Mutant of phenylalanine deaminase with anabaena source

A technology of phenylalanine deaminase and mutants, applied in the field of protein engineering, can solve the problems of enzyme application limitations and achieve good pH stability

Inactive Publication Date: 2017-03-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the extreme environment in the intestinal tract, the application of this enzyme is limited

Method used

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  • Mutant of phenylalanine deaminase with anabaena source
  • Mutant of phenylalanine deaminase with anabaena source
  • Mutant of phenylalanine deaminase with anabaena source

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] (1) Construction of mutant E75L:

[0020] Using pET28a-PAL as a template, using the primers shown in Table 1 and the conditions shown in Table 2, the recombinant vector pET28a-PAL / E75L carrying the gene encoding the mutant was obtained by PCR. For the construction method of pET28a-PAL, see Moffitt, M.C., Louie, G.V., Bowman, M.E., Pence, J., Noel, J.P. and Moore, B.S. (2007) Discovery of twocyanobacterial phenylalanine ammonia lyases: Kinetic and structural characterization. Biochemistry-Us, 46 ,1004-1012.

[0021] Table 1 Primers

[0022]

[0023] Table 2 Whole plasmid PCR amplification reaction system

[0024]

[0025]

[0026] The PCR amplification reaction conditions are:

[0027]

[0028] PCR products were identified by agarose gel electrophoresis. Then the PCR product was purified and digested and transferred to BL21 host.

[0029] (2) Inoculate recombinant Escherichia coli BL21 / pET28a-PAL / E75L in 4 mL of LB medium (peptone 10 g / L, yeast extract 5...

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Abstract

The invention discloses a mutant of phenylalanine deaminase with an anabaena source and belongs to protein engineering. The phenylalanine deaminase E75L obtained in the invention has trypsin degradation resistance and low pH resistance which are greatly improved compared with those of wild-type enzymes. The phenylalanine deaminase E75L provided by the invention achieves the effect of promoting application of the phenylalanine deaminase in oral medication of phenylketonuria (PKU) to a certain degree.

Description

technical field [0001] The invention relates to a mutant of phenylalanine deaminase derived from anabaena, which belongs to protein engineering. Background technique [0002] Phenylalanine deaminase (PAL) can be used for the effective and long-term treatment of phenylketonuria (PKU). Because of its convenience and effectiveness, oral administration of this enzyme has attracted increasing attention. However, due to the extreme environment in the intestine, the application of this enzyme is limited. A mutant of PAL (Av-PAL) derived from Anabaena variabilis can improve the application of this enzyme in this respect. Contents of the invention [0003] The present invention aims to provide a phenylalanine deaminase mutant with improved acid resistance and heat resistance, encoded by the nucleotide sequence shown in SEQ ID NO.1. [0004] The present invention also provides a method for recombinantly expressing the phenylalanine deaminase mutant, which uses Escherichia coli BL...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21A61K38/51A61P25/28C12R1/19
CPCC12N9/88A61K38/00C12N15/70C12Y403/01005
Inventor 周哲敏张帆周丽崔文璟刘中美郭军玲陆灿杰
Owner JIANGNAN UNIV
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