Method for preparing immobilized arginine deiminase (ADI) and producing [14/15N]-L-citrulline

A fusion protein cipA-arc, wherein arginine deiminase (ADI) arc is immobilized on a protein crystalline inclusion of cipA to produce cipA-arc with a catalytic activity, wherein

• a specific activity of the cipA-arc is between 21.8 and 23.1 U/mg, wherein the definition of the specific activity is one unit of enzyme activity (1U) required to catalyze the conversion of [14/15N]-L-arginine into 1 μmol of citrulline per minute at 37° C. and pH 6.0;
• the catalytic activity catalyzes a conversion reaction of [14/15N]-L-arginine to produce [14/15N]-L-citrulline and the conversion reaction conducted at 37° C., pH of 6.5 for 5 h has a yield of 97.4% [14/15N]-L-citrulline;
• and
• the fusion protein cipA-arc can catalyze the conversion reaction for 50 times without a reduction in enzymatic activity more than 1.5%;
• and wherein
• the fusion protein cipA-arc is prepared by a method comprising the following steps:
• (1) preparing C. glutamicum competent cells;
• (2) transforming a recombinant plasmid pXMJ19-cipA-arc into the C. glutamicum competent cells prepared in step (1) through electric shock to obtain a genetically engineered recombinant strain, wherein the recombinant plasmid pXMJ19-cipA-arc is constructed by the following process:
• (a) obtaining a first target fragment comprising the gene sequence of SEQ ID NO:
• 1, and sequencing the first target fragment;
• subjecting each of the first target fragment and an expression vector pXMJ19 to double enzyme digestion using a Hindlll restriction enzyme site at the 5′ terminus relative to SEQ ID NO:
• 1 and a SaII restriction enzyme site at the 3′ terminus relative to SEQ ID NO:
• 1, recovering each of enzyme digestion products by gel, and ligating the first target fragment and the expression vector pXMJ19;
• and transforming a ligation product into Escherichia coli (E. coli) DH5α competent cells to obtain a positive transformant expressing a vector pXMJ19-cipA;
• and
• (b) obtaining a second target fragment comprising the gene sequence of SEQ ID NO:
• 2, and sequencing the second target fragment;
• subjecting each of the second target fragment and the vector pXMJ19-cipA to double enzyme digestion using a XhoI restriction enzyme site at the 5′ terminus relative to SEQ ID NO:
• 2 and a SacI restriction enzyme site at the 3′ terminus relative to SEQ ID NO:
• 2, recovering each of enzyme digestion products by gel, and ligating the second target fragment and the vector pXMJ19-cipA;
• and transforming a ligation product into E. coli DH5α competent cells to obtain a recombinant plasmid pXMJ19-cipA-arc;
• and
• (3) inducing expression of the fusion protein cipA-arc in the genetically engineered recombinant strain obtained in step (2) by culturing, and subjecting the resulting cells to ultrasonic disruption and centrifugation to obtain a precipitate, wherein the precipitate contains the fusion protein cipA-arc.