Efficient production of High-Purity manno-oligosaccharides from guar gum by citric acid and enzymatic hydrolysis.

Currently, the production of manno-oligosaccharides (MOS) from guar gum faces challenges of low oligosaccharide enzymatic hydrolysis yield and complicated steps in separation and purification. In this work, a potential strategy to address these issues was explored. By combining citric acid pretreatment (300 mM, 130 °C, 1 h) with ß-mannanase hydrolysis, an impressive MOS yield of 61.8 % from guar gum (10 %, w/v) was achieved. The key success lay in the optimizing conditions that completely degraded other galactomannans into monosaccharides, which could be easily removable through Saccharomyces cerevisiae fermentation (without additional nutrients). Following ion exchange chromatography for desalination, and concluding with spray drying, 4.57 g of solid MOS with a purity of 90 % was obtained from 10 g of guar gum. This method offers a streamlined and effective pathway for obtaining high-yield and high-purity MOS from guar gum by combining citric acid pretreatment and enzymatic hydrolysis.

Economical synthesis of γ-cyclodextrin catalyzed by oriented cyclodextrin glycosyltransferase displayed on bacterial polyhydroxyalkanoate nanogranules.

BACKGROUND: The advantages of γ-cyclodextrin (γ-CD) include its high solubility, ability to form inclusion complexes with various poorly water-soluble molecules, and favorable toxicological profile; thus, γ-CD is an attractive functional excipient widely used in many industrial settings. Unfortunately, the high cost of γ-CD caused by the low activity and stability of γ-cyclodextrin glycosyltransferase (γ-CGTase) has hampered large-scale production and application. RESULTS: This study reports the in vivo one-step production of immobilized γ-CGTase decorated on the surface of polyhydroxyalkanoate (PHA) nanogranules by the N-terminal fusion of γ-CGTase to PHA synthase via a designed linker. The immobilized γ-CGTase-PHA nanogranules showed outstanding cyclization activity of 61.25 ± 3.94 U/mg (γ-CGTase protein) and hydrolysis activity of 36,273.99 ± 1892.49 U/mg, 44.74% and 18.83% higher than that of free γ-CGTase, respectively. The nanogranules also exhibited wider optimal pH (cyclization activity 7.0-9.0, hydrolysis activity 10.0-11.0) and temperature (55-60 °C) ranges and remarkable thermo- and pH-stability, expanding its utility to adapt to wider and more severe reaction conditions than the free enzyme. A high yield of CDs (22.73%) converted from starch and a high ratio (90.86%) of γ-CD in the catalysate were achieved at pH 9.0 and 50 °C for 10 h with 1 mmol/L K+, Ca2+, and Mg2+ added to the reaction system. Moreover, γ-CGTase-PHA beads can be used at least eight times, retaining 82.04% of its initial hydrolysis activity and 75.73% of its initial cyclization activity. CONCLUSIONS: This study provides a promising nanobiocatalyst for the cost-efficient production of γ-CD, which could greatly facilitate process control and economize the production cost.

Cost-Effective Production of L-DOPA by Tyrosinase-Immobilized Polyhydroxyalkanoate Nanogranules in Engineered Halomonas bluephagenesis TD01.

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).

Functionalized polyhydroxyalkanoate nano-beads as a stable biocatalyst for cost-effective production of the rare sugar d-allulose.

d-Allulose is a promising low-calorie sweetener especially for diabetes and obesity patients. The functionalized polyhydroxyalkanoate (PHA) nano-beads decorated with d-tagatose 3-epimerase (DTE) was produced in recombinant endotoxin-free ClearColi, whereby the expression, purification, and immobilization of the active DTE were efficiently combined into one step. The immobilized DTE exhibited remarkable enzyme activity of 649.3 U/g beads and extremely high stability at a harsh working condition (pH 7.0-8.0, 65 °C). When DTE-PHA beads were subjected to enzymatic synthesis of d-allulose, a maximum conversion rate of 33% can be achieved at pH 7.0 and 65 °C for 3 h, and DTE-PHA beads retained about 80% of its initial activity after 8 continuous cycles. Moreover, the d-allulose/d-fructose binary mixture can be simply separated by a single cation exchange resin-equipped chromatography. Taken together, DTE-PHA beads are promising and robust nano-biocatalysts that will remarkably simplify the production procedures of d-allulose, contributing to its cost-effective production.

