Decapods belong to Pancrustacea, all are of ecological and economic importance since many species are profitable either harvested in the wild or produced in aquafarming. The description of ...biochemical physiology is paramount for both, ecology and economy. Being protein the most expensive ingredient in fabricated feeds, explaining food protein digestion is a chief step in understanding nutrition.
The review deals with techniques used for the study of peptidases, enzymes hydrolyzing peptide bonds in proteins, with an emphasis in the study of food protein digestion in decapods.
Such techniques have shown what, who, and where peptidases are, and the importance of proteins in nutrition and hence in protein digestion. The methods used in the research of digestive peptidases in decapods are enlisted and some remarks are given, such techniques can be applied in other Crustacea and even Insecta species yet to be studied and decodified.
•Decapoda and Hexapoda belongs to Pancrustacea.•Decapods are of ecological and economical importance and hence their biology.•Techniques are available to study how decapods digest food protein.•A plethora of Decapoda and even Insecta species are to be studied.
In vitro assays used porcine or bovine trypsin as models of exogenous enzymes to determine functioning in the presence of enzymatic extracts from the digestive gland of whiteleg shrimp Penaeus ...vannamei. Using electrophoresis and zymograms, when enzymes from the shrimp were mixed in the absence of protein substrate, they hydrolysed the trypsin from bovine or porcine origin. Porcine or bovine trypsin, when mixed with shrimp enzymes in pH‐stat assays in the presence of shrimp commercial feed, fish meal, or casein, there was added activity to hydrolyse the protein substrate. Hydrolysis of protein substrate was twofold to threefold stronger if exogenous enzymes were added. Results suggest that porcine or bovine trypsin could be used as feed supplements for whiteleg shrimp P. vannamei to enhance hydrolysis of proteins in feeds, because the commercial enzymes contributed to the hydrolysis of the protein in the three substrates in the presence of shrimp enzymes.
To test the efficacy of adding enzyme supplements to feeds, an in vitro study was conducted by mixing bovine trypsin or proteinases from the gastric juice of the Cortez swimming crab Callinectes ...bellicosus with an enzyme extract from the digestive gland of the whiteleg shrimp Penaeus vannamei. Enzymes alone and mixtures were tested for hydrolysing proteinaceous natural substrates (bovine casein, bovine haemoglobin, and bovine serum albumin). All enzyme preparations hydrolysed casein. Shrimp enzymes hydrolysed haemoglobin but not serum albumin. Bovine trypsin and crab proteinases hydrolysed serum albumin but not haemoglobin. The mixture of shrimp and crab enzymes generated more hydrolytic products of serum albumin than shrimp enzymes alone. Shrimp enzymes mixed with bovine trypsin did not hydrolyse albumin because the bovine trypsin vanished; shrimp enzymes hydrolysed bovine trypsin. Results indicated that it is naive to assume that proteinolytic enzymes from different species will add their catalytic capabilities if mixed; here, we demonstrated that they may act as proteinases and will hydrolyse available protein regardless of its function. Our conclusion is that enzyme supplements should be tested in vitro before intending them as exogenous proteinases in feeds. This technique can be used to assess the compatibility and additivity of proteinases when mixed for biotechnological purposes. Besides, the technique can demonstrate who hydrolyses whom.
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.
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.
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.
EFFECT OF pH AND TEMPERATURE ON JUMBO SQUID PROTEINS DE LA FUENTE-BETANCOURT, G; GARCÍA-CARREÑO, F; NAVARRETE DEL TORO, M.A ...
Journal of food biochemistry,
April 2009, Letnik:
33, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Evaluation of the effect of pH (2 to 13) and temperature (0 to 50C) on functional properties of jumbo squid proteins was performed, followed by a 2 x 3 factorial design for producing squid protein ...hydrolysates bearing useful functional properties. In particular, the effects of pH (8, 9 and 10) and temperature (30, 35, 40C) were evaluated. Alcalase and papain were tested on each treatment. The protein recovery, whippability and emulsifying capacity of the hydrolysates were evaluated. Almost 80% of the proteins were recovered in water-soluble form after hydrolysis with papain at pH 10 at the three temperatures. The highest values of whippability (245 ± 17.7%), foam stability (100%), emulsion-forming capacity (27 ± 0.97%) and stability (99.99 ± 8.8%) occurred with papain-produced hydrolysates. When squid protein was treated at 50C and pH 8, the highest whippability value (390.0 ± 0.1%) and foam stability (100%) were obtained when no enzyme was added. This paper assesses how process variables, particularly temperature and pH, affect the functional properties of squid proteins. Making use of such process variables will produce more useful and efficient processes, as the application of a hydrolysis system. The processing of jumbo squid protein to obtain proteins bearing adequate functional properties would be an inexpensive way to provide added value to this marine resource and the production of a high-quality protein ingredient. These results hold promise for jumbo squid proteins as useful food ingredient because of their functional properties.
Over the long course of evolution and under the selective pressure exerted by pathogens and parasites, animals have selectively fixed a number of defense mechanisms against the constant attack of ...intruders. The immune response represents a key component to optimize the biological fitness of individuals. Two decades ago, prevention and control of diseases in crustacean aquaculture systems were considered priorities in most shrimp-producing countries, but knowledge was scarce and various pathogens have severely affected aquaculture development around the world. Scientific contributions have improved our understanding of the crustacean immune response. Several studies confirm the central role played by proteases in the immune response of animals, and the cooperative interaction of these enzymes in a wide variety of organisms is well known. This review summarizes the current information regarding the role of cysteine proteases in the immune system of Crustacea and points to aspects that are needed to provide a better integration of our knowledge.
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.
The present study describes the activity and some characteristics of proteinases in the hepatopancreas of red shrimp
Pleoticus
muelleri during the different stages of the molting cycle. Proteolytic ...activity was highest between pH 7.5 and 8. The hepatopancreatic protein content in the premolt stage was higher than in the other stages of the molting cycle (
P<0.05). No significant differences were found in total proteolytic activity in the hepatopancreas when comparing molting stages. The proteolytic activity of the
P. muelleri hepatopancreas enzyme preparations is the main responsibility of serine proteinases. TLCK, a trypsin inhibitor, reduced azocasein hydrolysis between 26% (intermolt) and 37% (premolt). TPCK, a chymotrypsin inhibitor, did not decrease hydrolytic activity, except for in postmolt. Low trypsin and chymotrypsin activities were found during intermolt, and increased in postmolt. The electrophoretogram of the enzyme extracts shows 12 bands of activity during intermolt (from 16.6 to 53.1 kDa). Some fractions were not detected in the postmolt and premolt stages. Three low molecular weight trypsin forms (17.4, 19.1 and 20 kDa) were found in all molting stages. One band of chymotrypsin (21.9 kDa) was observed in all molting stages. High molecular mass active bands (66–205 kDa) could not be characterized with inhibitors. Comparison of the protease-specific activity of the hepatopancreas of some species indicated a relationship between digestive enzyme activity and feeding habits of the shrimp. Omnivorous shrimp, such as
Penaeus
vannamei (syn:
Litopenaeus vannamei) and
Penaeus
monodon, showed higher protease activity than the carnivorous shrimp,
Penaeus
californiensis (syn:
Farfantepenaeus californiensis) and
P.
muelleri. In fact, the enzymatic activity in the hepatopancreas of
P.
muelleri showed variations in relation to feeding habit and molting cycle.
