Modified sarcosine oxidase, and gene and production method therefor

A modified sarcosine oxidase selected from the following:

• (a) a sarcosine oxidase wherein, when an amino acid sequence of the sarcosine oxidase is aligned with the amino acid sequence of SEQ ID NO:
• 1, at least one position of the following (i) to (vi) is substituted with the following amino acids, and having reduced relative activity to L-proline, wherein
• (i) the amino acid at the position corresponding to position 320 in SEQ ID NO:
• 1 is asparagine,
• (ii) the amino acid at the position corresponding to position 324 in SEQ ID NO:
• 1 is glycine,
• (iii) the amino acid at the position corresponding to position 348 in SEQ ID NO:
• 1 is tyrosine,
• (iv) the amino acid at the position corresponding to position 222 in SEQ ID NO:
• 1 is alanine or histidine,
• (v) the amino acid at the position corresponding to position 260 in SEQ ID NO:
• 1 is isoleucine, and/or
• (vi) the amino acid at the position corresponding to position 314 in SEQ ID NO:
• 1 is serine, and
• wherein relative activity to L-proline is reduced relative to the wild type;
• (b-1) a protein having one or more amino acid substitutions of (a), comprising an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO:
• 1, and having sarcosine oxidase activity with reduced reactivity to L-proline compared to that of a protein prior to modification;
• (b-2) a modified sarcosine oxidase that is a protein having one or more amino acid substitutions of (a), comprising an amino acid sequence having 75% or more full-length amino acid sequence identity with the amino acid sequence of SEQ ID NO:
• 1, and having sarcosine oxidase activity with reduced reactivity to L-proline compared to that of a protein prior to modification, and wherein the homologous region consisting of positions 6 to 86, positions 88 to 92, positions 94 to 97, positions 101 to 102, positions 104 to 109, positions 112 to 114, position 118, positions 120 to 121, positions 127 to 128, positions 130 to 131, positions 133 to 134, positions 136 to 137, positions 139 to 142, positions 144 to 154, positions 156 to 166, positions 168 to 178, positions 181 to 182, position 184, position 186, positions 188 to 189, positions 191 to 192, positions 194 to 195, positions 197 to 213, positions 216 to 233, positions 235 to 238, positions 242 to 249, positions 252 to 262, positions 264 to 270, positions 272 to 274, positions 276 to 286, positions 288 to 292, position 294, positions 296 to 300, positions 302 to 306, positions 308 to 311, positions 313 to 332, positions 335 to 358, positions 360 to 362, positions 364 to 375, positions 377 to 378, and positions 380 to 382 of SEQ ID NO:
• 1 has 95% or more amino acid sequence identity with the homologous region consisting of positions corresponding respectively to the modified sarcosine oxidase;
• (c-1) a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence of SEQ ID NO:
• 2, 3, or 4, and having sarcosine oxidase activity with reduced reactivity to L-proline compared to that of a protein prior to modification;
• (c-2) a modified sarcosine oxidase that is a protein comprising an amino acid sequence having 75% or more full-length amino acid sequence identity with the amino acid sequence of SEQ ID NO:
• 2, 3, or 4, and having sarcosine oxidase activity with reduced reactivity to L-proline compared to that of a protein prior to modification, and wherein the homologous region consisting of positions 6 to 86, positions 88 to 92, positions 94 to 97, positions 101 to 102, positions 104 to 109, positions 112 to 114, position 118, positions 120 to 121, positions 127 to 128, positions 130 to 131, positions 133 to 134, positions 136 to 137, positions 139 to 142, positions 144 to 154, positions 156 to 166, positions 168 to 178, positions 181 to 182, position 184, position 186, positions 188 to 189, positions 191 to 192, positions 194 to 195, positions 197 to 213, positions 216 to 233, positions 235 to 238, positions 242 to 249, positions 252 to 262, positions 264 to 270, positions 272 to 274, positions 276 to 286, positions 288 to 292, position 294, positions 296 to 300, positions 302 to 306, positions 308 to 311, positions 313 to 332, positions 335 to 358, positions 360 to 362, positions 364 to 375, positions 377 to 378, and positions 380 to 382 of SEQ ID NO:
• 1 has 95% or more amino acid sequence identity with the homologous region consisting of positions corresponding respectively to the modified sarcosine oxidase;
• (d) a protein comprising the amino acid sequence of SEQ ID NO:
• 2, 3, 4, or 22;
• and
• (e) a protein comprising an amino acid sequence wherein 1 or several amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO:
• 2, 3, 4, or 22 at positions other than the positions corresponding to positions 222, 260, 314, 320, 324, 343, and 348 in SEQ ID NO:
• 1, and having sarcosine oxidase activity with reduced reactivity to L-proline compared to that of a protein prior to modification.