dextranase-producing microbial screening and enzyme purification.Other assay procedures worth mentioning include a spectrophotometric method with the use of Blue Dextran developed by Kauko K. Makinen and Illika K. Paunio (2004) who recommend it for column chromatography 62, and a method based on simple titration, developed by Eggleston and Gillian (2005) for easy use at the sugar cane factory 19.Fluorometric assays are based on measuring the fluorescence of the samples and the results are often compared to a series of standards of Penicillium sp. A very sensitive fluorometric assay using amino-dextran-70 coupled with fluorescent dye BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sindacene-3-propionic acid, succinimidyl ester) as the substrate was described by M. Zhou et al. (1998). The BODIPY FL dye-labelled dextranase substrate is an amine-containing dextran derivative that is labelled with the pH-insensitive, s pirt fluorescent BODIPY FL dye, resulting in almost total quenching of the conjugates fluorescence. The increase of the fluorescent degradation products of BODIPY FL dextran is proportional to the amount of dextranase activity 102.A suspension of Sephadex in a buffer is supplemented with agar, sterilized, and poured in Petri dishes, and after the wells are filled with the test solution, they are left to incubate. The dextranase activity can be evaluated by the extent of halos nearly the holes due to the opalescence of Sephadex. Milson and Evans (2007), measured dextranase activity using SDS PAGE as described by Laemmli (1970), using both mini-gel and Protean II electrophoresis systems, and stain using Coomassie Blue 68, 56. Molecular weight markers were used to construct a calibration curve, from which molecular weights of dextranase were determined. Native gel electrophoresis was performed, but the loading buffer and the gel lacked SDS and -mercaptoethanol and the samples were no t heated prior to loading on the gel. In the same study, dextranase activity was estimated in SDS gels, without extraction, by a plate modified from the method of Lawman and Bleiweis (1991) 57.FL versus DNS assay methodThe classic method (DNS) for measuring glycosidases through release of reducing activity is simple and inexpensive and, as cited above, has been modified in several studies so as to suit the researchers needs. It may, however, have some pitfalls. The reaction taking place is the followingaldheyde group oxidation carboxyl group3,5-dinitrisalicylic acid reduction- 3-amino,5-nitrosalycilic acid(Nam Sun Wang, University of Maryland)The above reaction scheme shows that 1 mole of sugar reacts with 1 mole of 3,5-dinitrisalicylic acid. However, it is suspected that there are many side reactions, and the authentic stoichiometry is more complicated than that previously described. Different reduced sugars yield different colour intensities thus it is necessary to calibrate for each sugar. Apart from the oxidation, other side reactions may compete for the availability of 3,5-dinitrisalicylic acid. Consequently, the calibration curve may be affected and the intensity of the developed colour may be enhanced. Therefore, the method has low specificity and one must run blanks diligently if the colorimetric results are to be interpreted correctly and accurately 96.Another obstacle to be dealt with when using DNS is non-linearity. unmatchable cause of non-linearity could be the common practice of diluting reaction products before quantification of reducing compounds and another is the insufficiency of substrates.The fluorometric assay (FL), seems to gain ground in the most recent studies as faster and more accurate and it seems to leave space for modifications and combined use with other methods (see 1.3.1). A standard curve is constructed from Penicillium sp. and then compared with the one derived from Lipomyces starkeyi.As described in the previous paragraph d extranase activity is estimated by the increase of the fluorescent products of dextran degradation. However, if too many fluoro are conjugated to the dextran molecule unsought may come up.Molecular Probes TM seems to overcome this problem by removing as much of the free dye as possible and then assaying the fluorescent dextran by (TLC) to ensure that it is free of low molecular weight dyes. So, in general, FL seems to yield accurate curves. Millson and Evans (2007), used an assay of dextranase activity which was a variation on that reported by Zhou et al. (1998). In that study, fluorescence vs. dextranase activity produced a linear log 68, 102.Purification of L. starkeyi dextranaseDialysis tubingDialysis tubing is typically used for