title: Purification and characterization of heat-stable alkaline proteinase from bigeye snapper (Priacanthus macracanthus) muscle.
abstract: Heat-stable alkaline proteinase was purified from bigeye snapper (Priacanthus macracanthus) ordinary muscle by heat-treatment and a series of chromatographies including Phenyl-Sepharose 6 Fast Flow, Source 15Q and Superose 12 HR 10/30. It was purified to 5180-fold with a yield of 0.8%. The molecular weight of purified proteinase was estimated to be 72 kDa by gel filtration. The proteinase appeared as two proteinase activity bands with molecular weights of 66 and 13.7 kDa on non-reducing SDS-substrate gel. Accordingly, it was found to consist of two different subunits. The optimum pH and temperature for casein hydrolysis were 8.5 and 60 degrees C, respectively. The proteolytic activity was strongly inhibited by soybean trypsin inhibitor and partially inhibited by ethylenediaminetetraacetic acid, while pepstatin A and E-64 showed no inhibition. Purified proteinase was able to hydrolyze Boc-Phe-Ser-Arg-MCA, but rarely hydrolyzed Z-Phe-Arg-MCA and Z-Arg-Arg-MCA. In addition, it mainly degraded myosin heavy chain, not actin. These results suggest that purified proteinase was serine proteinase, which is probably involved in gel weakening of bigeye snapper surimi.
authors.affiliation: Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, 90112, Songkhla, Thailand. bsoottaw@ratree.psu.ac.th
authors.forename: Soottawat
authors.initials: S
authors.lastname: Benjakul
authors.forename: Wonnop
authors.initials: W
authors.lastname: Visessanguan
authors.forename: Kittima
authors.initials: K
authors.lastname: Leelapongwattana
mesh_terms: D000818:Animals; D002364:Caseins / Q000378:metabolism; D004791:Enzyme Inhibitors; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000737:chemistry*; D010473:Perciformes; D021122:Protein Subunits; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002364:Caseins; D004791:Enzyme Inhibitors; D021122:Protein Subunits; D012697:Serine Endopeptidases