Trypsin from pyloric caeca of Monterey sardine was purified by fractionation with ammonium sulfate, gel filtration, affinity and ionic exchange chromatography. Fraction 102, obtained from ionic ...exchange chromatography, generated one band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The molecular mass of the isolated trypsin was 25 kDa and showed esterase-specific activity on
Nα-
p-tosyl-
l-arginine methyl ester (TAME) that was 4.5 times greater than amidase-specific activity on
N-benzoyl-
l-arginine-
p-nitroanilide. The purified enzyme was partially inhibited by the serine-protease phenyl-methyl-sulfonyl fluoride (PMSF) inhibitor and fully inhibited by the soybean trypsin inhibitor (SBTI) and benzamidine, but was not inhibited by the metallo-protease inactivator EDTA or the chymotrypsin inhibitor tosyl-
l-phenylalanine chloromethyl-ketone. The optimum pH for activity was 8.0 and maximum stability was observed between pH 7 and 8. A marked loss in stability was observed below pH 4 and above pH 11. Activity was optimum at 50 °C and lost activity at higher temperatures. The kinetic trypsin constants
K
m and
k
cat were 0.051 mM and 2.12 s
−1, respectively, while the catalytic efficiency (
k
cat/
K
m) was 41 s
−1 mM
−1. General characteristics of the Monterey sardine trypsin resemble those of trypsins from other fish, especially trypsins from the anchovy
Engraulis japonica and
Engraulis encrasicholus and the sardine
Sardinops melanostica.
Nine fish species,
Gillichthys seta,
Oligoplites saurus,
Eucinostomus entomelas,
Synodus scituliceps,
Diplectrum pacificum,
Pseudopeneus grandisquamis,
Xenistius californiensis,
Arius seemanni, and
...Orthopristis reddinig, caught as bycatch by shrimp commercial fleet of the Guaymas, Sonora, México, were processed to produce protein concentrates using three drying processes (freeze-drying and heating at 65 and 110
°C). The soluble protein content from each process and species was evaluated. Significant differences were observed among processes. The composition of protein, observed by electrophoresis, was affected by temperature. Raw and freeze-dried protein from
S. scituliceps showed high proteolytic activity and unwent auto-hydrolysis. The highest proteolytic activity was at 65
°C and its optimum pH was 7.5. When assayed in the presence of several protease inhibitors, the enzymes were completely inactivated by soybean trypsin inhibitor. The in vitro digestibilities of the protein concentrates were higher when obtained by low-temperature methods.
Lipase activity of the midgut gland during larval and postlarval stages of
Penaeus vannamei was assayed to determine its capability to digest lipids from feed and involvement in digesting lipids from ...reserves during fasting conditions. Lipase activity was detected at all larval stages, increasing from nauplii to protozoea. Lipase isoenzymes at larval stages were evaluated by SDS-PAGE using 4-methylumbelliferone butyrate as the substrate. Results showed that shrimp larvae possess a nearly complete set of lipases starting with the first larval stage. In addition, to understand the effects of fasting conditions as a stress factor on lipase activity, intermolt shrimp were fasted up to 5
days, a period corresponding to the normal time that shrimp starve during molting, in which they cannot eat. Digestive lipases were affected by fasting, increasing in activity after 24
h of treatment, suggesting that lipid is used as an energy reserve during fasting. Proteins with lipase activity were identified and characterized by zymograms; the presence of more than one lipase enzyme could be one way to hydrolyze triacylglycerides more efficiently as the first step of fat assimilation and to obtain energy from fatty acids under fasting conditions.
