Production of intracellular recombinant bromelain by Escherichia coli BL21-AI using shake flask and stirred tank bioreactor

The optimization of recombinant protein production by Escherichia coli (E. coli) is an important first step to ensure economic feasibility and realization towards a sustainable future commercialization prospects. Bromelain, a protease native to pineapple plant species, is one of the commercially important therapeutic enzymes. In the field of bromelain research, production of recombinant Bromelain (recBromelain) using E. coli BL21-AI is one of the most recent novel developments to provide a more robust alternative as compared to conventional extraction approaches. Based on previous studies, the recBromelain indeed had proteolytic potency as effective as commercial bromelain. However, judging from the level of protein expression, the recBromelain productivity using E. coli BL21-AI expression system was too low to be considered for commercialization. It has been well established that suboptimal fermentation medium and process parameters which directly affect the growth of E. coli are among primary causes behind low recombinant protein expression. Optimization studies on these two aspects were therefore warranted and sought to fill the gap left unexplored from previous studies. Statistical design of experiments based on Plackett-Burman, Factorial design, One-Factor-at-a-Time and Response Surface Methodology were employed to identify significant few of E. coli growth parameters that affected recombinant bromelain productivity. Studier autoinduction medium was used as the basis for fermentation medium optimization as the medium produced highest crude recBromelain specific activity during preliminary media screening in shake flask. Analysis of variance (ANOVA) shows that concentration of tryptone, NH4Cl, and CoCl2 affected specific activity the most where at 14% (w/v), 153.4 mM and 0.4 μM, respectively, they significantly improved specific activity by 350% as compared to original autoinduction medium performance. Tryptone and NH4Cl ensured abundant nitrogenous elements used as the building blocks to sustain cell growth. CoCl2 was a vital metal cofactor in E. coli cellular enzymatic machinery but inevitably became toxic at higher concentration. A more economical alternative which used a low level of tryptone (4% w/v) was significantly reduced the medium cost by 68% at the expense of a reduced degree of improvement (130%) in crude specific activity. The effects of fermentation process parameters were investigated using 2-L stirred tank bioreactor using the improved media composition. ANOVA shows that dissolved oxygen tension (DOT), aeration, and reduction temperature had the most significant effect on specific activity. The highest level of specific activity was found at 35% DOT, 1 vvm, pH 7.4 and 30°C where the value was comparable as in optimized shake flask culture. At these conditions, a lower specific growth rate was achieved where it promoted better protein folding as compared to higher growth rate. Finally, attempt to increase recBromelain production volume further using 30-L bioreactor was made using constant impeller tip speed and constant oxygen transfer coefficient (kLa) to determine the best range for impeller tip speed. A range of impeller tip speed range between 0.47 – 0.86 m/s under constant kLa produced a more comparable specific activity as obtained from 2-L stirred tank bioreactor production. This confirmed that oxygen transfer was a limiting factor in aerobic culture owing to the low solubility of oxygen in the fermentation broth and insufficient mixing when dealing with high cell density cultures of fast-growing E. coli

Common Laboratory Procedure

This chapter presents all common laboratory procedures or protocols that researchers normally perform during studies of recombinant enzyme production. The principle and theory behind each experiment is presented to help the reader understand each step that is conducted in an experiment so the reader will be able to select and modify the procedures according to the type of enzyme they are working with. Examples of procedures and the results obtained during recombinant bromelain production are used to develop readers’ understandin

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

Optimization on cell disruption of E. coli BL21-AI expressing recombinant bromelain

Recent advancements in recombinant DNA technology proved to be a promising and effective approach for more sustainable large scale productions of many therapeutic proteins. Nevertheless, since this approach involves expression of proteins in a nonnative host microorganism, the overall production processes are not straight-forward due to several common challenges, such as protein degradation, especially during cell disruption stage. As the process has been subjected to both protein and host-specific, a systematic process conditioning for maximal production of recombinant protein is therefore required. In this study, a simple approach to determine optimal conditions for cell disruption using ultrasonication to isolate recombinant bromelain from E. coli BL21- AI is reported. Suspension cells were lysed using ultrasonication which transmit sound wave in other to break the cell wall. An optimized condition was obtained by response surface methodology (RSM). A three factor face-centered central composite design (FCCD) was applied to obtain the optimal process conditions consisting of amplitude, cycle and bursting period. The prediction model was further validated. Therefore, under the optimal conditions, having 20% amplitude, 0.5s cycle, and 1 minute bursting period in three times process, the specific enzyme activity of the recombinan

Bromelain enzyme assay using Casen as substrate

This procedure is following Sigma's non-specific protease activity. In this assay, casein acts as a substrate to the reaction. When protease digest casein, the amino acid tyrosine is liberated along with other amino acids and peptide fragments. Folin & Ciocalteus Phenol, or Folin's reagent primarily reacts with free tyrosine to produce a blue colored chromophore, which is quantifiable and measured as an absorbance value on the spectrophotometer at 660 nm absorbance. The more tyrosine that is released from the casein, the more chromophores are generated and the stronger the activity of the protease

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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

Effects of operating conditions in spray drying of recombinant bromelain

The aim of the present study is to investigate the effect of operating parameter of a Buchi B290 mini spray dryer towards specific activity of recBromelain. Using Response Surface Methodology (RSM), face centred central composite design (FCCCD) was tabulated for optimization. Optimization process was conducted by varying the inlet air temperature (°C), gas flow height (mm) and pump settings (%) of the laboratory Buchi B290 Mini Spray Dryer to obtained optimal spray drying operating processes recovering high recBromelain specific activity as response. Results showed that the optimized process parameter having 126 °C air inlet temperature, 42 mm gas flow height and 12 % feed pump setting resulted in 0.119 ± 0.003 U/mgprotein of spray dried recBromelain with R2 of 90.12% thus this model was found to be significance and reliable

Analysis of active pharmaceutical ingredients in 20 species of traditional Malay midwifery postnatal bath

Midwifery is the practice of assisting a woman through childbirth using natural procedures.In Malay midwifery practices, traditional bath is one of the most important treatments during the confinement periods. However, today in Malay community, midwifery traditional knowledge of herbal medicine is disappearing and extinct. The facts are Malay midwives are becoming rare and the more crucial is medicinal plants are over-harvested. Therefore the aim of the research is to investigate the active pharmaceutical ingredients content in 20 selected species used in Malay midwifery practices during traditional bath. Thus, there is a solid need to analyse the potential of these natural bioactive compounds particularly carotenoids to be fully utilised and commersialised especially in halal market and health advantages. Through HPLC analysis all 20 species were found to have at least four individual carotenoid pigments with a relatively high concentration of lutein and β-carotene and lower concentrations of zeaxanthin. Surprisingly in every of 20 malay midwifery species samples used in traditional postnatal bath a relatively high lutein and β-carotene were detected. Selection of the right species with the right composition to accumulate lutein and β-carotene still remain mystery and mystic. The significant outcome of the research will be new findings of new natural bioactive compound sources as health promoting agents which covers not only the Shariah requirement, but also safety aspects