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Sreedharan, S.K., Verma, C., Caves, L.S.D., Brocklehurst, S.M., Gharbias, S.E., Shah, H.N. and Brocklehurst, K. (1996) Demonstration that 1-trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) is one of the most effective low Mr inhibitors of trypsin-catalysed hydrolysis, Characterization by kinetic analysis and by energy minimization and molecular dynamics simulation of the E-64-13-trypsin complex. Biochemical Journal, 316, 777- 786.

has been cited by the following article:

• TITLE: Kinetic studies on recombinant stem bromelain

  AUTHORS: Muntari Bala, Maizirwan Mel, Mohamed Saedi Jami, Azura Amid, Hamzah Mohd Salleh

  KEYWORDS: Protease; Recombinant Bromelain; Substrates; Inhibitors; Activators

  JOURNAL NAME: Advances in Enzyme Research, Vol.1 No.3, September 16, 2013

  ABSTRACT: Stem bromelain is a plant thiol protease with several industrial and therapeutic applications. This current work presents kinetic studies of recombinant bromelain (recBM) expressed in Escherichia coli BL21-AI on foursynthetic substrates, N-α-carbobenzoxy-L-alanyl-p-nitrophenylester (ZANPE), N-α-carbobenzoxy-L-arginyl-L-ar-ginine-p-nitroanilide (ZAANA), N-α-carbobenzo-xy-L-phenylalanyl-L-valyl-L-arginine-p-nitroanili-de (ZPVANA) and L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA). Hydrolytic activities of recBM at various pH and temperature conditions were compared to that of commercial bromelain (cBM). Both enzymes demonstrated high activities at 45o C and pH 5 - 8 for recBM and pH 6 - 8 for cBM. recBM showed marginally lower Kmand slightly higher kcat/Kmfor ZAANA, ZANPE and ZPVANA in comparison to cBM.trans Epoxysuccinyl-L-leucylamido {4- guanidino}butane (E-64) severely affected recBM and cBM hydrolysis of the synthetic substrates by competitive inhibition with Kivalues of 3.6 - 5.1 μM and 5.5 - 6.9 μM for recBM and cBM, respectively. The evaluated properties of recBM including temperature and pH optima, substrate specificity and sensitivity to inhibitors or activators, satisfy the requisites required for food industries.