Subtilisin purification

Tween 85 is used extensively for RME [84]. Russell and coworkers [234] used Tween 85/isopropanol microemulsions in hexane to solubilize proteins and not only showed >80% solubilization of cytochrome C at optimum conditions, but also proved that Tween 85 does not have a detrimental effect on the structure, function, and stability of subtilisin and cytochrome C. There are other reports available on the extraction and purification of proteins using Tween 85-RMs and also on the stability of protein activity in these systems [234]. It has also been shown that Tween 85-RMs can solubilize larger amounts of protein and water than AOT. Tween 85 has an HLB of 11, which indicates that it is soluble in organic solvents. In addition, it is biodegradable and can be successfully used as an additive in fertihzers [235,236]. Pfammatter et al. [35] have demonstrated that RMs made of Tween 85 and Span 80 can be successfully used for the solubilization and growth of whole cells. Recently, Hossain et al. [84] showed an enhanced enzymatic activity of Chromobacterium viscosum Hpase in AOT/Tween 85 mixed reverse micellar systems when compared to that in classical AOT-RMs. This is due to the modification of the interface in AOT-RMs caused by the co-adsorption of Tween 85, and increased availability of the oHve oil molecules (substrate) to the enzyme. 

Enzymatic digestion with subtilisin A, liq-liq partns, HCI addn (pH 1), EtOAc extn, propylester derivative, LC purification on Hypersil-ODS, 3... 

A different semisynthetic method involves the acylation of an amino alcohol with a peptide ester and the resulting amino alcohol is subsequently oxidized to the aldehyde 40 The acylation of H-Phe[CH2OH] with the peptide ester Z-Ala-Ala-Leu-OMe is carried out in 5% DMF/MeCN with the subtilisin distributed on the surface of macroporous silica gel. The resulting peptide alcohol is oxidized under mild conditions using anhydrous dimethyl sulfoxide and 20-fold excess of acetic anhydride with purification via flash chromatography 40] Z-Phe[CH2OH] has been oxidized under these conditions and the optical rotation indicates little epimerization as compared to literature values 11 40 ... 

Jayakumar A, Kang Y, Mitsudo K, et al. Expression of LEKTI domains 6-9 in the baculovirus expression system Recombinant LEKTI domains 6-9 inhibit trypsin and subtilisin A. Protein Expr Purif 2004 35 93-101. 

Subtilisin BPN was prepared through a series of protein purification steps applied to the fermentation broth. These steps included ultrafiltration ethanol precipitation DEAE (diethyl-aminoethyl) Tris Acryl batch anionic exchange SP (sulfopropyl) Tris Acryl column cationic exchange and, concentration with an Amicon stirred cell. The enzyme purity was determined to be -951 via spectroscopic assays that measure the ratio of active enzyme to total protein. In addition, purity was verified via HPLC and SDS-page (sodium dodecyl sulfate polyacrylamide gel electrophoresis). 

The purification and characterization of BmSI-7 and BmSI-6, two subtilisin inhibitors from Boophilus microplus (BmSI) was reported by Sasaki et al. (2008). The inhibitors were found to exhibit significant inhibition on the activity of purified Prl proteases from M. anisopliae. 

Knight, E., Jr. Fathey, D. Human fibroblast interferon an improved purification. J. Biol. Chem. 1981,256, 3609-3611. Wells, J.A. Powers, D.B. In vivo formation and stability of engineered disulfide bonds in subtilisin. J. Biol. Chem. 1986, 261, 6564-6570. 

Secretion into the culture medium is preferred for some industrial enzymes because it can facilitate purification. This is especially true if the protein reaches high concentrations. Schellenberger s group at Genencor devised a method to select for improved subtilisin-secreting mutants in Bacillus1 7"1. Mutants secreting up to five times as much enzyme were found after one round of error-prone PCR and selection. 

Its volume practically does not change on variation of pH, ionic strength, and in the presence of some organic solvents. It may be sterilized. It cannot be used, of course, in the presence of cellulases. For the immobilization of bacitracin on the cellulose we used processes that give rise to a stable bond of the ligand to the polysaccharide activation of the cellulose with 2, 4, 6-trichlorotriazine or benzoquinone directly or by diazo-tization of a 2-(4-aminophenylsulfonyl) ethyl derivative of cellulose. Bacitracin-celluloses were used for the purification of subtilisin DY and for isolation of alkaline proteinase from the culture medium of Bacillus subtllis. The best results were obtained with a biospecific adsorbent prepared by the attachment of bacitracin to bead cellulose activated by 2,4,6-trichlorotriazine. 

Purification of subtilisin DY and isolation of alkaline proteinase from culture medium of Bacillus subtilis. 

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