Vol.1, No.3, 52-60 (2013) Advances in Enzyme Research http://dx.doi.org/10.4236/aer.2013.13006 Kinetic studies on recombinant stem bromelain Muntari Bala1,2, Maizirwan Mel1,3, Mohamed Saedi Jami1, Azura Amid1,3, Hamzah Mohd Salleh1,3* 1Bioprocess and Molecular Engineering Research Unit, Department of Biotechnology Engineering, International Islamic University Malaysia, Kuala Lumpur, Malaysia; *Corresponding Author: [email protected] 2Department of Biochemistry, Bayero University, Kano, Nigeria 3International Institute for Halal Research & Training (INHART) International Islamic University Malaysia, Kuala Lumpur, Malaysia Received 22 May 2013; revised 30 June 2013; accepted 8 July 2013 Copyright © 2013 Muntari Bala et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT cause they represent about 60% of all commercial en- zymes worldwide. They are widely used in food, phar- Stem bromelain is a plant thiol protease with maceutical and detergent industries [1]. Plant proteases several industrial and therapeutic applications. have been gaining unique attention in the field of bio- This current work presents kinetic studies of technology and medicine due to their exploitable proper- recombinant bromelain (recBM) expressed in ties. The most recognized plant proteases with greater Escherichia coli BL21-AI on four synthetic sub- commercial values are papain from Carica papaya, ficin strates, N-α-carbobenzoxy-L-alanyl-p-nitrophenyl from Ficus spp. and bromelain from Ananas comosus ester (ZANPE), N-α-carbobenzoxy-L-arginyl-L-ar- [2]. ginine-p-nitroanilide (ZAANA), N-α-carbobenzo- Bromelain is a crude, aqueous extract from the stems xy-L-phenylalanyl-L-valyl-L-arginine-p-nitroanili- and fruits of pineapples (Ananas comosus) derived from de (ZPVANA) and L-pyroglutamyl-L-phenylala- Bromeliaceae family. It contains a mixture of different nyl-L-leucine-p-nitroanilide (PFLNA). Hydrolytic proteases as well as phosphatase, glucosidases, peroxi- activities of recBM at various pH and tempera- dases, cellulases and glycoproteins [3]. Stem bromelain ture conditions were compared to that of com- mercial bromelain (cBM). Both enzymes dem- (EC 3. 4. 22. 32) is the major protease present in extracts onstrated high activities at 45˚C and pH 5 - 8 for of pineapple stem while fruit bromelain (EC 3. 4. 22. 33) recBM and pH 6 - 8 for cBM. recBM showed is the major enzyme present in pineapple fruit juice [3]. Some other minor thiol endopeptidases—ananain and marginally lower Km and slightly higher kcat/Km for ZAANA, ZANPE and ZPVANA in comparison comosain—are also present in the pineapple stem bro- to cBM. trans-Epoxysuccinyl-L-leucylamido {4- melain. All commercially available bromelain are de- guanidino} butane (E-64) severely affected recBM rived from the stem. Stem bromelain is activated by cys- and cBM hydrolysis of the synthetic substrates teine while hydrogen sulphide and sodium cyanide are less effective [4]. The enzyme is inhibited by heavy met- by competitive inhibition with Ki values of 3.6 - 5.1 µM and 5.5 - 6.9 µM for recBM and cBM, re- als such as mercury and silver as well as trans-epoxysu- spectively. The evaluated properties of recBM ccinyl-L-leucylamido {4-guanidino} butane, commonly including temperature and pH optima, substrate known as E-64. specificity and sensitivity to inhibitors or acti- Bromelain has numerous therapeutic, industrial and vators, satisfy the requisites required for food other applications. It has been widely used in food indus- industries. try for baking processes, meat tenderization, clarification of beer, as food supplement and in prevention of brown- Keywords: Protease; Recombinant Bromelain; ing of apple juice [5]. Additionally, it is used as active Substrates; Inhibitors; Activators ingredient to provide mild peeling effects in cosmetic industries [6]. Furthermore, it has also been used in lea- 1. INTRODUCTION ther industries for skin pre-tanning, softening and bating [7]. In textile industries, bromelain is used for improving Proteases are very significant industrial enzymes be- the dyeing qualities of protein fibers, decomposing or Copyright © 2013 SciRes. OPEN ACCESS M. Bala et al. / Advances in Enzyme Research 1 (2013) 52-60 53 partially solubilizing protein fiber from silk and wool [8]. 