Abstract:
Proteases present in shrimp processing waste are factors in the
technology of shrimp. The distribution of proteolytic activity in
shrimp parts was determined using hemoglobin and casein as substrates. The effects of various parameters upon activity of proteases
in the inedible portion were determined using muscle protein as a
substrate. Low pH active proteases from the hepatopancreas were
characterized using column chromatography and inhibitors.
A crude homogenate of shrimp processing waste showed maximum
proteolytic activity on casein at pH 6.25, although activity was
relatively uniform between pH 5.75 and 7.5. Hemoglobin digestion
was greatest between pH 3 and 3.65.
Specific activity was greatest in the foregut followed by the
hepatopancreas and remainder of the inedible portion. Shrimp which
contained food in their foreguts had greatest total activity in the foregut
followed by the hepatopancreas. The order was reversed for
shrimp which had not been feeding. Total activity was lowest in the
inedible portion with digestive organs removed. Only negligible
activity was detected in the muscle.
Autolytic changes in a model system were studied where shrimp
processing waste was the major protease source and muscle protein
served as the major substrate. Activity data showed a major maximum
at pH 3 and a minor broad maximum between pH 7 and 9. Maximum
autolytic activity occurred at 50°C for pH 3 and at 55°C for pH
7.4. Incubation of the inedible portion at 65°C for 30 min was sufficient
to inactivate the proteases. Proteases were unstable at low pH
and 10 min on ice at pH 1.8 were required for inactivation. Autolysis
at pH 3 was completely prevented by 10% NaCl while the inhibitory
effects were less at pH 7.4. Protein solubility was decreased by
NaCl at pH 3 and increased at pH 7.4. Heat-denatured muscle proteins
were less susceptible to hydrolysis, possibly through a reduction in
solubility. Changes occurred in the electrophoretic profile of soluble
proteins (pH 7.4) from a muscle mixture which was incubated at 50°C. Changes also occurred when incubation mixtures contained inedible
portion and these changes were more rapid than when inedible portion was absent.
Low pH active proteases from the hepatopancreas were studied
using hemoglobin as a substrate at pH 3.5. Gel filtration of a preparation
from the hepatopancreas on Sephadex G-150 separated hemoglobin
digesting activity in two distinct fractions. Chromatography
on DEAE-cellulose also separated activity into two fractions and
provided further evidence for the existence of at least two enzymes.
Fractions from chromatography were unaffected by phenylmethyl
sulphonylfluoride, unaffected or slightly activated by KCN, and
greatly inactivated by p-chloromercuribenzoate. EDTA greatly
activated all fractions. The result indicated that -SH groups are
important for enzyme activity. A crude homogenate of the hepatopancreas
and the major fraction from ion-exchange chromatography
were most active on hemoglobin between pH 3 and 4.
