SEQUENCE OF THE GENE ENCODING A HIGHLY THERMOSTABLE NEUTRAL PROTEINASE FROM BACILLUS SP STRAIN EA1 - EXPRESSION IN ESCHERICHIA-COLI AND CHARACTERIZATION

The gene for a highly thermostable neutral proteinase (Npr) was isolat ed from Bacillus sp. strain EA1 by the polymerase chain reaction using consensus primers based on the sequences of npr genes from related sp ecies. The gene was sequenced and shown to be closely related to a neu tral proteinase gene from Bacillus caldolyticus strain YP-T; the matur e form of the enzyme differing by only a single amino acid. Enzyme sam ples were prepared from both the native organisms and also from recomb inant Escherichia coli expressing the two npr genes. The proteinase fr om strain EA1 was shown to be significantly more thermostable than tha t from B. caldolyticus and that this difference is the result of a sin gle amino acid substitution which is situated proximal to a region of the enzyme known to be crucial to conferring thermal stability. The ph ylogenetic relationship of EA1 to other Bacilli is also described.