RESUMEN
Amphiphilic protein has lipophilic and hydrophilic domains, displaying the potential for development as a biosurfactant. The polyhydroxyalkanoate (PHA) surface binding protein derived from Bacillus is a type of protein that has not been studied for its emulsifying properties. In this study, PHA granule-associated protein (PhaP), PHA regulatory protein (PhaQ), and PHA synthase subunit (PhaR) derived from an alkali-tolerant PHA-producing Bacillus cereus HBL-AI were found and heterologously expressed in E. coli and purified to investigate their application as biosurfactants. It showed that the emulsification ability and stability of three amphiphilic proteins were higher than those of widely used chemical surfactants in diesel oil, vegetable oil, and lubricating oil. In particular, the PhaQ protein studied for the first time can form a stable emulsion layer in vegetable oil at a lower concentration (50 µg/mL), which greatly reduced the amount of protein used in emulsification. This clearly demonstrated that the PHA-binding protein of HBL-AI can be well applied as an environmentally friendly biosurfactants.
Asunto(s)
Bacillus , Polihidroxialcanoatos , Polihidroxialcanoatos/metabolismo , Bacillus/genética , Bacillus/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de la Membrana , Tensoactivos/metabolismo , Escherichia coli/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
A polyhydroxyalkanoate (PHA) magnetic microsphere was designed for one-step purification and immobilization of a novel carbonyl reductase (RLSR5) from recombinant Escherichia coli lysate. The hydrophobic core of this microsphere was composed of a highly biocompatible polymer, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), in which magnetic Fe3O4 particles were embedded during solvent evaporation. The hydrophilic shell of the fusion protein formed by PHA particle-binding protein (PhaP) and RLSR5 (PR) was expressed in recombinant E. coli. The magnetic core of Fe3O4@PHBHHx directly purified the hydrophilic shell from the E. coli lysate, and the two self-assembled to form Fe3O4@PHBHHx-PR through hydrophobic and hydrophilic interactions, eliminating the separation of the fusion protein. The microstructure, magnetic properties, morphology, size, and dispersion of Fe3O4@PHBHHx-PR were investigated by XRD, VSM, SEM, TEM, elemental mapping and DLS. It was found that Fe3O4@PHBHHx-PR correctly assembled, with a well dispersed spherical structure at the nanoscale and superparamagnetism properties. The amount of RLSR5 immobilized on PHA microspheres reached 121.9 mg/g. The Fe3O4@PHBHHx-PR was employed to synthesize (R)-tolvaptan with 99 % enantiomeric excess and 97 % bioconversion efficiency, and the catalyst maintained 78.6 % activity after 10 recovery cycles. These PHA magnetic microspheres are versatile carriers for enzyme immobilization and demonstrate improved stability and reusability of the free enzyme.
Asunto(s)
Polihidroxialcanoatos , Polihidroxialcanoatos/metabolismo , Microesferas , Escherichia coli/genética , Escherichia coli/metabolismo , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Fenómenos MagnéticosRESUMEN
An efficient cofactor regeneration system has been developed to provide a hydride source for the preparation of optically pure alcohols by carbonyl reductase-catalyzed asymmetric reduction. This system employed a novel glucose dehydrogenase (BcGDH90) from Bacillus cereus HBL-AI. The gene encoding BcGDH90 was found through the genome-wide functional annotation. Homology-built model study revealed that BcGDH90 was a homo-tetramer, and each subunit was composed of ßD-αE-αF-αG-ßG motif, which was responsible for substrate binding and tetramer formation. The gene of BcGDH90 was cloned and expressed in Escherichia coli. The recombinant BcGDH90 exhibited maximum activity of 45.3 U/mg at pH 9.0 and 40 °C. BcGDH90 showed high stability in a wide pH range of 4.0-10.0 and was stable after the incubation at 55 °C for 5 h. BcGDH90 was not a metal ion-dependent enzyme, but Zn2+ could seriously inhibit its activity. BcGDH90 displayed excellent tolerance to 90% of acetone, methanol, ethanol, n-propanol, and isopropanol. Furthermore, BcGDH90 was applied to regenerate NADPH for the asymmetric biosynthesis of (S)-(+)-1-phenyl-1,2-ethanediol ((S)-PED) from hydroxyacetophenone (2-HAP) with high concentration, which increased the final efficiency by 59.4%. These results suggest that BcGDH90 is potentially useful for coenzyme regeneration in the biological reduction.