Elucidation of different cold-adapted Atlantic cod (Gadus morhua) trypsin X isoenzymes

被引:8
作者
Stefansson, Bjarki [1 ]
Sandholt, Gunnar B. [2 ]
Gudmundsdottir, Agusta [1 ,2 ]
机构
[1] Zymetech, Fiskislod 39, IS-101 Reykjavik, Iceland
[2] Univ Iceland, Div Hlth Sci, Fac Food Sci & Nutr, IS-101 Reykjavik, Iceland
来源
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS | 2017年 / 1865卷 / 01期
关键词
Biomedicine; Trypsin; Cold-adapted; Atlantic cod; Kinetics; Trypsin X; STREPTOMYCES-GRISEUS PROTEASE-3; BIOTECHNOLOGICAL APPLICATIONS; ALPHA-CHYMOTRYPSIN; ANIONIC TRYPSIN; ACTIVE-CENTERS; GREENLAND COD; ENZYMES; PURIFICATION; TEMPERATURE; ADAPTATION;
D O I
10.1016/j.bbapap.2016.10.005
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Trypsins from Atlantic cod (Gadus morhua), consisting of several isoenzymes, are highly active cold-adapted serine proteases. These trypsins are isolated for biomedical use in an eco-friendly manner from underutilized seafood by-products. Our group has explored the biochemical properties of trypsins and their high potential in biomedicine. For broader utilization of cod trypsins, further characterization of biochemical properties of the individual cod trypsin isoenzymes is of importance. For that purpose, a benzamidine purified trypsin isolate from Atlantic cod was analyzed. Anion exchange chromatography revealed eight peaks containing proteins around 24 kDa with tryptic activity. Based on mass spectrometric analysis, one isoenzyme gave the best match to cod trypsin I and six isoenzymes gave the best match to cod trypsin X. Amino terminal sequencing of two of these six trypsin isoenzymes showed identity to cod trypsin X. Three sequence variants of trypsin X were identified by cDNA analysis demonstrating that various forms of this enzyme exist. One trypsin X isoenzyme was selected for further characterization based on abundance and stability. Stepwise increase in catalytic efficiency (k(cat)/K-m) of this trypsin X isoenzyme was obtained with substrates containing one to three amino acid residues. The study demonstrates that the catalytic efficiency of this trypsin X isoenzyme is comparable to that of cod trypsin I, the most abundant and highly active isoenzyme in the benzamidine cod trypsin isolate. Differences in pH stability and sensitivity to inhibitors of the trypsin X isoenzyme compared to cod trypsin I were detected that may be important for practical use. (C) 2016 Published by Elsevier B.V.
引用
收藏
页码:11 / 19
页数:9
相关论文
共 48 条
[31]  
MALE R, 1995, EUR J BIOCHEM, V232, P677, DOI 10.1111/j.1432-1033.1995.677zz.x
[32]   Molecular characterization and gene expression of six trypsinogens in the flatfish Senegalese sole (Solea senegalensis Kaup) during larval development and in tissues [J].
Manchado, Manuel ;
Infante, Carlos ;
Asensio, Esther ;
Crespo, Aniela ;
Zuasti, Eugenia ;
Pedro Canavate, Jose .
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY, 2008, 149 (02) :334-344
[33]   Biotechnological applications of psychrophiles [J].
Margesin, Rosa ;
Feller, Georges .
ENVIRONMENTAL TECHNOLOGY, 2010, 31 (8-9) :835-844
[34]   Trypsinogen expression during the development of the exocrine pancreas in winter flounder (Pleuronectes americanus) [J].
Murray, HM ;
Perez-Casanova, JC ;
Gallant, JW ;
Johnson, SC ;
Douglas, SE .
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR & INTEGRATIVE PHYSIOLOGY, 2004, 138 (01) :53-59
[35]   Temperature and pH sensitivity of trypsins from Atlantic salmon (Salmo salar) in comparison with bovine and porcine trypsin [J].
Outzen, H ;
Berglund, GI ;
Smalas, AO ;
Willassen, NP .
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY, 1996, 115 (01) :33-45
[36]   Evolutionary divergence of substrate specificity within the chymotrypsin-like serine protease fold [J].
Perona, JJ ;
Craik, CS .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1997, 272 (48) :29987-29990
[37]  
Rawlings ND, 2013, HANDBOOK OF PROTEOLYTIC ENZYMES, VOLS 1 AND 2, 3RD EDITION, P2491, DOI 10.1016/B978-0-12-382219-2.00559-7
[38]   The molecular evolution of the vertebrate trypsinogens [J].
Roach, JC ;
Wang, K ;
Gan, L ;
Hood, L .
JOURNAL OF MOLECULAR EVOLUTION, 1997, 45 (06) :640-652
[39]   Cold and hot extremozymes: industrial relevance and current trends [J].
Sarmiento, Felipe ;
Peralta, Rocio ;
Blamey, Jenny M. .
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, 2015, 3
[40]   Some like it hot, some like it cold: Temperature dependent biotechnological applications and improvements in extremophilic enzymes [J].
Siddiqui, Khawar Sohail .
BIOTECHNOLOGY ADVANCES, 2015, 33 (08) :1912-1922