Partial characterization of digestive proteases in juveniles of Microphis brachyurus (short-tailed pipefish) (Syngnathiformes: Syngnathidae)

ABSTRACT Short-tailed pipe fish (Microphis brachyurus) is a freshwater organism with high economic potential for the aquarium hobby, so it is necessary to implement methods to promote its culture through studies of digestive physiology. General activities of acid and alkaline proteases were evaluated, as well as the effect of pH, temperature and inhibitors. The optimal pH of stomach proteases was 2, while the optimal pH of intestinal proteases was 10. Optimal temperature for the acidic proteases was 35 ºC, while for alkaline proteases it was 45 ºC. Thermal stability showed high resistance at 35 ºC for both acid and alkaline proteases (above 100% residual activity). Acid proteases are resistant at pH 2 (50% of residual activity), meanwhile alkaline proteases were highly resistant at pH 10 (90% of residual activity). Acid proteases were inhibited by 80% with pepstatin A and alkaline proteases were inhibited with TLCK and TPCK for trypsin (75%) and chymotrypsin (80%), respectively. Finally, metallo-proteases were 75% partially inhibited some serine proteases by 75% with EDTA. In conclusion, M. brachyurus has a good digestive capacity, since they can degrade a wide variety of proteins due to their greater proteolytic activity.

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Comparative characterization of digestive proteases in redhead cichlid (Vieja melanurus) and twoband cichlid (Vieja bifasciata) (Percoidei: Cichlidae)

Neotropical Ichthyology ◽

10.1590/1982-0224-2020-0095 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Carlos Alfonso Frías-Quintana ◽

Emyr Saul Peña-Marín ◽

Carlos David Ramírez-Custodio ◽

Rafael Martínez-García ◽

Luis Daniel Jiménez-Martínez ◽

...

Keyword(s):

Meat Quality ◽

Alkaline Proteases ◽

Optimal Temperature ◽

Digestive Capacity ◽

Digestive Proteases ◽

Ph Values ◽

Specific Diet ◽

Specific Inhibitors ◽

Optimal Ph

ABSTRACT In the Southeast of Mexico, there are many native cichlids with commercial interest such as redhead cichlid (Vieja melanurus) and twoband cichlid (V. bifasciata), which have a great local demand and excellent meat quality. However, it is necessary to implement their culture based on nutrition studies and digestive biochemistry. This study’s objective was to characterize these two cichlids’ digestive proteases (pH, temperature, and inhibitors) through biochemistry techniques. Results showed that V. melanurus and V. bifasciata have a digestive capacity analogous to other omnivore fishes, where the optimal pH values of stomach proteases (4 and 2, respectively) and intestinal proteases (6 and 12, respectively), the optimal temperature of acid (35°C and 55°C, respectively) and alkaline proteases (45°C and 55°C, respectively) are quite similar. Both species presented high thermal and pH stabilities. Inhibition showed that V. melanurus is more sensitive to specific inhibitors for alkaline proteases than V. bifasciata. In conclusion, V. bisfasciata and V. melanurus have different digestive protease patterns. Both species can hydrolyze different protein ingredients to formulate a specific diet. Nevertheless, V. bifasciata is more resistant to the presence of inhibitors, which allow it to include vegetable proteins in its diet.

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Partial characterization of digestive proteases in Pacific red snapper Lutjanus peru Nichols & Murphy, 1922 (Perciformes: Lutjanidae)

Latin American Journal of Aquatic Research ◽

10.3856/vol49-issue3-fulltext-2624 ◽

2021 ◽

Vol 49 (3) ◽

pp. 442-450

Author(s):

Emyr S. Peña-Marín ◽

Leonardo Ibarra-Castro ◽

Juan M. Martínez-Brown ◽

Iris A. Hernández-López ◽

Dariel Tovar-Ramírez ◽

...

Keyword(s):

