Simple Preparation of Pacific Cod Trypsin for Enzymatic Peptide Synthesis

Trypsin from the pyloric caeca of Pacific cod (Gadus macrocephalus) was easily prepared by affinity chromatography on Benzamidine Sepharose 6B and gel filtration on Superdex 75. Pacific cod trypsin was composed of three isozymes, and their molecular masses were estimated 23,756.34 Da, 23,939.62 Da, and 24,114.81 Da by desorption/ionization time-of-flight mass spectroscopy (MALDI/TOF-MS) and their isoelectric points (pIs) were approximately 5.1, 6.0, and 6.2, respectively. The isolated Pacific cod trypsin showed high similarity to other frigid-zone fish trypsins. The kinetic behavior of tryptic hydrolysis toward N-p-tosyl-L-arginine methyl ester hydrochloride (TAME), N-benzoyl-L-arginine p-nitroanilide hydrochloride (BAPA), and p-amidinophenyl ester were also analyzed. In addition, the cod trypsin-catalyzed dipeptide synthesis was investigated using twelve series of “inverse subdtrates” that is p- and m-isomer of amidinophenyl, guanidinophenyl, (amidinomethyl)phenyl, (guanidinomethyl)phenyl, and four position isomers of guanidinonaphtyl esters derived from N-(tert-butoxycarbonyl)amino acid as acyl donor components. They were found to couple with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptide in the presence of the trypsin. All inverse substrates tested in this study undergo less enantioselective coupling reaction. The p-guanidinophenyl ester was most practical substrate in twelve series tested. The enzymatic hydrolysis of the resulting products was negligible

การใช้เปปซินในการสกัดคอลลาเจนและเจลาตินจากหนังปลาตาหวาน

Thesis (M.Sc., Food Technology)--Prince of Songkla University, 200

การทำบริสุทธิ์ การแยกส่วน และการใช้เปปซินจากปลาเพื่อการสกัดคอลลาเจนและการผลิตโปรตีนไฮโดรไลเสต

Thesis (Ph.D., Food Technology)--Prince of Songkla University, 201

Structural properties of trypsin from cold-adapted fish, arabesque greenling (Pleurogrammus azonus)

A cDNA clone encoding trypsin (AG-T) was isolated from the pyloric ceca of cold-adapted fish, arabesque greenling (Pleurogrammus azonus). The cDNA was composed of 892 bp with an open reading frame of 729 bp at nucleotide positions 25-753. Similar to all the known trypsin, the AG-T seemed to be synthesized as preproenzyme that contains a hydrophobic signal peptide, an activation pentapeptide and a mature trypsin of 222 amino acid residues. The AG-T also completely conserved the major structural features common to trypsin such as the catalytic triad (His57, Asp102, and Ser195), the obligatory Asp189 and twelve Cys residues. On the other hand, the AG-T possessed the deletion of Tyr151 and substitution of Pro152 for Gly in the autolysis loop when aligned with the sequence of tropical-zone fish and bovine trypsins. In addition, Val75 concerned in a combination with calcium ion was exchanged for Ala in the AG-T, and the content of positively charged amino acid residues at the calcium-binding site of the AG-T was three times higher than those of tropical-zone fish trypsins. Moreover, the ratio between charged and hydrophobic amino acid residues in the N-terminal region of the AG-T was also higher than those of temperate-zone fish and tropical-zone fish trypsins. Such structural properties of the AG-T would contribute to its low thermostability

Mackerel Trypsin Purified from Defatted Viscera by Supercritical Carbon Dioxide

Viscera of mackerel (Scomber sp.) were defatted by supercritical carbon dioxide (SCO2) treatment. Trypsin (SC-T) was then extracted from the defatted powder and purified by a series of chromatographies including Sephacryl S-200 and Sephadex G-50. The purified SC-T was nearly homogeneous on SDS-PAGE, and its molecular weight was estimated as approximately 24,000 Da. N-terminal twenty amino acids sequence of SC-T was IVGGYECTAHSQPHQVSLNS. The specific trypsin inhibitors, soybean trypsin inhibitor and TLCK, strongly inhibited the activities of SC-T. The pH and temperature optimums of SC-T were at around pH 8.0 and 60∘C, respectively, using Nα-p-tosyl-L-arginine methyl ester as a substrate. The SC-T was unstable below pH 5.0 and above 40∘C, and it was stabilized by calcium ion. These enzymatic characteristics of SC-T were the same as those of other fish trypsins, especially spotted mackerel (S. borealis) trypsin, purified from viscera defatted by acetone. Therefore, we concluded that the SCO2 defatting process is useful as a substitute for organic solvent defatting process

