Enzymatic biosynthesis of D-galactose derivatives: Advances and perspectives.

D-Galactose derivatives, including galactosyl-conjugates and galactose-upgrading compounds, provide various physiological benefits and find applications in industries such as food, cosmetics, feed, pharmaceuticals. Many research on galactose derivatives focuses on identification, characterization, development, and mechanistic aspects of their physiological function, providing opportunities and challenges for the development of practical approaches for synthesizing galactose derivatives. This study focuses on recent advancements in enzymatic biosynthesis of galactose derivatives. Various strategies including isomerization, epimerization, transgalactosylation, and phosphorylation-dephosphorylation were extensively discussed under the perspectives of thermodynamic feasibility, theoretical yield, cost-effectiveness, and by-product elimination. Specifically, the enzymatic phosphorylation-dephosphorylation cascade is a promising enzymatic synthesis route for galactose derivatives because it can overcome the thermodynamic equilibrium of isomerization and utilize cost-effective raw materials. The study also elucidates the existing challenges and future trends in enzymatic biosynthesis of galactose derivatives. Collectively, this review provides a real-time summary aimed at promoting the practical biosynthesis of galactose derivatives through enzymatic catalysis.

One-pot sustainable synthesis of glucosylglycerate from starch and glycerol through artificial in vitro enzymatic cascade.

Glucosylglycerate (R-2-O-α-D-glucopyranosyl-glycerate, GG) is a negatively charged compatible solution with versatile functions. Here, an artificial in vitro enzymatic cascade was designed to feasibly and sustainably produce GG from affordable starch and glycerol. First, Spirochaeta thermophila glucosylglycerate phosphorylase (GGP) was carefully selected because of its excellent heterologous expression, specific activity, and thermostability. The optimized two-enzyme cascade, consisting of alpha-glucan phosphorylase (αGP) and GGP, achieved a remarkable 81 % conversion rate from maltodextrin and D-glycerate. Scaling up this cascade resulted in a practical concentration of 58 g/L GG with a 62 % conversion rate based on the added D-glycerate. Additionally, the production of GG from inexpensive starch and glycerol in one-pot using artificial four-enzyme cascade was successfully implemented, which integrates alditol oxidase and catalase with αGP and GGP. Collectively, this sustainable enzymatic cascade demonstrates the feasibility of the practical synthesis of GG and has the potential to produce other glycosides using the phosphorylase-and-phosphorylase paradigm.

Sustainable cellobionic acid biosynthesis from starch via artificial in vitro synthetic enzymatic biosystem.

Cellobionic acid (CBA), a kind of aldobionic acid, offers potential applications in the fields of pharmaceutical, cosmetic, food, and chemical industry. To tackle the high cost of the substrate cellobiose in CBA production using quinoprotein glucose dehydrogenase, this study developed a coenzyme-free and phosphate-balanced in vitro synthetic enzymatic biosystem (ivSEBS) to enable the sustainable CBA synthesis from cost-effective starch in one-pot via the CBA-synthesis module and gluconic acid-supply module. The metabolic fluxes of this artificial biosystem were strengthened using design-build-test-analysis strategy, which involved exquisite pathway design, meticulous enzyme selection, module validation and integration, and optimization of the key enzyme dosage. Under the optimized conditions, a remarkable concentration of 6.2 g/L CBA was achieved from initial 10 g/L maltodextrin with a starch-to-CBA molar conversion rate of 60 %. Taking into account that the biosystem simultaneously accumulated 3.6 g/L of gluconic acid, the maltodextrin utilization rate was calculated to be 93.3 %. Furthermore, a straightforward scaling-up process was performed to evaluate the industrial potential of this enzymatic biosystem, resulting in a yield of 21.2 g/L CBA from 50 g/L maltodextrin. This study presents an artificial ivSEBS for sustainable production of CBA from inexpensive starch, demonstrating an alternative paradigm for biomanufacturing of other aldobionic acids.

Identification of Two Novel Fluorinases From Amycolatopsis sp. CA-128772 and Methanosaeta sp. PtaU1.Bin055 and a Mutant With Improved Catalytic Efficiency With Native Substrate.

