Case Study: Recombinant Bromelain Selection

This chapter presents an investigation that we performed prior to the decision to proceed cloning and producing recombinant bromelain. The criteria that we examined were the following: (1) easy access to a DNA source; (2) broad application; (3) an enzyme size amenable to the cloning strategy; (4) available data in free online databases; (5) broad industrial application

Case study: recombinant Bromelain cloning, characterization and upstream processes

This chapter presents the recombinant stem bromelain cloning procedure followed by its characterization and finally upstream processing. Most of the procedures had patents filed in Malaysia, Europe and the United State of America with application numbers PI 20095434, 10015711.4, and 12968766, respectively. Portions of the experimental procedures were also presented in our publication (Amid et al., Process Biochem 46:2232–2239, 2011). The entire cloning process, characterization and the upstream processing of the recombinant bromelain production are briefly explained

Bromelain production: Current trends and perspective

Bromelain is a mixture of proteolytic enzymes derived from the stems and juice of pineapples. It has been extensively used in food industry; for meat tenderization, baking processes, and in prevention of browning of apple juice. Stem bromelain is a highly accepted phytotherapeutic agent. It has anti-tumor and antiinflammatory effects, use in wound healing, as digestive aids, etc. This review aims at presenting a detailed account on the level of achievement on bromelain production from the conventional methods. Specifically, the isolation, purification and the biochemical properties of bromelain are discussed. The study also focused on the current trends and the progress made in the production of recombinant stem bromelain in Escherichia coli. This involved cloning of stem bromelain gene and its expression, optimization of labscale fermentation conditions for E. coli harboring recombinant bromelain as well as the enzyme thermal and storage stability measured using differential scanning calorimetry. It is hoped that this review would provide relevant information to contemporary researchers who are active in the field of bromelain and other related proteases

Differential Scanning Calorimetry as tool in observing thermal and storage stability of recombinant Bromelain

It is important to know the thermal and storage behavior of produced protein for the purpose of storage, transport and shelve life during industrial application. Differential Scanning Calorimetry (DSC) is used to measure the thermal and storage stability of recombinant bromelain produced in our laboratory. Recombinant bromelain thermal and storage stability were measured and compared to the commercial bromelain. The purified recombinant bromelain was powderized using freeze drying method and its transition midpoint, crystallinity and enthalpy were recorded. Recombinant bromelain is more stable than commercial bromelain at higher temperature but the stability reduced after 7 days of storage at 4oC although its activity is lower than commercial at day 1 and 7. Higher energy is needed to break the bond between amino acid chains in recombinant bromelain showing by the enthalpy recorded suggesting that recombinant bromelain has good protein structure and conformation compared to commercial

Recombinant-enzyme fermentation

This chapter presents an overview of the issues involved in the fermentation of E. coli harboring a transgene. The successful production of a heterologous enzyme requires the thoughtful integration and optimization of media formulations, physical factors associated with fermentation, host specificities and induction strategies

Expression, purification, and characterization of a recombinant stem bromelain from ananas comosus

Commercially available bromelain is prepared by performing a tedious and costly purification method that provides multiple degrees of purified bromelain. In the current study, a gene encoding stem bromelain from Ananas comosus was amplified using polymerase chain reaction (PCR). This bromelain gene was initially cloned into pENTR/TEV/D-TOPO before being sub-cloned into the expression vector pDEST17. DNA sequencing of the amplified products exhibited a high level of homology to the corresponding gene from the NCBI public database. Protein expression was conducted in the Escherichia coli, BL21-AI. The recombinant bromelain was then purified in a single step using a Ni-NTA spin column, an example of immobilized metal affinity chromatography. Purified recombinant bromelain was detected by Western blotting. In addition, the purified enzyme exhibited hydrolytic activity towards gelatin and a synthetic substrate, LNPE. The purified recombinant bromelain exhibited optimum activity at pH 4.6 and 45oC

Identification of potential biomarkers of porcine gelatin

This chapter discusses the identification of potential biomarkers of porcine gelatin using peptide mass fingerprinting (PMF) for the development of anti-peptide polyclonal antibodies. The selection of antigens is a prerequisite for the development of enzyme-linked immunosorbent assay (ELISA). A synthetic peptide is one of the prevalent antigens for ELISA development. It enables the produced antibodies to be targeted at small regions of the protein with fine specificity. It has been used to solve food authenticity issues