Substrate Conversion Rate Research Articles

Efficient cleavage of Cα[sbnd]Cβ bonds in lignin using tetrabutylammonium tetrafluoroborate as both the catalyst and auxiliary electrolyte

Lignin is a promising alternative to fossil resources due to its abundance of benzene ring monomers. However, the stability of the CαCβ bond in lignin has hindered its efficient depolymerization. Electrochemical methods for breaking this bond are not well-studied. This paper presents a novel approach for catalytic depolymerization of lignin to produce acetals under mild conditions, without the need for additional catalysts. Under room temperature and in an air atmosphere, the combination of tetrabutylammonium tetrafluoroborate (TBABF4) as an auxiliary electrolyte and methanol (MeOH) as a solvent has shown high selectivity in catalyzing the cleavage of CαCβ bonds in lignin. Over 90.0 % of the resulting products are acetals, with the optimal conditions being a substrate concentration of 0.02 M, TBABF4 concentration of 0.008 M, a constant current of 30 mA, and a reaction time of 3 h. This led to a substrate conversion rate of 95.8 % and a product yield of 98.0 % for benzaldehyde dimethyl acetal (Bda). The mechanism study reveals that the tributyl ammonium radical cation decomposed by TBABF4 is adsorbed on the electrode surface. Subsequently, the adsorbed O2 is activated to form superoxide anion radical active species through single electron transfer, which plays a crucial catalytic role. TBABF4 acts as both an auxiliary electrolyte and a catalyst in this process. This research introduces a novel approach for electrocatalytic depolymerization of inert CαCβ bonds in lignin, leading to the selective conversion into acetal chemicals.

Study on the efficient electrocatalytic depolymerization of α-O-4 bond in lignin assisted by simple electrolyte

Lignin is anticipated to serve as a replacement for dwindling fossil fuel resources owing to its abundant sources and renewable nature. The electrochemical oxidation technique for depolymerizing lignin has garnered significant interest for its environmentally friendly and mild operating conditions. Nevertheless, the current utilization of auxiliary electrolytes, predominantly organic bases, ionic liquids, and other specialized substances, poses a constraint on the widespread adoption of this approach. Furthermore, there is a scarcity of instances where electrochemical technology has been employed to depolymerize the α-O-4 bond in lignin for the production of highly selective acetals. In this study, a sodium chloride/methanol (NaCl/MeOH) system was utilized for the direct depolymerization of the α-O-4 bond in a lignin model molecule, specifically benzyl phenyl ether (BPE). The optimal conditions resulted in a 95.2 % conversion rate of the BPE substrate and a high yield of 94.5 % for the main product, benzaldehyde dimethyl acetal(Bda). This research offers a promising approach for the electrocatalytic depolymerization of α-O-4 bonds in lignin, leading to the selective production of acetal chemicals using a common auxiliary electrolyte at room temperature in just 2 h.

Effect of long-term nitrogen addition on the kinetics of phosphatases in a subtropical forest in southern China

Numerous studies have explored the effects of nitrogen (N) addition on the maximum rate of substrate conversion (Vmax) of phosphatase activity, whereas how N fertilization affects the substrate affinity (the Michaelis constant, Km) of phosphatases is not well understood. Here, we examined a recently-proposed novel hypothesis suggesting that N addition enhances the adaptability of soil microorganisms to low phosphorus (P) conditions by both increasing phosphatase abundance (i.e., increasing the Vmax of phosphatases) and enhancing the substrate-binding affinity of phosphatases (i.e., diminishing the Km of phosphatases). Contrary to the hypothesis, our study found that no level of N addition led to changes in either Vmax or Km of phosphatases in our study site. Furthermore, we observed a positive correlation between Vmax and Km, which challenges the proposed hypothesis, as this positive relationship contradicts the condition suggested by the hypothesis that the adaptive response of increasing phosphatase abundance (higher Vmax) should be associated with increased substrate affinity (lower Km). In conclusion, the present study indicates that N addition does not necessarily enhance the substrate affinity of phosphatases.

