Purification strain

Generally, for most fermentation processes to yield a good quality product at a competitive price, at least six key criteria must be met. (/) Fermentation is a capital intensive business and investment must be minimised. (2) The raw materials should be as cheap as possible. (J) Only the highest yielding strains should be used. (4) Recovery and purification should be as rapid and as simple as possible. (5) Automation should be employed to minimise labor usage. (6) The process must be designed to minimise waste production and efftciendy use all utilities (26,27). 

Purification. Enzyme purity, expressed in terms of the percent active enzyme protein of total protein, is primarily achieved by the strain selection and fermentation method. In some cases, however, removal of nonactive protein by purification is necessary. The key purification method is selective precipitation of the product or impurities by addition of salt, eg, sodium sulfate, or solvent, eg, ethanol or acetone by heat denaturation or by isoelectric precipitation, ie, pH adjustments. Methods have been introduced to produce crystalline enzyme preparations (24). 

The purification of j8-poly(L-malic acid) from A o-basiae has been reported involving methanol precipitation of the polymer in the form of the Ca salt [5]. This is possible because a high concentration of CaCOs is present in the growth medium. Unfortunately, the polymer acid is not soluble in aceton thus missing an additional purification step. In our hands, purification of jS-poly(L-malate) from several Aureobasidiae strains was unsatisfactory because of low yields and resisting impurities. 

Daubner, S. C., Astorga, A. M., Leisman, G. B., and Balwin, T. O. (1987). Yellow light emission of Vibrio barveyi strain Y-l purification and characterization of the energy-accepting yellow fluorescent protein. Proc. Natl. Acad. Sci. USA 84 8912-8916. 

O Kane, D. J., and Lee, J. (1986). Purification and properties of lumazine proteins from Photobacterium strains. Method. Enzymol. 133 149-172. 

It is often desirable to recover product and to choose a suitable strain of microorganism which produces extracellular rather than intracellular product. If the product stays inside the cells, the cells must be ruptured, so freeing intracellular enzyme, after which extraction or purification is performed to recover the valuable product. The fermentation broth has to be processed, and pass through several stages for separation and purification. The product requites a sequence of operations for high purification. Hie usual steps to follow are as follows. 

Purification and characterisation of galactose-induced pectinases from the exo-1 mutant strain of Neurospora crassa... 

A. tubingensis strain NW756 was cultivated for 55 h at 30 C in minimal medium according to Pontecorvo et al [1] supplemented with yeast extract (2g/l) and 10 g/1 endo-PG II digested polygalacturonic acid (PGA). Complete hydrolysis of PGA by endo-PG II resulted in a mixture of mono, di and trigalacturonate [2]. Four subsequent colunm chromatographic steps were used for purification of the enzyme for which the data are summarized in Table 1. 

Latus M, H-J Seitz, J Eberspacher, E Lingens (1995) Purification and characterization of hydroxyquinol 1,2-dioxygenase from Azotobacter sp. strain GPl. Appl Environ Microbiol 61 2453-2460. 

Allen JR, SA Ensign (1997) Purification to homogeneity and reconstitution of the individual components of the epoxide carboxylase multiprotein enzyme complex from Xanthobacter strain Py2. J Biol Chem 272 32121-32128. 

Beadle TA, ARW Smith (1982) The purification and properties of 2,4-dichlorophenol hydroxylase from a strain of Acinetobacter species. Eur J Biochem 123 323-332. 

Doukyu N, R Aono (1998) Purification of extracellular cholesterol oxidase with high activity in the presence of organic solvents from Pseudomonas sp. strain ST-200. Appl Environ Microbiol 64 1929-1932. 

Lei B, S-C Tu (1996) Gene overexpression, purification, and identification of a desulfurization enzyme from Rhodococcus sp. strain IGTS8 as a sulfide/sulfoxide monooxygenase. J Bacteriol 178 5699-5705. 

Schweizer D, A Markus, M Seez, HH Ruf, F Lingens (1987) Purification and some properties of component B of the 4-chlorophenylacetate 3,4-dioxygenase from Pseudomonas sp. strain CBS3. J Biol Chem 262 9340-9346. 

Wu J-F, C-W Sun, C-Y Jiang, Z-P Liu, S-J Liu (2005) A novel 2-aminophenol 1,6-dioxygenase involved in the degradation of p-chloronitrobenzene by Comamonas strain CNB-1 purification, properties, genetic cloning and expression in Escherichia coli. Arch Microbiol 183 1-8. 

Kengen SWM, GB Rikken, WR Hagen, CG van Ginkel, ALM Stams (1999) Purification and characterization of (per)chlorate reductase from the chlorate-respiring strain GR-1. J Bacteriol 181 6706-6711. 

Li T, JPN Rosazza (1997) Purification, characterization, and properties of an aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646. J Bacteriol 179 3482-3487. 

Itoh N, R Morihama, J Wang, K Okada, N Mizuguchi (1997) Purification and characterization of phenylacet-aldehyde reductase from a styrene-assimilating Corynebacterium strain, STIO. Appl Environ Microbiol 63 3783-3788. 

Jahns T, R Schepp, H Kalytwasser (1997) Purification and characterization of an enzyme from a strain of Ochrobactrum anthropi that degrades condensation products of urea and formaldehyde (ureaform). Can J Microbiol AE. 1111-1117. 

Sasaki M, A Akahira, L-i Oshiman, T Tsuchid, Y Matsumura (2005) Purification of cytochrome P450 and ferredoxin involved in bisphenol. A degradation from Sphingomonas sp. strain AOl. Appl Environ Microbiol 71 8024-8030. 

Uetz T, R Schneider, M Snozzi, T Egli (1992) Purification and characterization of a two-component monooxygenase that hydroxylates nitrilotriacetate from Chelatobacter strain ATCC 29600. J Bacteriol 174 1179-1188. 

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