Abstract:Alkaline polygalacturonate lyase (PGL), one of the pectinolytic enzymes, has been widely used for the bioscouring of cotton fibers, biodegumming, and biopulp production. In our study, PGL from Bacillus subtilis was successfully immobilized on the surface of polyhydroxyalkanoate (PHA) nanogranules by fusing PGL to the N-terminal of PHA synthase from Ralstonia eutropha via a designed linker. The PGL-decorated PHA beads could be simply achieved by recombinant fermentation and consequent centrifugation. The fused … Show more
“…The results indicated that the target fused proteins were immobilized on the PHA granules, comprising approximately 5% of the total attached protein (Table 1). The ratio of fused protein was higher than 0.985% described previously [6]. Fig.…”
Section: Resultsmentioning
confidence: 51%
“…These surface proteins have attracted much attention and inspired investigations to explore them as anchoring tags to attach proteins and peptides to the surface of BNPs. The engineered BNPs are exciting prospects for drug targeting [5], bio-catalysis [6], biochemical separation [7] and bioremediation [8]. Compared with existing chemical immobilization technologies [9, 10], surface engineering of BNPs is highly advantageous.…”
Background
Polyhydroxyalkanoate (PHA) are nano-granules naturally produced by bacteria. Two types of proteins, PHA synthase (PhaC) and phasins (PhaPs), are attached to the PHA surface by covalent and hydrophobic interactions. Utilizing these anchored proteins, functionalized PHA nano-granules displaying proteins of interest can be easily prepared by fermentation.
Results
In this study, a one-step fabrication method was developed for stable and efficient immobilization of an organophosphorus degrading enzyme on PHA nano-granules. The nano-biocatalysts were produced in recombinant Escherichia coli cells into which the polyhydroxyalkanoate synthesis pathway from Cupriavidus necator had been introduced. Two different strategies, covalent attachment and hydrophobic binding, were investigated by fusing bacterial organophosphorus anhydride hydrolase (OPAA4301) with PhaC and PhaP, respectively. Using both methods, the tetrameric enzyme successfully self-assembled and was displayed on the PHA surface. The display density of the target fused enzyme was enhanced to 6.8% of total protein on decorated PHA by combination of covalent and non-covalent binding modes. Immobilization of the enzyme on PHA granules resulted in higher catalytic efficiency, increased stability and excellent reusability. The kcat values of the immobilized enzymes increased by threefold compared to that of the free enzyme. The pH stability under acidic conditions was significantly enhanced, and the immobilized enzyme was stable at pH 3.0–11.0. Furthermore, more than 80% of the initial enzyme activity retained after recycling ten times.
Conclusions
This study provides a promising approach for cost-efficient in vivo immobilization of a tetrameric organophosphorus degrading enzyme. The immobilization process expands the utility of the enzyme, and may inspire further commercial developments of PHA nano-biocatalysts. As revealed by our results, combination of covalent and non-covalent binding is recommended for display of enzymes on PHA granules.
“…The results indicated that the target fused proteins were immobilized on the PHA granules, comprising approximately 5% of the total attached protein (Table 1). The ratio of fused protein was higher than 0.985% described previously [6]. Fig.…”
Section: Resultsmentioning
confidence: 51%
“…These surface proteins have attracted much attention and inspired investigations to explore them as anchoring tags to attach proteins and peptides to the surface of BNPs. The engineered BNPs are exciting prospects for drug targeting [5], bio-catalysis [6], biochemical separation [7] and bioremediation [8]. Compared with existing chemical immobilization technologies [9, 10], surface engineering of BNPs is highly advantageous.…”
Background
Polyhydroxyalkanoate (PHA) are nano-granules naturally produced by bacteria. Two types of proteins, PHA synthase (PhaC) and phasins (PhaPs), are attached to the PHA surface by covalent and hydrophobic interactions. Utilizing these anchored proteins, functionalized PHA nano-granules displaying proteins of interest can be easily prepared by fermentation.
Results
In this study, a one-step fabrication method was developed for stable and efficient immobilization of an organophosphorus degrading enzyme on PHA nano-granules. The nano-biocatalysts were produced in recombinant Escherichia coli cells into which the polyhydroxyalkanoate synthesis pathway from Cupriavidus necator had been introduced. Two different strategies, covalent attachment and hydrophobic binding, were investigated by fusing bacterial organophosphorus anhydride hydrolase (OPAA4301) with PhaC and PhaP, respectively. Using both methods, the tetrameric enzyme successfully self-assembled and was displayed on the PHA surface. The display density of the target fused enzyme was enhanced to 6.8% of total protein on decorated PHA by combination of covalent and non-covalent binding modes. Immobilization of the enzyme on PHA granules resulted in higher catalytic efficiency, increased stability and excellent reusability. The kcat values of the immobilized enzymes increased by threefold compared to that of the free enzyme. The pH stability under acidic conditions was significantly enhanced, and the immobilized enzyme was stable at pH 3.0–11.0. Furthermore, more than 80% of the initial enzyme activity retained after recycling ten times.
