Novel D-Amidase from Arthrobacter sp. KNK1101J for High-Activity Production
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Solution Overview
Problem
Current methods for producing D-α-amino acids via fermentation are inefficient and lack effective microorganisms with high amidase activity, particularly those belonging to the genus Arthrobacter, which are not well-characterized in terms of their amidase enzyme's amino acid and nucleotide sequences.
Innovation Solution
Isolation and characterization of a novel D-amidase from Arthrobacter sp. KNK1101J, including its amino acid and nucleotide sequences, and the development of a method for producing the enzyme using recombinant DNA technology to enhance amidase production in host microorganisms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional amidase-producing microorganisms are used, then amidase production is possible, but the hydrolytic activity is low and cannot be said to be satisfactory for industrial applications
Solution Approach 1:
The patent identifies and utilizes specific amino acid sequence parameters (SEQ ID NO: 1 and variants) to create amidase with improved hydrolytic activity. By changing the molecular parameters of the enzyme through genetic modification, the patent achieves both high productivity and reliability for industrial applications.
Solution Approach 2:
The patent creates recombinant DNA copies of the amidase gene from Arthrobacter sp. KNK1101J and introduces them into host microorganisms. This copying approach allows mass production of the high-activity amidase while maintaining the original enzyme's superior hydrolytic properties.
2Productivity
If amidase production methods are developed, then D-α-amino acid production is possible, but the yield and optical purity need improvement
Solution Approach 1:
The patent utilizes specific amino acid sequence parameters (SEQ ID NO: 1 and variants) to create amidase with improved hydrolytic activity. By changing the molecular parameters of the enzyme through genetic modification, the patent achieves both high productivity and reliability for industrial applications.
Solution Approach 2:
The patent employs screening methods that measure hydrolytic activity and optical purity to select and optimize amidase variants. This feedback loop allows continuous improvement of both yield and optical purity through iterative selection of superior enzyme variants.
3Productivity
If genetic engineering is used to modify microorganisms, then amidase production can be enhanced, but the complexity of the process increases
Solution Approach 1:
The patent extracts the essential amidase gene from Arthrobacter sp. KNK1101J and separates it from the complex native genetic system. This extraction allows the gene to be transferred to simpler host systems like E. coli, reducing overall process complexity while maintaining high productivity.
Solution Approach 2:
The patent creates a universal amidase production system where the same gene can be expressed in multiple host organisms (Arthrobacter sp., E. coli, and other bacteria). This universality simplifies the genetic engineering process by allowing selection of the most convenient host without changing the core enzyme sequence.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables efficient production of D-amino acids from amino acid amides or esters, improving the yield and optical purity of D-α-amino acids, addressing the limitations of previous methods by utilizing a well-defined and optimized amidase-producing microorganism.
Implementation Method 1
Amidase (enzyme No. [EC3.5.1.4]) is an enzyme, which hydrolyzes a carboxylic amide to carboxylic acid and ammonia
Implementation Method 2
amidase catalyzes hydrolysis of an ester as a substrate
Data Source
AI summary
It is an object of the present invention to provide a novel amidase that is useful for production of an optically active amino acid, and in particular, a D-amino acid, and a production method thereof.The present invention relates to a novel D-amidase isolated and purified from the Arthrobacter sp. KNK1101J, a gene encoding the above amidase, a recombinant plasmid comprising the above gene, and a transformant into which the above amidase gene has been introduced. In addition, the present invention also relates to a method for producing the amidase, comprising culturing the Arthrobacter sp. KNK1101J or the above transformant, and collecting the above amidase.