changing the buffering solution of a protein and is also a method for concentrating protein solutions by dialysis against a hygroscopic environment (e.g. PEG, Sephadex). The protein solution is contained within a membrane which permits solute exchange w ith a contact solution and whose pore size prevents the protein from escaping. Except for small volumes, this method is time-consuming 11.Filtration UltrafiltrationUltrafiltration (UF) is a variety of membrane filtration in which hydrostatic pressure forces a liquid against a semi-permeable membrane. Suspended solids and solutes of high molecular weight are retained, while low molecular weight solutes pass through the membrane. UF is not fundamentally different from microfiltration or nanofiltration, except in terms of the size of the molecules it retains. 11, 77.SDS-PAGEPurification of Lipomyces starkeyi dextranase is carried out mainly by running a SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) analysis. The solution of proteins to be analyse is first mixed with SDS, an anionic detergent which denatures secondary and non-disulfide-linked tertiary structures, and applies a negative charge to each protein in proportion to its mass. SDS binds in a ratio of a pproximately 1.4g SDS per 1.0g protein. The size of the protein is directly related to the distance it migrates through the gel. Dextranase molecules migrate as bands based on size. Each band can be observe using stains such as Coomassie blue dye 77.Modifications to the polypeptide backbone, such as N- or O- linked glycolylisation, however have a significant impact on the apparent molecular weight. Thus, the apparent molecular weight is not a true reflection of the mass of the polypeptide chain.In most cases, SDS-polyacrylamide gel electrophoresis is carried out with a discontinuous buffer system in which the buffer in the reservoirs is of a different pH and ionic strength from the buffer used to tender the gel. After migrating through a stacking gel of high porosity the SDS-polypeptide complexes are deposited in a very thin zone (or stack) on the surface of the result gel. The discontinuous buffer system that is most widely used was originally devised by Orstein (1964) and Dvis (1964) 77. The sample and the stacking gel contain Tris Cl (pH 6.8), the upper and lower buffer reservoirs contain Tris-glycine (pH 8.3) and the resolving gel contains Tris Cl (pH 8.8). All components of the system contain 0.1% SDS 56. foolhardiness methods of proteinsPrecipitation is widely used in downstream processing of biological products, especially proteins. It serves to concentrate and fractionate the target product from various contaminants, as in biotechnology industry where precipitation helps to eliminate contaminants commonly contained in blood. The cardinal mechanism of precipitation is to alter the solvation potential of the solvent and thus lower the solubility of the solute by addition of a reagent.Precipitation is usually induced by any of the following methods 11Salting outIsoelectric point precipitationPrecipitation with organic solventsNon-ionic hydrophilic polymersFlocculation by polyelectrolytesPolyvalent metallic ionsSalting outThis the most common type of p recipitation. Normally a neutral salt is added, such as ammonium sulphate, which compresses the solvation layer and increases protein protein interactions. As the salt concentration of a solution is increased, more of the bulk water is associated with the ions. Consequently, less water is available to partake in the solvation layer rough the protein, which exposes hydrophobic interactions, aggregate and precipitate from solution.Isoelectric point precipitationThe isoelectric point (pI) is the pH of a solution at which the net primary charge of a protein causes zero. At a solution pH that is above the p the surface of the protein is principally negatively charged and therefore like-charged molecules will exhibit repulsive forces. At a solution pH that is below the pI, the surface of the protein is primarily positively charged and repulsion between proteins occurs. At the pI, the negative and positive charges cancel, repulsive electrostatic forces are reduced and the dispersive fo rces predominate, and will, therefore, cause aggregation and precipitation. The pI of most proteins lies in the pH range of 4-6. Mineral acids, such as hydrochloric and sulphuric acid are used as precipitants. The greatest disadvantage to isoelectric point precipitation is the irreversible denaturation caused by the mineral acids. For this reason isoelectric point precipitation is most often used to precipitate contaminant proteins, rather than