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

COMPARISON OF DIFFERENT CELL DISRUPTION METHODS AND CELL EXTRACTANT BUFFERS FOR RECOMBINANT BROMELAIN EXPRESSED IN E.COLI BL21-A1

The often-encountered problem such as protein degradation has driven various methods of cell lysis in obtaining recombinant protein post fermentation. In this paper, we compare methods such as homogenization, sonication, sonication with lysozyme and chemical lysis using B-PER reagent with lysozyme to extract the recombinant bromelain from E. coli BL21-AI. The sonication process is found to be the most effective in releasing recombinant bromelain without any pre-treatment. To obtain the high quality of protein from sonication method, the influence of different extractant buffer was investigated including phosphate buffer saline (PBS), PBS containing cysteine and EDTA (PBS-CE), and sodium phosphate buffer containing cysteine and EDTA (EB-CE). The highest specific enzyme activity was obtained when it was extracted with EB-CE buffer. Under sodium dodecyl sulfate polyacrylamide gel electrophoresis, the recombinant bromelain showed protein band at 55kDa. In conclusion, the sonication method with extractant buffer containing 100mM phosphate buffer pH7.0 with 15 mM cysteine and 2 mM EDTA (EB-CE) was shown to give high specific activity of recombinant bromelain

PLA Degradation and PLA-Degrading Bacteria: A Mini-Review Journal: Key Engineering Materials

Polylactic acid (PLA) is not new to the world of science, since the application of PLA can be found in various industries such as biomedical, agricultural, and packaging. Despite the amazing properties shown by PLA, it still has a setback in terms of waste disposal of PLA. Since PLA is more resistant towards bacterial attack, it prolonged the decomposition of PLA disposed in the environment. Therefore, PLA microbial degradation and enzymatic degradation needs to be highlighted since most PLA waste will end up in the landfill. Most PLA-degrading can be found in the genus family Amycolatopsis, and a few can be found in the genus Lentzea, Kibdelosporangium, Paecilomyces, Thermomonospora, and Thermopolyspora. The enzymatic degradation of PLA is mostly studied relating to enzyme proteinase K, serine protease, and even hydrolase. This review paper aims to discuss the microbial degradation mechanism of PLA as well as the types of microorganisms and enzymes that involve in the biodegradation of PLA

Case Study: Recombinant Bromelain Downstream Processing

This chapter presents details on the purification, formulation and drying of recombinant bromelain. The wide range of applications of recombinant bromelain has increased interest in finding viable purification techniques for large-scale production. An affinity chromatography technique was developed by Amid and co-workers (Expression, purification, and characterization of a recombinant stem bromelain from Ananas comosus, Process Biochem 46:2232–2239, 2011) to purify recombinant bromelain. However, this technique presented low recovery and small sample loading capacity and thus is not suitable as a purification tool in the largescale production of recombinant bromelain. An aqueous two-phase system is one alternative method that we use to purify recombinant bromelain, as it reliable and easy to scale up and has a low cost. As part of avoiding cysteine degradation, spray drying the purified recombinant protein with maltodextrin as an excipient provides the possibility of preserving its activity and creating fine particles that are suitable for end-product application. The processes of the purification, formulation and spray drying of recombinant bromelain are explained briefly

Rhodopseudomonas palustris collagen-like recombinant protein purification using an aqueous two-phase system

The potential use of recombinant collagen-like protein (recCLP) extracted from bacteria as disease-free collagen has been studied over the past decade. However, the complexity of the downstream processing generates high demand for an efficient and low-cost purification method. Aqueous two-phase system (ATPS) was adopted as a new approach to the recovery of biomolecules due to its simple, benign, and straightforward process. This study aimed to purify recombinant collagen-like protein from Rhodopseudomonas palustris using ATPS formed by a polymer/salt system. Recombinant collagen-like protein from R. palustris was partitioned in ATPS composed of polyethylene glycol (PEG) and potassium phosphate and several factors that influence the protein partitioning such as volume ratio, system pH, the concentration of polymer and salt were studied. Then, optimization of the selected ATPS conditions (PEG and salt concentration) were performed using response surface methodology (RSM). Results showed that the optimum conditions were found in ATPS with 24.80% (w/w) PEG 2000 and 29.23% (w/w) potassium phosphate with recCLP concentration of 3.23 ± 0.12 mg/mL with purification factor 7.48 ± 0.3. In comparison with the affinity chromatography method, ATPS was found to be low-cost, and time-saving with a higher protein recovery. Hence, this study demonstrated the potential application of ATPS in the recovery of recombinant CLPs for large-scale downstream processin

