Combination of Thermostability Related Mutations

To produce multiple mutants with combinations of two or three thermostability-related amino acid mutations, restriction fragments containing the mutations were used to replace the corresponding mutation-free DNA fragments (16). In the DCase of Agrobacterium sp. KNK712, the mutation of the 57th amino acid is located in a Ndel-SacI DNA fragment of about 190 bp; the 203rd amino acid mutation is in a SaK-Clal DNA fragment of about 170 bp (hereinafter this fragment is referred to as fragment A); and the 236th amino acid mutation is in a Clal-SphI DNA fragment of about 75 bp. These fragments were replaced and the thermostabilities of the enzymes were then mea-

1. GVKRRFNTSILVDKSGKIVGKYRKIHLPGHKEYEAYRPFQHLEKRYFEPGD--LGFPVYDVDAAKMGMFICNDRRWPEAWRVMGLRGAEI I

2. GVKRRFNTSILVDKSGKIVGKYRKIHLPGHKEYEAYRPFQHLEKRYFEPGD--LGFPVYDVDAAKMGMFICNDRRWPETWRVMGLKGAEI I

3. GRKRRFNTSILVDRSGR IVGKYRKVHLPGHKEPQPGRKHQHLEKRYFEPGD--LGFGVWRAFDGVMGMCICNDRRWPETYRVMGLQGVEMV

4. PNKPPYNTLILIDNKGEIVQRYRKI-LPWCP----------IEGWY--PGD--TTYVTEGPKGLKISLI ICDDGNYPEIWRDCAMKGAELI

5. PRKAPYNTLVLIDNNGEIVQKYRKI - IPWCP----------IEGWY--PGG--QTYVSEGPKGMKISLI ICDDGNYPEIWRDCAMKGAELI

6. GV— LWNTAVVI SNSGLVMGKTRKNHIPRVG--------DFNESTYYMEGN--LGHPVFQTQFGR I AVN ICYGRHHPLNWLMYSVNGAEI I

7. YT — LYCTALFFSPQGQFLGKHRKL-MP-----------TSLERC IWGQGDG-STIPVYDTPIGKLGAA ICWENRMPLYRTALYAKGI ELY

8. YT — LYCTVLFFSPQGQFLGKHRKL-MP-----------T S L ERC I WGQGDG - ST I PVYDTP I GKLGAA I CWENRMP L YRT A L Y AKG I ELY

9. AS — RYL SQVFIDQNGDIVANRRKL-KP-----------THVERTIYGEGNG-TDFLTHDFGFGRVGGLNCWEHFQPLSKYMMYSLNEQIH

10. GS--LYMTQLVIDADGQLVARRRKL-KP-----------THVERSVYGEGNG-SDI SVYDMPFARLGALNCWEHFQTLTKYAMYSMHEQVH

11. RT--LYMSQMLIDADGITKIRRRKL-KP-----------TRFERELFGEGDG-SDLQVAQTSVGRVGALNCAENLQSLNKFALAAEGEQIH

12. GS — LYLGQCL I DDKGQMLWS RRKL - KP-----------THVERTV F GEG Y A - RD L I V S DTE L GRVG AL C CWEHL S P L S K Y A L Y S QHE A I H

13. AT--LYLTQVLI SPLGDVINHRRKI-KP-----------THVEK LV YGDG S GDSFEPVTQTEIGRLGQLNCWENMNPFLKSLAVARGEQIH

Figure 4. Optimal alignment with the CLUSTAL W program of the high-scoring segments in the predicted amino acid sequences of DCases and related enzymes. Identical amino acid residues in all enzymes are indicated by asterisks. 1, Agrobacterium sp. KNK712 DCase (104-192); 2, Agro-bacterium radiobacter NRRL B11291 DCase (104-192); 3, Pseudomonas sp. KNK003A DCase (104192); 4, Brevibacterium sp. R312 aliphatic amidase, PIR Ac. NO. JC1174 (104-186); 5, Pseudomonas aeruginosa aliphatic amidase, PIR Ac. No. A26741 (115-186); 6, rat beta-alaninesynthetase, PIR Ac. No. S27881 (176-252); 7, Arabidopsis thaliana nitrilase, PIR Ac. No. S31969 (126-199); 8, Arabidopsis thaliana nitrilase, PIR Ac. No. S22398 (133-206); 9, Rhodococcus rhodochrous aliphatic nitrilase, PIR Ac. No. A43470 (117-190); 10, Rhodococcus rhodochrous nitrilase, PIR Ac. No. A45070 (112-185); 11, Klebsiela ozaenae nitrilase, PIR Ac. No. A28658 (108-181); 12, Alcali-genes faecalis nitrilase, PIR Ac. No. A47181 (110-183); 13, Gloeocercospora sorghi cyanide hydra-tase, PIR Ac. No. JQ1613 (109-183).

Table S. Nucleotide Analysis of Mutagenized DCase Genes

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