Serine Proteases ◽

Acid Protease ◽

Red Snapper ◽

Alkaline Proteases ◽

Digestive Proteases ◽

Optimum Ph ◽

Commercial Species ◽

Electrophoretic Techniques ◽

Pepstatin A

Pacific red snapper (Lutjanus peru) is an important commercial species in Mexico with great aquaculture potential; however, digestive physiology is still unknown. Therefore, the objective of the present work was to characterize the digestive proteases of L. peru juvenile using biochemical and electrophoretic techniques. Results showed a higher acid protease activity than the alkaline proteases, trypsin, chymotrypsin, and leucine aminopeptidase (LAP). The optimum temperature for acid proteases was between 30 to 40°C. Trypsin activity showed two maximum peaks of temperature (30 and 50°C), while alkaline proteases, chymotrypsin, and LAP had optimum temperatures of 50, 50 to 60, and 40°C, respectively. Moreover, the optimum pH of acid proteases was between 2 and 3. Also, alkaline proteases, trypsin, chymotrypsin showed pH optimums at pH 6, 9, and 5, respectively, although LAP showed two optimum pH values at 6 and 9. Acid protease zymogram showed three isoforms, totally inhibited by pepstatin A. Alkaline protease zymogram revealed six bands (125.4, 67.2, 57.9, 48.6, 29.8, and 26.9 kDa), which were inhibited by specific serine-proteases and metalloproteases inhibitors. In conclusion, the main digestion in L. peru depends on stomach proteases, which are characteristic of carnivorous fish, followed by intestinal digestion supported mainly by chymotrypsin.

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Kinetic Characterization of Anticarsia gemmatalis Digestive Serine- Proteases and the Inhibitory Effect of Synthetic Peptides

Protein and Peptide Letters ◽

10.2174/0929866524666170918103146 ◽

2018 ◽

Vol 24 (11) ◽

Cited By ~ 1

Author(s):

Adriana M. Patarroyo-Vargas ◽

Yaremis B. Merino-Cabrera ◽

Jose C. Zanuncio ◽

Francelina Rocha ◽

Wellington G. Campos ◽

...

Keyword(s):

Synthetic Peptides ◽

Serine Proteases ◽

Inhibitory Effect ◽

Anticarsia Gemmatalis ◽

Kinetic Characterization

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Characterization of digestive physiology in Pacific bluefin tuna Thunnus orientalis juveniles fed a raw fish feed and a commercial diet

Aquaculture ◽

10.1016/j.aquaculture.2021.736562 ◽

2021 ◽

pp. 736562

Author(s):

Koji Murashita ◽

Hiroshi Hashimoto ◽

Toshinori Takashi ◽

Takeshi Eba ◽

Kazunori Kumon ◽

...

Keyword(s):

Bluefin Tuna ◽

Fish Feed ◽

Pacific Bluefin Tuna ◽

Digestive Physiology ◽

Thunnus Orientalis ◽

Commercial Diet ◽

Raw Fish

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Immobilization and Characterization of Tannase and its Haze-removing

Food Science and Technology International ◽

10.1177/1082013209352919 ◽

2009 ◽

Vol 15 (6) ◽

pp. 545-552 ◽

Cited By ~ 9

Author(s):

Erzheng Su ◽

Tao Xia ◽

Liping Gao ◽

Qianying Dai ◽

Zhengzhu Zhang

Keyword(s):

Kinetic Parameter ◽

High Activity ◽

Green Tea ◽

Storage Stability ◽

Residual Activity ◽

Black Tea ◽

Optimum Temperature ◽

Oolong Tea ◽

Optimum Ph

Tannase was effectively immobilized on alginate by the method of crosslinking-entrapment-crosslinking with a high activity recovery of 76.6%. The properties of immobilized tannase were investigated. Its optimum temperature was determined to be 35 ° C, decreasing 10 °C compared with that of free enzyme, whereas the optimum pH of 5.0 did not change. The thermal and pH stabilities of immobilized tannase increased to some degree. The kinetic parameter, Km, for immobilized tannase was estimated to be 11.6 × 10-4 mol/L. Fe2+ and Mn2+ could activate the activity of immobilized tannase. The immobilized tannase was also applied to treat the tea beverage to investigate its haze-removing effect. The content of non-estern catechins in green tea, black tea and oolong tea increased by 52.17%, 12.94% and 8.83%, respectively. The content of estern catechins in green tea, oolong tea and black tea decreased by 20.0%, 16.68% and 5.04%, respectively. The anti-sediment effect of green tea infusion treated with immobilized tannase was significantly increased. The storage stability and reusability of the immobilized tannase were improved greatly, with 72.5% activity retention after stored for 42 days and 86.9% residual activity after repeatedly used for 30 times.

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Recombinant Deg/HtrA proteases from Synechocystis sp. PCC 6803 differ in substrate specificity, biochemical characteristics and mechanism

Biochemical Journal ◽

10.1042/bj20102131 ◽

2011 ◽

Vol 435 (3) ◽

pp. 733-742 ◽

Cited By ~ 20

Author(s):

Pitter F. Huesgen ◽

Helder Miranda ◽

XuanTam Lam ◽

Manuela Perthold ◽

Holger Schuhmann ◽

...