Bromelain: an overview of industrial application and purification strategies

Abstract This review highlights the use of bromelain in various applications with up-to-date literature on the purification of bromelain from pineapple fruit and waste such as peel, core, crown, and leaves. Bromelain, a cysteine protease, has been exploited commercially inmany applications in the food, beverage, tenderization, cosmetic, pharmaceutical, and textile industries. Researchers worldwide have been directing their interest to purification strategies by applying conventional and modern approaches, such as manipulating the pH, affinity, hydrophobicity, and temperature conditions in accord with the unique properties of bromelain. The amount of downstream processing will depend on its intended application in industries. The breakthrough of recombinant DNA technology has facilitated the large-scale production and purification of recombinant bromelain for novel applications in the future

Polymer-salt interaction

Up to this moment, an uprising demand in downstream processing by incorporate innovative, effective, and reliable extraction methods as plenty effort and progression in the upstream production have led to advancement in the biomanufacturing industry. Previously, deployment of conventional downstream technologies has been a hindrance due to high cost, time-consuming, low yield, detrimental, noneco-friendly, lack of simplicity, and difficulties in scaling-up. A new emerging liquid–liquid extraction technology by means the polymer–salt aqueous two phases system for the recovery of biomolecules such as proteins, DNA, and nucleic acids, virus-like particles, and drug residues in food and water. Moreover, the partition coefficient of polyethylene glycol (PEG) 6000-phosphate aqueous two-phase system (ATPS) increased by 62-fold by adding NaCl which results in the recovery of proteins from 90% to 95% in a single purification step. Besides, plasmid DNA successfully recovered by 67% in the PEG-rich phase and managed to partition the contaminants toward the salt-rich phase. The total recovery of 90% from the separation of adenoviral vectors in HEK 293 cells using PEG-ammonium sulfate ATPS. In addition, the recovery of drug residues from food and water corresponds to recovery rate of 96%–100% which is safe for human consumption. This review discusses the following basic mechanism and working principles of ATPS. Factors affecting the partitioning behavior of biomolecules for instance, PEG molecular weight, PEG concentration, system pH, effect of temperature, hydrophobicity, and addition of salt are presented. Future consideration and modification to allow further improvement toward reliable large-scale extraction and purification of biomolecules

Sustainable Carrageenan/Nanocomposite Films Incorporated with Optimized Zingiber officinale Extracts for Active Packaging Systems

Optimized Bentong ginger (BG) extract using ultrasonic-assisted extraction (UAE) was incorporated in hybrid carrageenan/nanocomposite biopolymers for active packaging films. The design of experiments based on central composite design was applied for the study of the operating parameters (amplitude, sonication time, and solvent concentration) of UAE in the recovery of phenolic compounds from BG. Optimized extracts were analyzed for the content of 6-gingerols in liquid chromatography quadrupole time-of-flight mass spectrometry and formulated in carrageenan/nanocellulose hybrid films for active packaging applications. The mechanical properties of the films and release behavior of antioxidant compounds into food simulants were investigated. The optimized UAE BG extract was found efficacious as an antioxidant agent in active packaging

Effect of spray drying parameters on the physicochemical properties and oxidative stability of oil from menhaden (Brevoortia spp.) and Asian swampeel (Monopterus albus) oil extract microcapsules

This work investigated the effect of spray drying parameters on the physicochemical properties and oxidative stability of oil from menhaden (Brevoortia spp.) and Asian swamp eel (Monopterus albus) oil microcapsules. Different emulsion formulations (Maltodextrin - MD, Maltodextrin+Gum Arabic - MD+GA, and Maltodextrin+Starch - M+S), inlet air temperatures (180, 190, and 200 °C), and feed flow rates (280, 382, and 485 mL/h) were applied to microencapsulate Brevoortia spp. oil. The best operating parameters were then used to microencapsulate the Monopterus albus oil. The moisture content, MC (%), peroxide value, PV (mEq/kg), free fatty acid, FFA (%), acid value, AV (mg KOH/g) and the morphology of the microcapsules were then evaluated. The Brevoortia spp. oil microcapsules produced with the Maltodextrin+Gum Arabic emulsion formulation, inlet air temperature of 200 °C, and feed flow rate of 280 mL/h showed the lowest moisture content, peroxide value, free fatty acid, and acid value of 9.145%, 3.293 mEq/kg, 4.891%, and 2.981 mg KOH/g, respectively. Using similar parameters, the microencapsulation of the Monopterus albus oil extract recorded a moisture content, peroxide value, free fatty acid, and acid value of 8.432%, 2.713 mEq/kg, 4.911%, and 2.871 mg KOH/g, respectively. In conclusion, improved physicochemical properties and oxidative stability of Monopterus albus oil extract microcapsules were achieved using the Maltodextrin+Gum Arabic emulsion formulation and spray drying at a high air inlet temperature of 200 °C and a low feed flow rate of 280 mL/h