© 1999 Institute of Food Technologists NUTRITION SENSORY EVALUATION Food Chemistry and Toxicology 772 JOURNAL OF FOOD SCIENCE—Volume 64, No. 5, 1999 Purification and Characterization of Proteinase from Atlantic Menhaden Muscle Y.J. Choi, Y.-J. Cho, and T.C. Lanier ABSTRACT Two proteinases (A and B) were isolated from Atlantic menhaden muscle with molecular weights of 112,000 and 90,500 daltons, respectively. Proteinase B had higher activity than A for protein substrates except casein; proteinase B had no caseinolytic activity. Both proteinases hydrolyzed synthetic substrates such as Z-Phe-Arg-NMec and TAME, but not BAEE and BAPNA. Optimum Z-Phe-Arg-NMec hydrolyzing activity was shown at pH 7.4, 40 to 50 ° C for both proteinases A and B. Activities of A and B in the presence of 3.0% NaCl were reduced to 71.2% and 62.2%, respectively. Both proteinases were inhibited by 1 mM TLCK, 1 mM benzamidine, 1% egg white, and 1% bovine plasma hydrolysate. Proteinases A and B are most likely tryptic serine type proteinases. Key Words: Atlantic menhaden, proteinase, trypsin, serine tide with maximum activity near 55 °C (Sey- mour et al., 1994). Heat-stable proteinases have been identified in fish muscle (Iwata et al., 1974; Makinodan et al., 1987) that were composed of complex subunits (Hase et al., 1980). Heat-stable alkaline proteinases have been hypothesized to be responsible for the heat-induced softening of surimi gels at neu- tral pH near 55 to 60 °C (Hamann et al., 1990). The objective of this study was to investi- gate the presence and properties of additional proteinases from Atlantic menhaden muscle and their possible effects on degradation of menhaden actomyosin during heating. MATERIAL & METHODS ATLANTIC MENHADEN, BREVOORTIA TYR- annus (370 g and 27 cm average body length) were obtained at the North Carolina coast in a very fresh state (pound or gill net caught) and immediately transported to the laborato- ry on ice. Excised muscle was minced and homogenized with 2 volumes distilled water in a Sorvall Omni-mixer in the cold for 3 min. The homogenates were centrifuged at 10,000 ∞ g for 30 min. The supernatant was filtered through four layers of gauze, and filtrates were fractionated with 30% to 60% cold ac- etone. This fraction was then lyophilized and stored at -80 °C. Purification of proteinases The crude extract was fractionated with ammonium sulfate at 30% to 60% saturation. The precipitated fraction was dissolved in 0.1 M NaCl-20 mM sodium acetate, pH 5.6, and centrifuged at 6,000 ∞ g for 30 min. The su- pernatant was applied to a Sephacryl S-200 column (2.6 cm ∞ 80 cm) equilibrated and eluted with the same buffer. The active pro- teinase fractions were separated, and each fraction was dialyzed against 20 mM sodi- um acetate, pH 5.6. Each dialysate was ap- plied to a CM-Sephadex C-50 column (2.6 cm ∞ 35 cm) equilibrated with 20mM sodi- um acetate, pH 5.6, and eluted with a 0 to 0.5 M NaCl gradient in 20 mM sodium ace- tate, pH 5.6. The active fractions were pooled and concentrated by ultrafiltration in an Amicon stirred cell (Amicon Instruments, Lexington, MA) using a PM 10 membrane. The concentrated samples were applied to a Sephacryl S-200 column (2.6 cm ∞ 80 cm) equilibrated with the above buffer and washed with the same buffer. The purified proteinases were again concentrated by ul- trafiltration in an Amicon stirred cell using a PM 10 membrane and dialyzed against dis- tilled water. The final products were stored at -80 °C and used for further studies. Ab- sorbance at 280 nm was measured to moni- tor the protein peaks, and proteinase activi- ties for Z-Phe-Arg-NMec in each fraction from the chromatography column were de- termined at pH 5.6 by the method of Barrett and Kirschke (1981). Assay of proteolytic activity Z-Phe-Arg-NMec hydrolyzing activity was determined throughout the purification process, according to the method of Barrett and Kirschke (1981). The assay mixture was composed of 1.25 mL of assay buffer, 0.25 mL of 20 mM Z-Phe-Arg-NMec solution, and 50 μ L of enzyme. Fluorescence intensi- ty was measured by excitation at 375 nm and emission at 460 nm on a fluorometer (Per- kin-Elmer LS-2B Filter Fluorometer). The amount of free aminomethyl-coumarin (AMC) was determined from a standard curve. The instrument was set to read 1000 arbitrary units for a 0.5 M standard, an ex- perimental reading of 1000 units correspond- ing to 0.1 mM of activity. The proteolytic activity on casein and the degradation of ac- tomyosin were measured by the method of Pyeun and Kim (1986). The assay mixture was composed of 1.5 mL buffer solution, 0.5 mL 2% casein solution, and 0.1 mL enzyme solution. The reaction was stopped by 5% TCA (w/w) addition. The TCA-soluble ma- terial of the supernatant after centrifuging (3000 ∞ g, 20 min) was assayed according to the method of Lowry et al. (1951). From the amount of TCA-soluble material the mole of tyrosine was determined using a standard curve. The proteolytic activities on azocasein were measured by the method of Barrett and Kirschke (1981) and expressed as the change in absorbance at 366 nm. Author Choi is with the Division of Marine Bio- science, Institute of Marine Industry, Gyeong-Sang National Univ., Tong-Yeong, Korea, 650-160. Author Cho is with the Dept. of Food Science and Technol- ogy, Pukyeong Univ., Pusan, Korea, 607-791. Au- thor Lanier is with the Dept. of Food Science, North Carolina State Univ., Raleigh, NC 27695-7624. Ad- dress inquiries to Dr. T. C. Lanier (E-mail: tyre@unity.ncsu.edu). INTRODUCTION ATLANTIC MENHADEN HAS BEEN PRO- posed as a potential species for surimi pro- duction due to its abundance and unsuitabil- ity for other food products (Lanier, 1988). Several studies have reported development of technology to utilize fatty, dark-flesh fish in surimi manufacture (Lanier, 1988; Shimi- zu et al., 1992; Jiang et al., 1998) , but prob- lems remain. Fatty, dark-fleshed fish, such as menhaden, exhibit autolysis and hydroly- sis of muscle proteins during heating (Lanier, 1988). Two alkaline serine protein- ases that degraded actomyosin have been purified from menhaden muscle (Choi et al., 1999); properties of two trypsin-like enzymes distributed in the intestine of menhaden have been investigated (Pyeun et al., 1990). The most active proteinases in fish mus- cle are cathepsins and heat-stable alkaline proteinases (An et al., 1996). Cathepsin B and L have been shown to cause softening in chum salmon (Yamashita and Konagaya, 1990a, 1991). Cathepsin L was found to be the predominant proteinase involved in heat- induced degradation of the myofibrillar pro- tein in Pacific whiting surimi (An et al., 1994). Purified cathepsins, such as cathep- sin B from chum salmon muscle (Yamashita and Konagaya, 1990b), cathepsin L from Pacific whiting (Seymour et al., 1994), and cathepsin H from hepatopancreas of carp (Aranishi et al., 1992), generally have a low molecular weight ranging from 23,000 to 30,000. Cathepsin L consists of a single pep- JOURNAL OF FOOD SCIENCE FOOD CHEMISTRY AND TOXICOLOGY