2.2. Microbial Strain Similarly, bromelain is used as hydrolyzing agent for the The Escherichia coli BL21-AI strain (Invitrogen, USA) release of antimicrobial peptides of leatherjacket’s in- harboring stem bromelain gene used in this study was as soluble proteins [9]. described in our earlier study [12]. Specifically, the gene Stem bromelain is a highly accepted phytotherapeutic encoding stem bromelain was initially amplified from agent. It has anti-tumor and anti-inflammatory effects [4]. pineapple’s stem cloned into pENTR/TEV/D-TOPO be- In addition, it exerts several inhibitory effects on platelet fore being sub-cloned into the expression vector aggregation, bronchitis, angina pectoris, surgical traumas, pDEST17 (Invitrogen, USA). The expression vector sinusitis, thrombophlebitis and pyelonephritis. Moreover, containing stem bromelain gene was then transformed it enhances absorption of drugs, especially antibiotics into E. coli BL21-AI competent cells. [10]. The advances in molecular genetics and genetic engineering in the last decades have made it possible to 2.3. Enzyme Expression clone and express virtually any gene into a suitable mi- crobial host, so that new enzymes from other microor- Recombinant bromelain expression was conducted as ganisms and also from higher organisms can be produced described in our earlier studies [12-14]. E. coli BL21-AI in convenient microbial hosts like bacteria, yeasts and cells harboring recombinant bromelain gene were grown fungi [11]. in shake flasks overnight in LB media containing 100 Considering the numerous applications of stem bro- μg/ml ampicillin. The overnight culture was then diluted melain, it is imperative to produce the enzyme in recom- 50 folds in a fresh LB media and was grown in a 2 liter binant form so as to achieve improved purification, bio- bioreactor at 27˚C, air flow rate of 1.0 vvm with 250 rpm chemical characterization and formulation. In that regard, agitation until a cell density of OD600 nm = 0.6 was we have reported the cloning and expression of recom- reached. This was followed by addition of L-arabinose binant stem bromelain (recBM) [12], recovery from solu- (0.15% w/v, final concentration) and the induction was ble and insoluble enzyme forms [13], lab-scale optimiza- allowed to continue for 8 hours. Cells were harvested tion of some of its culture cultivation conditions [14] and from the spent media by centrifugation (6000 × g) at 4˚C the enzyme thermal and storage stability [15]. The pur- for 20 min and stored at −20˚C for further use. All the pose of the current research is to further characterize the experiments were performed in triplicates. enzyme by focusing on the kinetic studies of recBM us- ing four synthetic substrates; N-α-carbobenzoxy-L-phe- 2.4. Enzyme Purification nylalanyl-L-valyl-L-arginine-p-nitroanilide (ZPVANA), The harvested cells were subjected to sonication (so- N-α-carbobenzoxy-L-alanyl-p-nitrophenyl ester (ZANPE), nicator, 150 v/t model, Biologics, Inc. USA) on ice using N-α-carbobenzoxy-L-arginyl-L-arginine-p-nitroanilide 6 - 10 sec burst, with 10 sec interval at high amplitude. (ZAANA) and L-pyroglutamyl-L-phenylalanyl-L-leuci- This was followed by centrifugation (8000 × g) at 4˚C, ne-p-nitroanilide (PFLNA). The results are then com- for 30 min and the supernatant was collected and purified pared to that of commercial bromelain (cBM) as control. by AKTA purifier FPLC system (GE Healthcare Bio- Sciences, USA) attached with a glass column (0.7 × 10 2. MATERIALS AND METHODS cm, Bio-Rad, USA) that is packed with 4 ml Ni-NTA 2.1. Reagents His•bind resin (Novagen, Germany). Purification of recBM was conducted in accordance to the manufac- Synthetic amide and peptide substrates, N-α-carbo- turer’s instructions. The supernatant (10 ml) was loaded benzoxy-L-alanyl-p-nitrophenyl ester (ZANPE), N-α-car- onto a pre-equilibrated Ni-NTA His•bind column in ac- bobenzoxy-L-arginyl-L-arginine-p-nitroanilide (ZAANA), cordance to the manufacturer’s protocol. The FPLC sys- N-α-carbobenzoxy-L-phenylalanyl-L-valyl-L-arginine-p- tem was set at a flow rate of 1 ml/min throughout the nitroanilide (ZPVANA) and L-pyroglutamyl-L-phenyla- operation. Washing step was achieved by using a wash lanyl-L-leucine-p-nitroanilide (PFLNA) were purchased buffer (50 mM NaH2PO4 pH 8, 300 mM NaCl, 20 mM from Bachem, Germany. Luria Bertani (LB) was a prod- imidazole) while purified recBM was recovered using an uct of Merck, Germany. A single lot (Ref. No. 61391) of elution buffer (50 mM NaH2PO4 pH 8, 300 mM NaCl stem bromelain (EC3. 4. 22. 32) was purchased from MP and 500 mM imidazole). Biomedicals, LLC (France). Coomassie brilliant blue R-250, acrylamide, bisacrylamide and low-range mo- 2.4.1. SDS-PAGE and Western Blot lecular weight markers were obtained from Bio-Rad After each step of enzyme recovery and purification, (Hercules, CA). All other reagents were purchased from the protein fractions were tested by SDS-PAGE in 12.5% Sigma (St. Louis, MO). polyacrylamide gels as described earlier [12]. Visualiza- Copyright © 2013 SciRes. OPEN ACCESS 54 M. Bala et al. / Advances in Enzyme Research 1 (2013) 52-60 tion was conducted by Coomassie staining [16]. On the on the hand, for the remaining three substrates The protein bands on the gel were transferred to poly- (ZAANA, ZPVANA and PFLNA) changes in the ab- vinylidene fluoride (PVDF) membrane (Bio-Rad Labo- sorbance were measured at 410 nm for the p-nitroaniline ratory, Inc., USA) using Mini-Trans-Blot system (Bio- generated [19,20].