target protein.Precipitation with organic solventsEthanol or methanol, if added to a solution may cause the proteins of the solution to precipitate. As the organic solvent gradually displaces water from the surface of the protein and binds it in layers around the organic solvent molecules, the solvation layer around the protein decreases. In that state, the protein can aggregate by attractive electrostatic and dipole forces. Parameters to consider are temperature (should be less than 0C to avoid denaturation), pH and protein concentration of the solution. Miscible organic solvents decrease the dielectric constant of water, which in effect allows two proteins to come together. At the pI the relationship between the dielectric constant and protein solubility is given bylog S = k/e2 + log S0S0 is an extrapolated value of S, e is the dielectric constant of the mixture and k is a constant that relates to the dielectric constant of water 98.Non- ionic hydrophilic polymersDextrans, polyethylene glycols and other polymers are used in precipitation of proteins due to their low flammability and are less likely to denature biomaterials compared to pI precipitation. These polymers attract water molecules away from the salvation layer around the protein, which enforces protein-protein interactions and induces precipitation. For the case of polyethylene glycol, the following equation models precipitationln(S) +pS = X CC is the polymer concentration, P is a protein-protein interaction coefficient, is protein- polymer interaction coe fficient andX = ( i i0 )RT is the chemical potential of component I, R is the universal gas constant and T is the absolute temperature 98.Flocculation by polyelectrolytesPolyelectrolytes form extended networks between protein molecules in solution. These include alginate, carboxylmethylcellulose, polyacrylic acid, tannic acid and polyphosphates. The pH of the solution determines the effectiveness of these polyelectrolytes. Anionic polyelectrolytes are used at pH above the pI. Cationic polyelectrolytes are used at pH above the pI. The precipitate may dissolve back into the solution if an excess of polyelectrolytes is used.Polyvalent metallic ionsEnzymes and nucleic acids are precipitated with the use of metal salts at low concentrations. Most frequently polyvalent metallic ions used are Ca+, Mg+, Mn+ or Fe+.Precipitation reactorsIndustrial scaled reactors that are used to precipitate large amounts of proteins, such as recombinant DNA polymerases from a solution includeBatch reactors The agent is slowly added to the protein solution under variety, so the aggregating particles tend to be regular in shape. The protein particles are exposed to a wide range of shear stresses for a log period of time and become mechanically stable.Tubular reactorsThe precipitating reagent and the feed protein solution are contacted in an area of mixing and then added into enlongeted tubes where precipitation occurs. Plug flow is approached by the elements as they move along the tubes. The tubular reactor is inexpensive to be constructed but can become long and slow in case that aggregation of the particles occur slowly.Continuous stirred tank reactorsCSTR reactors also known as vat or back mix reactors, run at steady state with a continuous flow of reactants and products in a well-mixed tank. It is a type of reactor mainly used in chemical engineering. A CSTR often refers to a mathematical model which is used to estimate the key unit operation variables when using a continuous agit ated-tank reactor to reach a specified output. Perfect mixing is demanded.Precipitation of L. starkeyiThe most common precipitation methods in the case of L. starkeyi cited in literature areIsoelectric focusingKoening and Day (1988) used precast IsoGel agarose isoelectric focusing plates, pH 5.0-8.5. A standard mixture of proteins was applied in the lane next to each sample and the protein profile was quantified by densitometer scans. The enzyme activity in the gel was determined by slicing an unstained gel into 0.9 mm sections. Each section was placrd in a test tube with 1.0 ml 0.05 M citrate/phosphate (pH 5.5) buffer, allowed to elute overnight at 4oC and assayed for enzyme activity. This method separated the protein mixture into five isoelectric bands. All five forms were found to have dextranase activity and exhibited the same Km values.Organic solventsPolyethylene glycol precipitation is often used. Nishimura et al. (2002) used this method in an effort to prepare total DNA from L. starkeyi for taxonomy analysis. They added phenol solution (phenol anaesthetise isoamyl alcohol=25241) to a test tube of Tris-SDS. The