VIEW ARTICLE DOI: 10.1094/ASBCJ-55-0058
Purification, Identification, and Partial Characterization of a Barley Protein That Inhibits Green Malt Endoproteinases (1). Berne L. Jones (2), U.S. Department of Agriculture, Agricultural Research Service, Cereal Crops Research Unit, Madison, WI 53705 and Department of Agronomy, University of Wisconsin, Madison, WI 53706; Laurie A. Marinac, U.S. Department of Agriculture, Agricultural Research Service, Cereal Crops Research Unit, Madison, WI 53705. (1) Presented at the 62nd ASBC Annual Meeting, Chicago, IL, May 1996. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable. (2) Corresponding author. E-mail: <BLJONES@facstaff.wisc.edu> J. Am. Soc. Brew. Chem. 55(2):58-64. Accepted March 3, 1997. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. American Society of Brewing Chemists, Inc., 1997.
Endoproteinases control the rate of hydrolysis of storage proteins during barley germination and are thus critically important to the malting process. We have shown that endoproteinases comprising all four proteinase classes are present in green malt, with the cysteine proteinases probably being most important for hydrolyzing storage proteins during malting. Compounds from both barley and malt inhibit some of these cysteine proteinases. This article reports the purification and characterization of a 10-kDa barley protein, purified from both seed and beer extracts, that specifically inhibits green malt cysteine endoproteinases. Amino acid composition, matrix-assisted laser desorption/ionization mass spectrometric and N-terminal amino acid sequence data indicated that the inhibitor is identical to barley lipid transfer protein 1 (probable amylase/proteinase inhibitor, PAPI), a nonspecific lipid-transfer protein. The protein did not inhibit the activities of either papain or subtilisin but did suppress the activities of many of the green malt cysteine endoproteinase activities that are separated on a two-dimensional isoelectric focusing and polyacrylamide gel electrophoresis system. Some serine proteinases were also partially inhibited. The purified inhibitor totally inhibited the activity of a purified 31-kDa cysteine endoproteinase from green malt. In the absence of inhibitor, the 31-kDa enzyme rapidly hydrolyzed barley storage proteins. LTP1-PAPI may well play an important role in controlling protein hydrolysis during malting. Keywords: Brewing, Hordeum vulgare, Malting, Mashing, Protease, LTP, PAPI, Protein hydrolysis.
Las endoproteinasas controlan el índice de hidrólisis de proteínas almacenadas durante la germinación de la cebada lo que las hace de crítica importancia para el proceso de malteo. Hemos mostrado que endoproteinasas incluyendo las cuatro clases de proteinasas están presentes en malta verde, con las proteinasas de cisteina probablemente siendo las más importantes para hidrolizar las proteínas almacenadas durante el malteo. Compuestos presentes cebada y malta inhiben algunas de esas proteinasas de cisteina. Este artículo reporta la purificación y caracterización de una proteína de cebada de 10-kDa, purificada de extractos de grano y cerveza, que específicamente inhibe las endoproteinasas de cisteina de malta verde. La composición de aminoácidos, y la información de espectrometría de masas y secuencia de aminoácidos N-terminales indica que el inhibidor es idéntico a la proteína 1 de transferencia de lípido de cebada (probable inhibidor amilasa/proteinasa, LTPl-PAPI), una proteína de trasferencia de lipido no específica. La proteína no inhibió las actividades ya sea de papaina o subtilisina pero suprimió las actividades de muchas endoproteinasas de cisteinas en malta verde, actividades que fueron separadas en un sistema bidimensional de poliacrilamida gel electroforesis y enfocamiento isoeléctrico. Algunas proteinasas de serina también fueron parcialmente inhibidas. El inhibidor purificado inhibió totalmente la actividad de la endoproteinasa de cisteina de 31-kDa purificada de malta verde. En ausencia del inhibidor, la enzima 31-kDa hidrolizó rápidamente las proteínas almacenadas de la cebada. LTP1-PAPI puede jugar un papel importante en el control de la hidrólisis de proteínas durante el malteo.