Conclusions
This study provides a promising approach for cost-efficient in vivo immobilization of a tetrameric organophosphorus degrading enzyme. The immobilization process expands the utility of the enzyme, and may inspire further commercial developments of PHA nano-biocatalysts. As revealed by our results, combination of covalent and non-covalent binding is recommended for display of enzymes on PHA granules.
“…The bioscouring assay exhibited that PEL3 can obviously improve the wettability and softness of fabrics, suggesting its potential application in textile industry (Wang et al, 2018). The first case for enzyme immobilization of pectate lyases (PGL) from B. subtilis was reported by Ran et al (2017), PGL was successfully immobilized on the surface of polyhydroxyalkanoate (PHA) nanogranules by fusing PGL to the N-terminal of PHA synthase from Ralstonia eutropha via a designed linker. The activity of PGL-PHA compared to the free PGL was decreased almost 15%, and immobilization process did not affect the optimal pH and temperature of the free Pels, which was also observed with PEL3/Cu 3 (PO 4 ) 2 hybrid nanoflowers.…”
Pectate lyases (Pels) can be used in the textile industrial process for cotton scouring and ramie degumming, and its hydrolyzed products oligo galacturonic acid, are high-value added agricultural and health products. In our previous studies, an alkaline pectate lyase PEL168 mutant, PEL3, was obtained with improved specific activity and thermostability. Here, a facile and rapid method for preparing an immobilized PEL3-inorganic hybrid nanoflower was developed, as it could improve its biocatalytic performance. With 0.02 mg/mL (112.2 U/mL) PEL3 in PBS buffer, five different divalent ions, including Mn 2+ , Ca 2+ , Co 2+ , Zn 2+ , and Cu 2+ , were used as inorganic component. The results showed that PEL3/Cu 3 (PO 4) 2 hybrid nanoflowers presented the highest relative activity with 2.5-fold increase, compared to the free PEL3. X-ray diffraction analysis confirmed that the composition of PEL3/Cu 3 (PO 4) 2 hybrid nanoflowers were pectate lyase PEL3 and Cu 3 (PO 4) 2 •5H 2 O. The optimum temperature and pH of PEL3/Cu 3 (PO 4) 2 hybrid nanoflowers were ascertained to be 55 • C and pH 9.0, respectively, exhibiting subtle difference from the free PEL3. However, the PEL3/Cu 3 (PO 4) 2 hybrid nanoflowers maintained 33% residual activity after 24 h incubation at 55 • C, while the free PEL3 completely lost its activity after 18 h incubation at 55 • C. Furthermore, over 50% residual activity of the PEL3/Cu 3 (PO 4) 2 hybrid nanoflowers was remained, even after four times of repetitive utilization, demonstrating its promising stability for practical application.
“…Given the encouraging proof-of-concept results adapting the PHA particle technology for development of immobilized enzymes for uses in the food industry (Moldes et al, 2004;Rasiah and Rehm, 2009;Ran et al, 2017Ran et al, , 2019, production of commodity chemicals (Jahns and Rehm, 2015;Yang et al, 2015;Seo et al, 2016), production of fine chemicals Chen S. Y. et al, 2014;Tan et al, 2019), and bioremediation (Robins et al, 2013;Hooks and Rehm, 2015;Li et al, 2019), it is anticipated that research prototypes will be developed into industrial products.…”
Section: Potential Industrial Applications Of the Pha Particle Technomentioning
Enzymes function as biocatalysts and are extensively exploited in industrial applications. Immobilization of enzymes using support materials has been shown to improve enzyme properties, including stability and functionality in extreme conditions and recyclability in biocatalytic processing. This review focuses on the recent advances utilizing the design space of in vivo self-assembled polyhydroxyalkanoate (PHA) particles as biocatalyst immobilization scaffolds. Self-assembly of biologically active enzyme-coated PHA particles is a one-step in vivo production process, which avoids the costly and laborious in vitro chemical cross-linking of purified enzymes to separately produced support materials. The homogeneous orientation of enzymes densely coating PHA particles enhances the accessibility of catalytic sites, improving enzyme function. The PHA particle technology has been developed into a remarkable scaffolding platform for the design of cost-effective designer biocatalysts amenable toward robust industrial bioprocessing. In this review, the PHA particle technology will be compared to other biological supramolecular assembly-based technologies suitable for in vivo enzyme immobilization. Recent progress in the fabrication of biological particulate scaffolds using enzymes of industrial interest will be summarized. Additionally, we outline innovative approaches to overcome limitations of in vivo assembled PHA particles to enable finetuned immobilization of multiple enzymes to enhance performance in multi-step cascade reactions, such as those used in continuous flow bioprocessing.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
