PATENT CLAIM ANALYSIS

Application Number: 15907340
Application Type: Utility
Filing Date: 2018-02
Publication Date: 2018-09
Patent Classification: ["435", "188000"]

Abstract:
An arginine deiminase mutant with improved enzyme activity and temperature stability and application thereof were provided, belonging to the technical field of genetic engineering and enzyme engineering. The arginine deiminase mutant is proline, namely Gly292 Pro, mutated from glycine near an enzyme active center. A wild-type arginine deiminase arcA coding gene is molecularly modified by a site-directed mutation technique to obtain a mutant enzyme ADIG292P, which has glycine at position 292 of an amino acid sequence of the wild type arginine deiminase mutated to proline. The arginine deiminase, modified by site-directed mutation, of the present invention has 1.5 times of increase in enzyme activity and 5.43 times of increase in half-life period at 40° C. compared with the wild-type enzyme, which solves the problems of low catalytic ability and temperature stability during the catalytic synthesis of citrulline using arginine deiminase, and lays a foundation for industrial production of efficient synthesis of citrulline and medication application.

Claim (Index 14):
The application of  claim 13 , comprising constructing the recombinant strain by following steps:\n 1) designing a primer according to a gene sequence of arginine deiminase arcA of  Enterococcus faecalis  SK32.001; by taking  Enterococcus faecalis  SK32.001 comprising an arginine deiminase sequence as a template, obtaining a gene segment comprising arginine deiminase arcA by a PCR method to construct a recombinant plasmid with a connection to an expression vector pET-28a; 2) using B-FITTER software to recognize key amino acid residues that have adverse effects on temperature stability in enzyme molecules; then using SWISS-MODEL software to perform protein structure simulation on parent arginine deaminase, so as to obtain a tertiary structure of an arginine deaminase; through a comparative analysis, determining an amino acid site to be mutated is glycine at position 292; 3) designing a mutation primer, using a one-step PCR method to perform site-directed mutation on a nucleotide sequence of arginine deaminase, and replacing the amino acid at position 292 to obtain a recombinant vector comprising an arginine deaminase mutant gene sequence; and 4) enabling the recombinant vector comprising the arginine deaminase mutant gene sequence to enter competent cells of  Escherichia coli E. coli  BL21(DE3), inducing expression, collecting thalli, and using Ni-NTA for protein separation and purification after ultrasonication on cells to obtain the arginine deaminase mutant.

Metadata:
- Claim Count in Document: 1.0
- Percentile: 88.0
- Lexical Diversity: 1.85556
- Patent Class: 435.0
- Transitional Phrase Type: open
- Component Type: 1
- Foreign Priority: True
- Related Applications: ['13214398', '15876478', '14197236', '14981855', '10210115']

Analysis Scores:
- 35 USC 101 Eligibility (BERT): 0.6098965009673223
- 35 USC 102 Novelty (BERT): 0.5057211503466961
- Combined Prediction Score: 0.5994789659052597
- Mean Citation Score: 256.962756
- Max Citation Score: 277.8915
- Similarity Product: 193.7350881420672

Labels:
- Claim Label 101: 1
- Claim Label 102: 1
- Claim Label 103: 1
- Claim Label 112: 1
- Combined Label: 1
- Label 101 Adjusted: 1

Dataset: test