The invention discloses a glucose 6-
phosphate dehydrogenase mutant with NMN < + > preference and a preparation method of the glucose 6-
phosphate dehydrogenase mutant, and belongs to the field of
gene engineering and
enzyme engineering. According to the present invention, the existing TmG6PDH-R7
mutant is adopted as the starting
protein, the error-prone PCR technology is adopted to carry out
random mutation, the random mutant
library is constructed, the plate positive clone high-
throughput screening method is adopted to obtain the clone with the most obvious color development, the recombinant
bacteria are cultured, and the purification is performed to obtain the mutant; through three rounds of screening, the glucose 6-
phosphate dehydrogenase mutant with enhanced NMN < + > preference as shown in the
amino acid sequence of SEQ ID NO.6, SEQ ID NO.8 or SEQ ID NO.10 is finally obtained, the
specific enzyme activity of TmG6PDH with NMN < + > as a coenzyme is remarkably improved, and compared with a currently known R7 mutant with the highest
specific enzyme activity, the
specific enzyme activity of the mutant with the
amino acid sequence as shown in SEQ ID NO.10 is improved by more than 5 times at 37 DEG C, and the specific
enzyme activity of the mutant with the
amino acid sequence as shown in SEQ ID NO.8 or SEQ ID NO.10 is remarkably improved. The method has a good application prospect in industrial production.