Isolation Of Mung Bean Trypsin Inhibitor And Its Application In Aquatic Products Processing

Mung bean trypsin inhibitor (MBTI) was purified to homogeneity. The purificationprocedures consisted of soaking and extracting with0.1mol/L sulfuric acid, heat treatment,ultrafiltration, DEAE-Sepharose ion exchange column chromatography and Superdex75gel-filtration. The molecular mass of MBTI was approximately9ku, as determined bySDS-PAGE and8887.25u by MALDI-QIT-TOF MS. MBTI effectively inhibited the enzymaticactivity of silver carp myofibril-bound serine protease (MBSP) and porcine trypsin. Theinhibition type of MBTI to MBSP is reversible inhibition. MBTI is an effective inhibitor inpreventing myofibrillar protein (such as myosin heavy chain, MHC, actin and tropomyosin)degradation caused by MBSP and was thus supposed to be effective in preventing surimi geldegradation.On the other hand, with the increasing production of Pacific white shrimp in our country, thequality protection, especially muscle autolysis during storage should be notified. In the presentstudy, a serine proteinase from the hepatopancreas of Pacific white shrimp was purified by aseries of procedures, including ammonium sulfate precipitation, column chromatographies onDEAE-Sepharose, Phenyl-Sepharose, Hydroxyapatite and Superdex75. Purified serineproteinase revealed a single band on SDS-PAGE. The molecular weight was approximately28ku, The optimum pH and temperature of the enzyme were9.0and40℃, respectivety. Theenzyme was stable up to35C in the pH range from7.0to10.0. The N-terminal amino acidsequence of the enzyme as determined by Edman degradation method wasIVGGTDAKPGELPYQLSFQDI which revealed higher idetities to other serine proteinasesreported. Substrate specificity and inhibitor sensitivity experiments suggested that the enzyme isa trypsin-type serine proteinase. Kinetic constants of Kmwas0.69nmol/L, kcatwas0.33S-1andkcat/Kmwas5×10（8mol/L）-1S-1using Boc-Phe-Ser-Arg-MCA as substrate. The suppressing effectof MBTI on Pacific white shrimp muscular protein degradation strongly suggested that MBTIcan be used as an important ingredient as shrimp preservative.