1.14.11.26 A106T 80% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate 671443 1.14.11.26 A177V random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme -, 744105 1.14.11.26 A311V random mutagenesis 744105 1.14.11.26 C155Y 90% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate 671443 1.14.11.26 C155Y/Y184H/V275I/C281Y 580% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate 671443 1.14.11.26 C281Y 200% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate 671443 1.14.11.26 E16G/T90A/T304A random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 E16G/T90A/T304A site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 E209Q random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 E82D random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme -, 744105 1.14.11.26 F267L random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 G300V 410% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate 671443 1.14.11.26 G79E 90% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate 671443 1.14.11.26 H244Q 140% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate 671443 1.14.11.26 I193V random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 I305L 230% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate 671443 1.14.11.26 I305M 380% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate 671443 1.14.11.26 L236V random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 L277Q 270% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate 671443 1.14.11.26 M184I random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 M188I 90% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate 671443 1.14.11.26 M188V 150% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate 671443 1.14.11.26 M229V random mutagenesis 744105 1.14.11.26 M306I hydroxylation reaction of deacetoxycephalosporin C, EC 1.14.11.26, is abolished, 59% of wild-type ring expansion activity 659322 1.14.11.26 M73T 180% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate 671443 1.14.11.26 additional information mutagenic cefF library creation by random mutagenesis and screening of the mutant deacetylcephalosporin C synthase enzyme library, homology modeling of DACS structure and mapping of mutant positions. Process-level biotransformation reaction of cephalosporin G to deacetylcephalosporin G by mutants of deacetylcephalosporin C synthase. Deacetylcephalosporin G can be converted completely into hydroxymethyl-7-amino-cephalosporanic acid (HACA) in about 30 min by a subsequent reaction, thus facilitating scalability toward commercialization. Directed-evolution strategies such as random, semirational, rational, and computational methods are used for systematic engineering of DACS for improved activity with cephalosporin G -, 744105 1.14.11.26 additional information truncation of C-terminus to residue 310, 2fold enhancement of ring expansion reaction of penicillin G. Double mutant with truncation at residue 310 and M306I, selective catalyzation of ring expansion. Triple mutant with truncation at residue 310, M306I and N305L, selective catalization of ring expansion with improved kinetic parameters 659322 1.14.11.26 N304K 220% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate 671443 1.14.11.26 N305L 107% of wild-type ring expansion activity, 85% of wild-type hydroxylation activity 659322 1.14.11.26 N305L improved ability to convert penicillin analogs in ring expansion reaction of EC 1.14.20.1 661796 1.14.11.26 P186L random mutagenesis and site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 P72L random mutagenesis and site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 R182S random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 R182W random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 R308L improved ability to convert penicillin analogs in ring expansion reaction of EC 1.14.20.1 661796 1.14.11.26 S251F random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 S260G random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T273A random mutagenesis 744105 1.14.11.26 T90A random mutagenesis -, 744105 1.14.11.26 T90A/A311V site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/M184I/I193V/F267L site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/R182S site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/S251F site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/S260G site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V171L/F267L site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/A241V/V307A site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/G108D site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/N313D site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/R91G site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/R91G/A241V/V307A site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/T96S/A241V/V307A site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/T96S/G255D/A280S site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/V226I site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/A40V/M229I/T273A/V221P site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/F195L site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/P7L/A237V site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/P7L/T273A site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/R250L site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/V206I/A210V site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T90A/P72L/A311V/V206I/A210V/T273A site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 T91A 110% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate 671443 1.14.11.26 V150A random mutagenesis 744105 1.14.11.26 V171L random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 V171M random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 V221A random mutagenesis -, 744105 1.14.11.26 V221H site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 V221P site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 V221T random mutagenesis and site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 V249I random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme 744105 1.14.11.26 V275I 270% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate 671443 1.14.11.26 V275I/I305M 500% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate 671443 1.14.11.26 W82A 5.5% of wild-type ring expansion activity, 71% of wild-type hydroxylation activity 659322 1.14.11.26 W82A ring expansion reaction of EC 1.14.20.1 is reduced 659322 1.14.11.26 W82S 44% of wild-type ring expansion activity, 18% of wild-type hydroxylation activity 659322 1.14.11.26 Y184H 200% relative activity compared to the wild type enzyme using 1 mM penicillin G as substrate 671443 1.14.11.26 Y38C random mutagenesis 744105