A gene encoding the extracellular a-amylase of Aevomonas hydrophila MCC-1 was cloned and expressed using its own promoter on the recombinant plasmid pCA101. Subcellular fractionation of Eschevichia coli JA221 carrying pCAlOl revealed that approximately 60 YO of the amylase activity was localized in the periplasmic space. The extracellular amylase was purified to homogeneity, identified as an a-type and its amino-terminal sequence was determined. Nucleotide sequence analysis predicted a 443 amino acid ORF and 24 amino acids at the amino terminus of the sequence that are not found in the secreted protein. This 24 amino acid sequence has many of the characteristics common to known signal peptides. The predicted amino acid sequence has considerable similarity with mammalian, invertebrate and Streptomycete a-amylases. Most of the amino acid residues that are involved in catalytic activity, substrate binding and calcium binding in several a-amylases were also present in A . hydrophila a-amylase at the corresponding positions.

A genomic library of Pseudomonas putida DNA was constructed by using plasmid pBR322. Transformants ofEscherichia coli in combination with Proeus mirabiis cells grown on creatinase test plates were screened for creatinase activity; transformants were considered positive for creatinase activity if a red-pink zone appeared around the colonies. One creatinase-positive clone was further analyzed, and the gene was reduced to a 2.7-kb DNA fragment. A unique protein band (with a molecular weight of approximately 50,000) was obsernred in recombinant E. coli by minicell analysis.

The gene for the creatinase from Pseudomonas putida NTU-8 was sequenced and revealed an open reading frame (ORF) of 1209 base pairs encoding a polypeptide of 403 amino acids with a calculated molecular weight (M(r)) of 45,691. The deduced amino acid sequence is very similar to that of the creatinase of Pseudomonas putida and Flavobacterium sp. An overproduction system for the chitinase signal peptide--creatinase hybrid gene was constructed by using the pQE-51 expression vector in E. coli JM109. The amount of this fusion enzyme was about 50% exported into the periplasmic space of E. coli.