No 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 PGL occupied 0.985% of the total weight of purified PHA granules, which was identified by mass spectrometer-based quantitative proteomics. The activity of immobilized PGL (184.67 U/mg PGL protein) was a little lower than that of the free PGL (215.93 U/mg PGL protein). The immobilization process did not affect the optimal pH and the optimal temperature of the PGL, but it did enhance the thermostability as well as the pH stability at certain conditions, which will extend the practicability of the immobilized PGL-PHA beads in the alkaline and generally harsh bioscouring process. Furthermore, the immobilized PGL still retained more than 60% of its initial activity after 8 cycles of reuse. Our study provided a novel and promising approach for cost-efficient in vivo PGL immobilization, contributing to wider commercialization of this environmental-friendly biocatalyst.

3,4-dihydroxyphenyl-L-alanine (L-DOPA) is a preferred drug for Parkinson’s disease, with an increasing demand worldwide that mainly relies on costly and environmentally problematic chemical synthesis. Yet, biological L-DOPA production is unfeasible at the industrial scale due to its low L-DOPA yield and high production cost. In this study, low-cost Halomonas bluephagenesis TD01 was engineered to produce tyrosinase TyrVs-immobilized polyhydroxyalkanoate (PHA) nanogranules in vivo, with the improved PHA content and increased immobilization efficiency of TyrVs accounting for 6.85% on the surface of PHA. A higher L-DOPA-forming monophenolase activity of 518.87 U/g PHA granules and an L-DOPA concentration of 974.36 mg/L in 3 h catalysis were achieved, compared to those of E. coli. Together with the result of L-DOPA production directly by cell lysates containing PHA-TyrVs nanogranules, our study demonstrated the robust and cost-effective production of L-DOPA by H. bluephagenesis, further contributing to its low-cost industrial production based on next-generation industrial biotechnology (NGIB).

Poly­(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) is an intracellular biopolyester synthesized by various bacteria. Polyhydroxyalkanoate granule-binding protein (PhaP), a natural biomacromolecule symbiotic with PHBHHx, can be steadily adsorbed into the PHBHHx matrix through hydrophobic interactions. In this study, PHBHHx nanoparticles (NPs) and iRGD peptide fused PhaP (iRGD-PhaP) were used in conjunction to build a specific drug delivery system for targeted accumulation and tissue penetration in prostate tumors. A proper presentation and high surface density of iRGD could be ensured within 1 h through a convenient coincubation method using a PhaP-mediated modification strategy. iRGD-PhaP-NPs showed a satisfactory particle size (182.9 ± 4.9 nm) and slightly negative surface charge (−17.2 ± 0.3 mV), with a uniformly spherical shape. In human prostate cancer cell line PC3, iRGD-PhaP-NPs displayed remarkably improved cellular uptake compared to naked NPs, which was attributed to iRGD receptor-mediated active endocytosis. Enhanced targeted accumulation and retention of iRGD-PhaP-NPs in prostate tumors were found in both the ex vivo tumor spheroid assay and in vivo real-time imaging. Moreover, slices of the tumor deep region demonstrated the favorable tumor penetration ability of iRGD-PhaP-NPs after intravenous administration. These results highlight the specificity and efficiency of iRGD-PhaP-NPs in future clinical use.