Fluoride plays an important role in the fields of materials and medicine. Compared with chemical synthesis, fluorinases are natural catalysts with more application potential, which provide a green and effective way to obtain organofluorine. However, the application of fluorinases is limited by certain factors, such as the limited number of enzymes and their low activity. In this work, two new fluorinases from Amycolatopsis sp. CA-128772 and Methanosaeta sp. PtaU1.Bin055 were identified by gene mining and named Fam and Fme, respectively. The activities of these two enzymes were reported for the first time, and Fme showed good thermal stability, which was different from the reported fluorinases. In addition, the activity toward natural substrate of Fam was improved by site-directed mutagenesis, the catalytic efficiency (k cat /K m ) of the best mutant containing two amino acid substitutions (T72A and S164G) toward the substrate S-adenosyl-L-methionine was improved by 2.2-fold compared to the wild-type. Structural modeling analysis revealed that the main reason for the increased enzyme activity might be the formation of a new substrate channel. Experimental evidence suggests that the substrate channel may indeed play a key role in regulating the function of the fluorinases.

Whole-cell catalysis by surface display of fluorinase on Escherichia coli using N-terminal domain of ice nucleation protein.

BACKGROUND: Fluorinases play a unique role in the production of fluorine-containing organic molecules by biological methods. Whole-cell catalysis is a better choice in the large-scale fermentation processes, and over 60% of industrial biocatalysis uses this method. However, the in vivo catalytic efficiency of fluorinases is stuck with the mass transfer of the substrates. RESULTS: A gene sequence encoding a protein with fluorinase function was fused to the N-terminal of ice nucleation protein, and the fused fluorinase was expressed in Escherichia coli BL21(DE3) cells. SDS-PAGE and immunofluorescence microscopy were used to demonstrate the surface localization of the fusion protein. The fluorinase displayed on the surface showed good stability while retaining the catalytic activity. The engineered E.coli with surface-displayed fluorinase could be cultured to obtain a larger cell density, which was beneficial for industrial application. And 55% yield of 5'-fluorodeoxyadenosine (5'-FDA) from S-adenosyl-L-methionine (SAM) was achieved by using the whole-cell catalyst. CONCLUSIONS: Here, we created the fluorinase-containing surface display system on E.coli cells for the first time. The fluorinase was successfully displayed on the surface of E.coli and maintained its catalytic activity. The surface display provides a new solution for the industrial application of biological fluorination.

An Aminotransferase from Enhydrobacter aerosaccus to Obtain Optically Pure ß-Phenylalanine.

An aminotransferase ω-TAEn was identified from Enhydrobacter aerosaccus. The ω-TAEn was successfully expressed in Escherichia coli and the obtained enzyme showed activity toward ß-phenylalanine (ß-phe) at optimal conditions. For optically pure (R)-ß-phe, 50% yield was observed by kinetic resolution of racemic amino with pyruvate as the amino acceptor. To obtain (S)-ß-phe, the lipase/ω-TAEn catalytic system was adopted. The ω-TAEn showed strict stereoselectivity to the amino donor. The formation of (S)-ß-phe was observed using 3-aminobutyric acid as the amino donor, and (S)-ß-phe was obtained by asymmetric synthesis with a yield of 82%.

Comparing complications of vertebroplasty and kyphoplasty for treating osteoporotic vertebral compression fractures: a meta-analysis of the randomized and non-randomized controlled studies.

PURPOSE: To compare complications of percutaneous vertebroplasty (PVP) and balloon kyphoplasty (BKP) for the treatment of osteoporotic vertebral compression fractures (OVCFs). BACKGROUND: PVP and BKP are two minimally invasive procedures for treating OVCFs, while few studies emphases attention to intra- and post-operative complications about the two procedures. METHODS: Online databases were searched for studies comparing complications of PVP and BKP for OVCFs, the randomized controlled trials, clinical controlled trials and cohort studies that provided related data were identified. Demographic characteristics and complications related to procedures were extracted and analysed from all of the included studies. RESULTS: Nineteen studies encompassing 1,787 patients in total, of whom 887 received PVP and 900 received BKP, met the inclusion criteria. For subsequent fractures, our meta-analysis detected no significant difference between the two procedures, both for adjacent fractures (p = 0.29) and non-adjacent fractures (p = 0.37). For cement extravasations, there was no significant difference between the two interventions if considering disc spaces extravasations only (p = 0.24), while considering total extravasations and paravertebral extravasations, the cement leakage rate in the PVP group was significantly higher than the BKP group (total: p < 0.01; paravertebral: p < 0.01). CONCLUSIONS: The two procedures suffer from equal risk of subsequent spinal fractures; PVP has a significant higher cement leakage rate compared to BKP, mainly caused by a higher paravertebral leakage, patients with extremely poor pulmonary function or unstable haemodynamic are better candidates for BKP.