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Chemically modified enzymes

A technology for modifying enzymes and proteases, applied in chemical instruments and methods, enzymes, biochemical equipment and methods, etc., can solve the problems that molecular biology methods cannot be easily routinely applied or synthesized in large quantities

Inactive Publication Date: 2000-03-01
GENENCOR INT INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, modification of enzyme features by protein engineering is limited to natural amino acid substitutions, and molecular biology methods designed to overcome this limitation cannot be readily adapted for routine applications or large-scale synthesis.

Method used

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Examples

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Embodiment 1

[0084] The subtilisin (SBL) gene from Bacillus lentus was cloned into the phage M13mp19 vector for mutagenesis (US Patent 5,185,258). Mutagenesis of the indicated oligonucleotides was performed as described in Zoller et al. (1983) Methods in Enzymology, 100: 1468-500. The mutated sequences were cloned, excised and reintroduced into the expression vector GG274 in the Bacillus subtilis host. PEG (50%) was added as a stabilizer. First by using pH 5.2 buffer (20mM sodium acetate, 5mM CaCl 2 ) through Sephadex TM The resulting crude protein concentrate was purified on a G-25 desalting matrix to remove small molecular weight contaminants. The pooled desalted column fractions were then loaded onto a strong cation exchange column (SP Sepharose TM FF), and the SBL was eluted with a gradient of 0-200 mM CaCl acetate buffer (pH 5.2). Salt-free enzyme powder was obtained after dialysis against Millipone pure water followed by lyophilization. Passed with 0.1M HCl 2 The purity of th...

Embodiment 2

[0085] 1-Bromo-3-methylbutane (1.7520 g, 0.0116 mol) and sodium methanesulfonate (1.554 g, 0.0116 mol) in anhydrous DMF (5 ml) were heated at 50° C. for 2 hours. Water (15ml) was added at room temperature and the reaction mixture was extracted with ether (3x30ml). The combined ether extracts were washed twice with brine, dried, concentrated and the residue was subjected to flash column chromatography on silica gel with EtOAc-hexane (1:4). The product was obtained as a colorless liquid (1.4777 g, 70%). IR(film): 3030(w), 3011(w), 2958(st), 2932(st), 2873(st), 1468(m), 1420(w), 1388(w), 1367(w), 1319(st), 1136(st), 955(st), 748cm -1 (st); 1 H NMR (200MHz, CDCl 3 ): δ3.33(s, 3H, CH 3 SO 2 S); 3.19(t, J=7.1Hz, 2H, SCH 2 CH 2 ), 1.70-1.58 (m, 3H, SCH 2 CH 2 CHMe 2 ), 0.95(d, 5.3Hz, 6H, CHMe 2 );13 C NMR (50MHz, CDCl 3 ): δ 50.60, 38.19, 34.59, 27.40, 22.06. Neopentyl Methanesulfonate

[0086] Neopentylsulfonate (3.054g, 0.0154mol), sodium methanesulfonate (2.272g, 0....

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Abstract

Modified enzymes are provided in which at least one amino acid, such as asparagine, leucine, methionine or serine, of an enzyme is replaced with a cysteine and the thiol hydrogen is replaced with a substituent group providing a thiol side chain selected from the group consisting of: a) -SR<1>R<2>, wherein R<1> is an alkyl and R<2> is a charged or polar moiety; b) -SR<3>, wherein R<3> is a substituted or unsubstituted phenyl; c) -SR<4>, wherein R<4> is substituted or unsubstituted cyclohexyl; d) -SR<5>, wherein R<5> is C10-C15 alkyl. Also, methods of producing the modified enzymes are provided, as well as detergent and feed additives and a composition for the treatment of a textile. A method for using the modified enzymes in organic synthesis is additionally provided. Further, modified enzymes having improved activity, altered pH profile and / or wash performance are provided.

Description

Background of the invention [0001] Modification of enzyme properties by site-directed mutagenesis is limited to substitution of natural amino acids, but molecular biology strategies to overcome this limitation have recently been developed [Cornish, V.W. et al. (1995) Angew.chem., Int.Ed.Engl.34: 621]. But the latter approach is generally not easy to apply in most experiments. In contrast, the chemical modification of enzymes offers a broad potential for simple and flexible modification of enzyme structures, thereby opening up a wide range of possibilities for controllable regulation of enzyme specificity. [0002] In the past, it has been developed to change the enzyme properties by chemical modification. In 1966, it was developed by Bender (Polger, L., etc., 1966, Journal of the American Chemical Society, 88: 3153) and Koshland (Neet, K.E., etc., 1996, Proceedings of the National Academy of Sciences of the United States, 56:1606) group first reported that they created thios...

Claims

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Application Information

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IPC IPC(8): C11D3/386C12N9/00C12N9/54C12S3/00D06M16/00
CPCC12N9/00C12N9/54C11D3/38663
Inventor R·R·伯特T·P·格拉卡C·米特施逊G·德桑提斯J·B·琼斯
Owner GENENCOR INT INC
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