Mutated penicillin expandase and process for preparing 7-ADCA using the same

#The present invention relates to a mutated penicillin expandase having high substrate specificity to penicillin G, recombinant cells expressing the mutated expandase, and a process of preparing 7-aminodeacetoxycephalosporanic acid (7-ADCA) using the mutated expandase.

#7-aminodeacetoxycephalosporanic acid (7-ADCA) is one of the important intermediates for the production of cephalosporins cefalexin, cefradine, and cefadroxil which are antibiotic compounds commonly and long used in humans and animals. The industrial process to synthesize 7-ADCA mainly includes two steps: a chemical ring expansion of penicillin G to phenylacetyl-7-ADCA and an enzymatic side chain cleavage of phenylacetyl-7-ADCA. However, the chemical reaction of the ring expansion is complex and expensive, and the by-products and the organic solvents (such as pyridine and HBr) are toxic to the environment. Therefore, an enzymatic reaction is greatly desirable to replace such chemical reaction.

#It has been reported that a natural enzyme, deacetoxycephalosporain C synthase (DAOCS, or expandase), may be responsible for the catalysis of the expansion reaction. Streptomyces sp. (such as Streptomyces clavuligerus, Streptomyces ambofaciens and Streptomyces chartreusis) can produce the expandase. As illustrated in EP-A-0341892, the expandase can be obtained from Streptomyces clavuligerus, and has been cloned. The expandase has been well studied for its chemical and functional properties, see EP-A-0366354. Unfortunately, the native expandase has less substrate specificity to penicillin G than the normal substrate penicillin N (Rollins, M. J. et al., Can. J. Microbiol. 34: 1196-1202 (1988) and Crawford, L., et al., Bio/Technology, 13: 58-62 (1995)). Penicillin G is commercially available at a low cost. In contrast, penicillin N is expensive and not easily available. Furthermore, even though penicillin N is expanded, its side chain cannot be easily removed. Accordingly, the chemical synthesis of 7-ADCA, rather than an enzymatical synthesis, is still used in industrial production.

#FIG. 1 shows the SDS-PAGE results of the purified DAOCS of the invention. Lanes 1 to 9 represent molecular weight markers and the purified DAOCS of YS5, YS8, YS11, YS12, YS16, YS49, YS53 and YS59 mutants, respectively. Each loaded DAOCS dose was in an amount of 5 μg.

#FIG. 2 shows the SDS-PAGE results of the purified DAOCS of the invention. Lanes 1 and 2 represent molecular weight markers and the purified DAOCS of YS67 mutant, respectively. The loaded DAOCS dose was in an amount of 5 μg.

#FIG. 3 shows the SDS-PAGE results of the purified DAOCS of the invention. Lanes 1 represents molecular weight markers. Lanes 2 to 5 represent samples irrelevant to the invention. Lanes 6 and 7 represent the purified DAOCS of SC29 and SC39 mutants, respectively. The loaded DAOCS dose was in an amount of 5 μg.

#The primary aspect of the present is to provide a mutated penicillin expandase having a better substrate specificity to penicillin G, wherein the mutated penicillin expandase comprises an amino acid substitution at one or more residual positions corresponding to those in a wild-type expandase selected from the group consisting of methionine 73, serine 79, valine 275, leucine 277, cysteine 281, glycine 300, asparagine 304 and isoleucine 305, provided that the amino acid substitution at the residue position of asparagine 304 is not N304L. More specifically, the invention provides a mutated penicillin expandase comprising one or more amino acid substitutions selected from the group consisting of M73T, S79E, V275I, L277K, C281Y, G300V, N304K, I305L and I305M.

#A “wild type penicillin expandase”, as used herein, refers to the native penicillin expandase obtained from Streptomyces sp. Preferably, the wild-type expandase is obtained from Streptomyces clavuligerus. The native penicillin expandase and its corresponding gene (cefE gene) have been well characterized and described in the prior art (e.g., EP-A-0366354 and EP-A-0341892). Persons skilled in the art can readily obtain the nucleic acid sequences of the wild-type penicillin expandase and the corresponding amino acid sequences from the prior art.

#The mutated penicillin expandase of the invention comprises the functional equivalents of the same. As used herein, the “functional equivalents” of the mutated penicillin expandase may contain further amino acid mutations (e.g., deletions, additions or substitutions) located at positions other than those described above, wherein said further amino acid mutations result in silent changes and thus do not substantially affect the function (e.g., enzyme activity) of the mutated penicillin expandase. Furthermore, in the “functional equivalents” of the mutated penicillin expandase, the specific amino acid substitutions (i.e., selected from M73T, S79E, V275I, L277K, C281Y, G300V, N304K, I305L and I305M) may be exchanged with other amino acid substitutions of similar characteristics which result in a silent change. For example, a mutated penicillin expandase with an amino acid substitution of “S79D” is functional equivalent to that with an amino acid substitution of “S79E,” since the amino acid D (aspartic acid) and E (glutamic acid) are both classified as acid amino acids and are of similar characteristics.