Cold-adapted structural properties of trypsins from walleye pollock (Theragra chalcogramma) and Arctic cod (Boreogadus saida)

Complementary DNA clones encoding trypsins were isolated from pyloric ceca of cold-adapted fish, walleye pollock (Theragra chalcogramma) (WP-T) and Arctic cod (Boreogadus saida) (AC-T). The isolated full-length cDNA clones of WP-T and AC-T were 852 bp and 860 bp, respectively, and both cDNAs were contained an open reading frame of 726 bp. WP-T and AC-T seemed to be synthesized as preproenzyme that contains a signal peptide, an activation peptide, and a mature trypsin. Although the amino acid sequence identities of WP-T and AC-T to that of bovine trypsin were 64% and 63%, respectively, they completely conserved the structural features for catalytic function of trypsin. On the other hand, WP-T and AC-T possessed the four Met residues (Met135, Met145, Met175 and Met242) in their molecules and the deletion of Tyr151 and substitution of Pro152 for Gly in their autolysis loops when aligned with the sequences of tropical-zone fish and bovine trypsins. In addition, the contents of charged amino acid residues at the N-terminal regions (positions 20-50) of WP-T and AC-T were extremely higher than those of other fish and bovine trypsins. Moreover, one amino acid (Asn72) and two amino acids (Asn72 and Val75) coordinating with Ca2+ in bovine trypsin were exchanged for another amino acids in WP-T (His) and AC-T (His and Glu), respectively, and the contents of negative charged amino acids at their Ca^[2+]-binding regions were lower than those of tropical-zone fish and bovine trypsins. Therefore, it was considered that these structural characteristics of WP-T and AC-T are closely related to their lower thermo stability

Alpha-amylase inhibitory activity of collagen hydrolysate from Asian bullfrog skin and its application in dark chocolate

AbstractThe present study aimed to investigate the in-vitro antidiabetic activity of collagen hydrolysate (CH) from Asian bullfrog skin and its application as a bioactive compound in dark chocolate bars. Papain (3% [w/w]) was used to obtain CH with a simultaneous conjunction of ultrasound. CH possessed α-amylase inhibitory activity with an IC50 value of 3.60 mg/mL, while results from inhibition reaction kinetics revealed that CH inhibited α-amylase in a non-competitive mode. Different concentrations (0.5, 1.0, and 2.0% [w/w]) of CH were then fortified into dark chocolate bars, and the physicochemical characteristics, breaking strength, and rheological behaviour of the resulting chocolate were investigated and compared with a control (without CH addition). A higher level of CH significantly enhanced antidiabetic activity against α-amylase (p ≤ 0.05) in chocolate bar. Chocolate bar with 2% CH complied with the nutritional claim requirement for ‘source of protein’ with lower fat content and energy value, while sensory evaluation revealed good organoleptic properties

Characteristics, Functional Properties, and Antioxidant Activities of Water-Soluble Proteins Extracted from Grasshoppers, Patanga succincta and Chondracris roseapbrunner

Water-soluble proteins extracted from two species of grasshoppers, Patanga succincta (WSPP) and Chondracris roseapbrunner (WSPC), were characterized as well as their functional properties and antioxidant activities were investigated. The extraction yield, on a wet weight basis, was 7.35% and 7.46% for WSPP and WSPC, respectively. The most abundant amino acid in both proteins was glutamic acid, followed by aspartic, alanine, and leucine, in that order. The electrophoretic study revealed that proteins with MW of 29, 42, 50, 69, and 146 kDa were the major protein components in WSPP and WSPC. FTIR analysis showed that those proteins remained their structural integrity. The surface hydrophobicity at pH 7 of WSPC was higher than WSPP, but the sulfhydryl group content did not show significant difference between the proteins from two species. Both grasshopper proteins were mostly soluble in strong acidic and alkaline aqueous solutions with a minimum value at pH 4. Those proteins exhibited poor emulsifying properties and foaming capacity, but they had greater foaming stability compared with bovine serum albumin (BSA) (pThis article is published as Niphattha Chatsuwan, Sitthipong Nalinanon, Yuporn Puechkamut, Buddhi P. Lamsal, Praphan Pinsirodom; Characteristics, Functional Properties, and Antioxidant Activities of Water-Soluble Proteins Extracted from Grasshoppers, Patanga succincta and Chondracris roseapbrunner. Journal of Chemistry 2018, Article ID 6528312, DOI: 10.1155/2018/6528312. </p