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) Biopolyester Based Nanoparticles as NVP-BEZ235 Delivery Vehicle for Tumor Targeting Therapy.

As a biopolyester with excellent properties, the potential biomedical applications of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) have gained extensive attention. In this research, PHBHHx was fabricated into nanoparticles (NPs) to encapsulate NVP-BEZ235 (BEZ), an efficient kinase inhibitor/antitumor agent, for tumor targeting therapy. The resulting BEZ-NPs displayed a regularly spherical form with an appropriate diameter at 76.0 ± 3.6 nm. The encapsulation efficiency of BEZ was 83.7 ± 3.6%, and the sustained release profiles showed that almost 97% of BEZ could be gradually unrestricted from PHBHHx NPs within 72 h. The nanotoxicity studies revealed a satisfactory biosafety of PHBHHx NPs. PHBHHx NPs presented significantly improved cellular uptake in human prostate cancer cell line PC3, thereby enhancing the antiproliferation ability and kinase inhibitory activity of BEZ in vitro. More importantly, the in vivo real-time imaging demonstrated the adequate tumor targeting and accumulation capability of PHBHHx NPs. The remarkably delayed tumor growth, increased tumor necrosis, and reduced tumor proliferation in PC3 tumor xenograft mice further confirmed the antitumor efficacies of BEZ-loaded PHBHHx NPs. The above results suggest that PHBHHx NPs might be a promising drug delivery vehicle, safe and effective, for tumor targeting therapy.

Highly efficient biocatalytic synthesis of L-DOPA using in situ immobilized Verrucomicrobium spinosum tyrosinase on polyhydroxyalkanoate nano-granules.

L-DOPA (3,4-dihydroxyphenyl-L-alanine) is a preferred drug for Parkinson's disease, and is currently in great demand every year worldwide. Biocatalytic conversion of L-tyrosine by tyrosinases is the most promising method for the low-cost production of L-DOPA in both research and industry. Yet, it has been hampered by low productivity, low conversion rate, and low stability of the biocatalyst, tyrosinase. An alternative tyrosinase TyrVs from Verrucomicrobium spinosum with more efficient expression in heterologous host and better stability than the commercially available Agaricus bisporus tyrosinase was identified in this study. Additionally, it was prepared as a novel nano-biocatalyst based on the distinct one-step in situ immobilization on the surface of polyhydroxyalkanoate (PHA) nano-granules. The resulting PHA-TyrVs nano-granules demonstrated improved L-DOPA-forming monophenolase activity of 9155.88 U/g (Tyr protein), which was 3.19-fold higher than that of free TyrVs. The nano-granules also exhibited remarkable thermo-stability, with an optimal temperature of 50 °C, and maintained more than 70% of the initial activity after incubation at 55 °C for 24 h. And an enhanced affinity of copper ion was observed in the PHA-TyrVs nano-granules, making them even better biocatalysts for L-DOPA production. Therefore, a considerable productivity of L-DOPA, amounting to 148.70 mg/L h, with a conversion rate of L-tyrosine of 90.62% can be achieved by the PHA-TyrVs nano-granules after 3 h of biocatalysis under optimized conditions, without significant loss of enzyme activity or L-DOPA yield after 8 cycles of repeated use. Our study provides an excellent and robust nano-biocatalyst for the cost-effective production of L-DOPA.

A Specific Drug Delivery System for Targeted Accumulation and Tissue Penetration in Prostate Tumors Based on Microbially Synthesized PHBHHx Biopolyester and iRGD Peptide Fused PhaP.

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.

Immobilization of alkaline polygalacturonate lyase from Bacillus subtilis on the surface of bacterial polyhydroxyalkanoate nano-granules.

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.