Keyword(s):

Serine Proteases ◽

Protein Quality ◽

Biochemical Characterization ◽

Pdz Domain ◽

Control Mechanisms ◽

Ph Optimum ◽

Periplasmic Proteins ◽

Synechocystis Sp ◽

Pcc 6803

Cyanobacteria require efficient protein-quality-control mechanisms to survive under dynamic, often stressful, environmental conditions. It was reported that three serine proteases, HtrA (high temperature requirement A), HhoA (HtrA homologue A) and HhoB (HtrA homologue B), are important for survival of Synechocystis sp. PCC 6803 under high light and temperature stresses and might have redundant physiological functions. In the present paper, we show that all three proteases can degrade unfolded model substrates, but differ with respect to cleavage sites, temperature and pH optima. For recombinant HhoA, and to a lesser extent for HtrA, we observed an interesting shift in the pH optimum from slightly acidic to alkaline in the presence of Mg2+ and Ca2+ ions. All three proteases formed different homo-oligomeric complexes with and without substrate, implying mechanistic differences in comparison with each other and with the well-studied Escherichia coli orthologues DegP (degradation of periplasmic proteins P) and DegS. Deletion of the PDZ domain decreased, but did not abolish, the proteolytic activity of all three proteases, and prevented substrate-induced formation of complexes higher than trimers by HtrA and HhoA. In summary, biochemical characterization of HtrA, HhoA and HhoB lays the foundation for a better understanding of their overlapping, but not completely redundant, stress-resistance functions in Synechocystis sp. PCC 6803.

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Identification and functional characterization of legumain in amphioxus Branchiostoma belcheri

Bioscience Reports ◽

10.1042/bsr20090049 ◽

2009 ◽

Vol 30 (3) ◽

pp. 177-186 ◽

Cited By ~ 6

Author(s):

Lei Teng ◽

Hiroshi Wada ◽

Shicui Zhang

Keyword(s):

Functional Characterization ◽

Specific Substrate ◽

Glycosylation Sites ◽

Branchiostoma Belcheri ◽

Hen’S Egg ◽

Hind Gut ◽

Catalytic Dyad ◽

Pepstatin A

Legumain has been reported from diverse sources such as plants, parasites (animals) and mammals, but little is known in the lower chordates. The present study reports the first characterization of legumain cDNA from the protochordate Branchiostoma belcheri. The deduced 435-amino-acid-long protein is structurally characterized by the presence of a putative N-terminal signal peptide, a peptidase_C13 superfamily domain with the conserved Lys123-Gly124-Asp125 motif and catalytic dyad His153 and Cys195 and two potential Asn-glycosylation sites at Asn85 and Asn270. Phylogenetic analysis demonstrates that B. belcheri legumain forms an independent cluster together with ascidian legumain, and is positioned at the base of vertebrate legumains, suggesting that B. belcheri legumain gene may represent the archetype of vertebrate legumain genes. Both recombinant legumain expressed in yeast and endogenous legumain are able to be converted into active protein of ~37 kDa via a C-terminal autocleavage at acid pH values. The recombinant legumain efficiently degrades the legumain-specific substrate Z-Ala-Ala-Asn-MCA (benzyloxycarbonyl-L-alanyl-L-alanyl-L-asparagine-4-methylcoumaryl-7-amide) at optimum pH 5.5; and the enzymatic activity is inhibited potently by iodoacetamide and N-ethylmaleimide, partially by hen's-egg white cystatin, but not by E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane], PMSF and pepstatin A. In addition, legumain is expressed in vivo in a tissue-specific manner, with main expression in the hepatic caecum and hind-gut of B. belcheri. Altogether, these results suggest that B. belcheri legumain plays a role in the degradation of macromolecules in food.

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Characterization of three alternative splice variants associated with the Arabidopsis rhomboid protein gene At1g74130

Botany ◽

10.1139/cjb-2013-0173 ◽

2013 ◽

Vol 91 (12) ◽

pp. 840-849 ◽

Cited By ~ 4

Author(s):

Joshua Powles ◽

Katharine Sedivy-Haley ◽

Eric Chapman ◽

Kenton Ko

Keyword(s):

