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Abstract
<p class="first" id="d4329821e77">A screening system using enrichment culture has
been established with the aim of obtaining
a novel enzyme for protein modification that has not been previously reported. This
enzyme catalyzes deamidation of the side-chain amide group of asparagine in proteins.
Enrichment culture of 390 soil samples was carried out with Z-Asn-Gly as the sole
source of nitrogen, and the reaction product, Z-Asp-Gly, was detected in the culture
supernatant of 102 strains. Strains with particularly high activity were Leifsonia
sp., Luteimicrobium sp., Microbacterium sp., and Agromyces sp., all belonging to the
class Actinobacteria. Of these, a protein-asparaginase (PA) was obtained from the
culture supernatant of Luteimicrobium album 333B-h1, and its reactivity with different
substrates and its basic enzymatic characteristics were investigated. Addition of
the enzyme solution resulted in specific deamidation of only the asparagine residue
in insulin chain B. The enzyme showed no reactivity with free asparagine or asparagine
in low molecular weight peptides. It was demonstrated that the enzyme reacts with
various protein substrates. In particular, proteins that have open structures, such
as casein or gelatin, were good substrates. The activity and stability of PA at different
temperatures and pH values were investigated. It was found that a temperature of 37°C
and a roughly neutral pH are optimal conditions for the enzyme.
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