Phenylalanine dehydrogenase mutant with improved substrate specificity and application of phenylalanine dehydrogenase mutant

A technology of phenylalanine dehydrogenase and mutants, which is applied in the fields of enzyme engineering and microbial engineering, can solve problems such as phenylalanine concentration interference and phenylketonuria screening errors, and achieve improved substrate specificity, The effect of improving detection sensitivity and reducing specificity

Active Publication Date: 2019-12-24
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since phenylalanine dehydrogenase also has activity on amino acids other than phenylalanine, such as L-norvaline, L-leucine, L-valine, L-phenylglycine, etc., Therefore, the use of phenylalanine dehydrogenase to detect the concentration of phenylalanine in the blood of newborns is often interfered by other impurities in the blood, and there are also large errors in the screening of phenylketonuria.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Preparation and expression of different phenylalanine dehydrogenases

[0045] Specific steps are as follows:

[0046] Chemical synthesis coding amino acid sequence is as shown in the gene of phenylalanine dehydrogenase shown in SEQ ID NO.1 (the nucleotide sequence of gene is as shown in SEQ ID NO.3); The gene obtained and pET-28a (+ ) plasmids were ligated after double digestion (EcoR I, BamHI), and the ligated products were transformed into Escherichia coli E.coli BL21(DE3). Pick 5 transformants from the medium, insert them into LB liquid medium for culture, and extract the plasmid after culturing at 37°C for 10 hours. The plasmid is sequenced, and the recombinant plasmid pET28a-PheDH and recombinant Escherichia coli pET28a-PheDH are obtained if the sequence is correct. / E. coli BL21.

[0047] Using the whole plasmid PCR technology, the obtained recombinant plasmid pET28a-PheDH was used as a template for site-directed mutagenesis to obtain mutants L51S, T...

Embodiment 2

[0063] Example 2: Substrate specificity of different phenylalanine dehydrogenases to different substrates

[0064] Specific steps are as follows:

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Abstract

The invention discloses a phenylalanine dehydrogenase mutant with improved substrate specificity and application of the phenylalanine dehydrogenase mutant and belongs to the technical field of enzymeengineering and microorganism engineering. The substrate specificity of the phenylalanine dehydrogenase mutant disclosed by the invention upon phenylpyruvic acid is remarkably improved when being compared with that of a wild type, and meanwhile, the specificity of the mutant upon L-norvaline, L-leucine, L-valine and L-phenylglycine is remarkably degraded, so that when the phenylalanine dehydrogenase mutant disclosed by the invention is applied to detection on the content of phenylalanine in samples such as blood, interference of other amino acids can be eliminated, the detection sensitivity can be improved, and the mutant has very good application prospects in preparing phenylalanine detection kits and phenylketonuria detection kits.

Description

technical field [0001] The invention relates to a mutant of phenylalanine dehydrogenase with improved substrate specificity and application thereof, belonging to the technical fields of enzyme engineering and microbial engineering. Background technique [0002] Phenylketonuria (PKU), as an autosomal recessive genetic disease, is one of the most common inherited metabolic diseases. The main reason for its occurrence is the mutation of the gene encoding phenylalanine-4α-hydroxylase (phenylalaninehydroxy-lase, PAH) or the coenzyme tetrahydrobiopterin (tetrahydrobiopterin BH4) in patients, resulting in phenylalanine-4α- The lack or reduction of hydroxylase activity will lead to the catabolism of phenylalanine (Phe) in the patient's body, which will lead to a large accumulation of phenylalanine and its metabolites in the patient's body. [0003] According to the blood phenylalanine concentration and the degree of enzyme deficiency, phenylketonuria can be divided into mild hyperp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12Q1/32
CPCC12N9/0018C12Q1/32C12Y104/0102G01N2333/90622G01N2800/7076
Inventor 饶志明王雅玲周俊平杨套伟徐美娟张显邵明龙
Owner JIANGNAN UNIV
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