Alternative Splicing ◽

Alternative Splice ◽

Splice Variant ◽

Serine Proteases ◽

Transmembrane Domain ◽

Splice Variants ◽

Genes Encoding ◽

Different Characteristics ◽

Alternative Splice Variants

Rhomboid serine proteases are grouped into three main types — secretases, presenilin-like associated rhomboid-like (PARL) proteases, and “inactive” rhomboid proteins. Although the three rhomboid groups are distinct, the different types are likely to operate within the same cell or compartment, such as observed in the plastids of Arabidopsis. There are four distinct plastid rhomboid genes at play in Arabidopsis plastids, two for active types (At1g25290 and At5g25752) and two for inactive forms (At1g74130 and At1g74140). The number of working plastid rhomboids is further increased by alternative splicing, as reported for At1g25290. To understand how the plastid rhomboid system works, it is necessary to identify all rhomboid forms in play. To this end, this study was designed to examine the alternative splicing activities of At1g74130, one of the two genes encoding proteolytically “inactive” plastid rhomboids. The exon mapping and DNA sequencing results obtained here indicate the presence of three prominent alternative splice variants in the At1g74130 transcript population. The dominant splice variant, L, encodes the full-length protein. The other two splice variants, M and S, produce proteins lacking sections from the carboxyl transmembrane domain region. The splice variants M and S appear to be at levels with functional potential and appear to adjust relative to each other during development and in response to changes in the level of Tic40, a component of the plastid translocon. The splice variant proteins themselves exhibit different characteristics with respect to rhomboid protein–substrate interactions. These differences were observed in bacterial co-expression pull-down assays and in yeast mitochondrial studies. When considered together, the data suggest that the alternative splicing of At1g74130 bears functional significance in Arabidopsis and is likely to be part of a mechanism for diversifying plastid rhomboid function.

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Characterization of mitochondria isolated from the freezing-tolerant larvae of the goldenrod gall fly (Eurosta solidaginis): substrate preferences, salt effects, and pH effects on warm- and cold-acclimated animals

Canadian Journal of Zoology ◽

10.1139/z85-057 ◽

1985 ◽

Vol 63 (2) ◽

pp. 373-379 ◽

Cited By ~ 9

Author(s):

James S. Ballantyne ◽

Kenneth B. Storey

Keyword(s):

Salt Concentration ◽

Respiratory Control ◽

Ph Effects ◽

Respiratory Control Ratio ◽

Eurosta Solidaginis ◽

Optimal Range ◽

Salt Effects ◽

Substrate Preferences ◽

Optimal Ph

The mitochondria of the freezing-tolerant larvae of the goldenrod gall fly (Eurosta solidaginis) have been isolated and characterized. Proline is the preferred substrate of mitochondria from both warm- and cold-acclimated animals based on state 3 rates. Lipid is used as a substrate by warm- and cold-acclimated mitochondria assayed at 20 °C, but not by the mitochondria from cold-acclimated animals assayed at 1 °C. Cold-acclimated mitochondria assayed at 1 °C have a higher and broader optimal range of salt concentration for the oxidation of proline based on the respiratory control ratio (RCR) than those from warm-acclimated animals oxidizing the same substrate at 20 °C. The optimal pH for warm-acclimated mitochondria oxidizing proline at 20 °C is low (6.2) based on the RCR, but rises to pH 7.0 in cold-acclimated animals at 1 °C. It is suggested that the broad optimal salt concentration in the cold-acclimated animals and the very low optimal pH in warm-acclimated animals are adaptations for survival in this freezing-tolerant larva.

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Molecular characterization of a partial sequence encoding a novel Schistosoma mansoni serine protease

Parasitology ◽

10.1017/s0031182097001546 ◽

1997 ◽

Vol 115 (4) ◽

pp. 395-402 ◽

Cited By ~ 6

Author(s):

C. COCUDE ◽

C. PIERROT ◽

C. CETRE ◽

C. GODIN ◽

A. CAPRON ◽

...

Keyword(s):

Schistosoma Mansoni ◽

Serine Protease ◽

Serine Proteases ◽

Mrna Level ◽

Cross Reactivity ◽

Reading Frame ◽

Consensus Sequences ◽

Degenerate Oligonucleotide ◽

Sequence Encoding

A PCR strategy using degenerate oligonucleotide primers based upon consensus sequences of the active site of serine proteases yielded a 467 bp fragment from genomic DNA from Schistosoma mansoni cercariae. The sequence presented a continuous open reading frame and the deduced amino acid sequence (156 aa) presented homologies with various serine proteases, in particular the highest percentage identity was observed with a mammalian plasma kallikrein. The expression of this serine protease was studied first at the mRNA level and it was only detected by RT-PCR in cercariae and in adult worms. At the protein level we were able to detect it by Western blotting and by using antigen extracts from metabolically radio-isotope labelled worms. The absence of any positive signal in Northern blot and the detection of the protein suggest that the mRNA has a very short half-life, however the protein may be accumulated in the parasite. The significance of identity with mammalian kallikrein was confirmed by cross-immunoreactivity with a native porcine pancreatic kallikrein. However, no cross-reactivity was observed with S. mansoni elastase, another serine protease. Thus, we suggest that the serine protease described in this paper is a kallikrein-like protease.

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