An intracellular lipase present in the whiteleg shrimp
Litopenaeus vannamei was detected in pleopods. The lipase from pleopods was purified and characterized by biochemical and kinetic parameters. ...Purified intracellular lipase has a molecular mass of 196
kDa, the polypeptide is assembled by two monomers, 95.26 and 63.36
kDa. The enzyme lacks glycosylation, and it has an isoelectric point of 5.0. The enzyme showed the highest activity at a temperature range of 30–40
°C at pH 8.0–10.0. Activity was completely inhibited by tetrahydrolipstatin and diethyl
p-nitrophenyl phosphate, suggesting that the intracellular lipase is a serine lipase. The lipase hydrolyzes short and long-chain triacylglycerides, as well as naphthol derivatives at comparable rates in contrast to other sources of lipases. Specific activity of 930
U mg
−1 and 416.56
U mg
−1 was measured using triolein and tristearin at pH 8.0 at 30
°C as substrates, respectively. The lipase showed a
K
M,app of 41.03
mM and
k
cat/
K
M,app ratio of 4.88 using MUF–butyrate as the substrate. The intracellular lipase described for shrimp has a potential role in hydrolysis of triacylglycerides stored as fat body, as has been shown in humans.
Chymotrypsin was isolated from the viscera of Monterey sardine by ammonium sulphate fractionation, gel filtration, and ionic exchange chromatography. The approximate molecular weight was 26,000 and ...its isoelectric point was about 5. Identity as chymotrypsin was established by its catalytic specificity for amide or ester bonds on the synthetic substrates succinyl-
l-ala-ala-pro-
l-pheilalanine-
p-nitroanilide and benzoyl-
l-tyrosine-ethyl-ester, showing esterase activity 3.2-fold higher than amidase. It was inhibited by phenylmethylsulfonyl-fluoride and soybean trypsin inhibitor, partly inhibited by the specific chymotrypsin inhibitor
N-toluenesulfonyl-
l-phenylalanine chloromethyl-ketone, but not inhibited by EDTA or Benzamidine. Chymotrypsin showed its maximum activity at pH 8.0 and 50
°C for the hydrolysis of SAAPNA. The Michaelis–Menten constant was 0.074 mM with a catalysis constant of 18.6 seg
−1, and catalytic efficiency of 252 seg
−1
mM
−1. Results indicated that Monterey sardine chymotrypsin is a good catalyst and could be used as a biotechnological tool in food processing and using sardine industry wastes as a material for production of fine reagents.
The bacteria Microbacterium sp. 8L and Exiguobacterium mexicanum 8N are known to improve the culture of Artemia franciscana using baker's yeast as food. Using spectrophotometry, sodium dodecyl ...sulphate polyacrylamide gel electrophoresis (SDS-PAGE), substrate-SDS-PAGE and pH-stat in vitro-digestibility assays, the activity, molecular mass and hydrolysis on baker's yeast protein of proteases from extracellular polymeric substances (EPS) of the strains 8L and 8N along with the pathogenic strains Microbacterium sp. 8R and Vibrio parahaemolyticus 588 CECT (Vp) were studied. The EPSs of 8L and 8R showed one activity band, on which the serine inhibitor phenylmethylsulphonyl fluoride (PMSF) had no effect. The EPSs of 8N showed four bands; two were unaffected by PMSF, whereas one was affected, and the other was partially affected. The EPSs of Vp showed two bands, one partially inhibited by PMSF. No inhibitory effects from 1-chloro-3-tosylamido-7-amino-2-heptanone (trypsin inhibitor) were observed in the protease bands of the studied bacteria. The EPSs of 8L and 8N showed a similar degree of hydrolysis (pH-stat). The EPSs of 8L had the lowest Dice index of similarity of yeast protein profiles at 1 h of reaction. We conclude that the strain 8L could benefit A. franciscana by providing bacterial proteases for digestion of baker's yeast.
Total enzyme activity of whole viscera, and partial characterization of acidic proteases from Monterey sardine viscera are presented. Major proteolytic activity in alkali (pH 10) and minor activity ...in acid (pH 3) were detected. From purified acidic proteases, six fractions with high activity were selected. One fraction (42) showed one band on SDS–PAGE and two bands on isoelectrofocusing, with pI close to 4.0 and 4.5, respectively. The optimal pH for acidic protease activity was 2.5, with high stability in the acid range and marked loss of activity at neutral and alkaline pH. The optimum temperature was 45 °C, and activity was high at 10 °C, whereas denaturation occurred above 55 °C. Activity was inhibited by Pepstatin A but not by SBTI or EDTA. The general characteristics of these enzymes resemble those of the digestive enzymes of other fish. Because Monterey sardine is abundant in Mexico, it is a potential source for biological reagent production.