Extracellular Overexpression of a Neutral Pullulanase in Bacillus subtilis through Multiple Copy Genome Integration and Atypical Secretion Pathway Enhancement.

Neutral pullulanases, having a good application prospect in trehalose production, showed a limited expression level. In order to address this issue, two approaches were utilized to enhance the yield of a new neutral pullulanase variant (PulA3E) in B. subtilis. One involved using multiple copies of genome integration to increase its expression level and fermentation stability. The other focused on enhancing the PulA-type atypical secretion pathway to further improve the secretory expression of PulA3E. Several strains with different numbers of genome integrations, ranging from one to four copies, were constructed. The four-copy genome integration strain PD showed the highest extracellular pullulanase activity. Additionally, the integration sites ytxE, ytrF, and trpP were selected based on their ability to enhance the PulA-type atypical secretion pathway. Furthermore, overexpressing the predicated regulatory genes comEA and yvbW of the PulA-type atypical secretion pathway in PD further improved its extracellular expression. Three-liter fermenter scale-up production of PD and PD-ARY yielded extracellular pullulanase activity of 1767.1 U/mL at 54 h and 2465.1 U/mL at 78 h, respectively. Finally, supplementing PulA3E with 40 U/g maltodextrin in the multi-enzyme catalyzed system resulted in the highest trehalose production of 166 g/L and the substrate conversion rate of 83%, indicating its potential for industrial application.

Design of NAMPTs with Superior Activity by Dual-Channel Protein Engineering Strategy.

The nicotinamide phosphoribosyltransferase (NAMPT)-catalyzed substitution reaction plays a pivotal role in the biosynthesis of nucleotide compounds. However, industrial applications are hindered by the low activity of NAMPTs. In this study, a novel dual-channel protein engineering strategy was developed to increase NAMPT activity by enhancing substrate accessibility. The best mutant (CpNAMPTY13G+Y15S+F76P) with a remarkable 5-fold increase in enzyme activity was obtained. By utilizing CpNAMPTY13G+Y15S+F76P as a biocatalyst, the accumulation of β-nicotinamide mononucleotide reached as high as 19.94 g L-1 within 3 h with an impressive substrate conversion rate of 99.8%. Further analysis revealed that the newly generated substrate channel, formed through crack propagation, facilitated substrate binding and enhanced byproduct tolerance. In addition, three NAMPTs from different sources were designed based on the dual-channel protein engineering strategy, and the corresponding dual-channel mutants with improved enzyme activity were obtained, which proved the effectiveness and practicability of the approach.

Optimization of immobilized urease enzyme on porous polymer for enhancing the stability, reusability and enzymatic kinetics using response surface methodology

The study examines the immobilization of the urease enzyme on a range of High Internal Phase Emulsion (polyHIPE) materials, assessing characteristics, efficiency, and performance. It also investigates the impact of polyHIPE type, quantity, incubation time, and various parameters on the process and enzyme activity. Surface morphology and functional groups of polyHIPE materials were determined through scanning electron microscopy (SEM) and fourier transform infrared spectroscopy (FT-IR) analyses, revealing significant alterations after modification with polyglutaraldehyde (PGA). The maximum immobilization efficiency of 95% was achieved by adding PGA to polyHIPE materials with an incubation period of 15 h. The optimized conditions for immobilized enzyme using a Box-Behnken design (BBD) of response surface methodology (RSM) were as follows: temperature (40.8 °C), pH (7.1) and NaCl concentration (0.007 g/L). Furthermore, the immobilized enzyme demonstrated remarkable reusability, retaining 75% of its initial activity after six cycles, and sustained shelf-life stability, retaining over 40% activity after 10 days at room temperature. Kinetic analyses revealed that immobilized urease exhibited higher affinity for the substrate, but lower rate of substrate conversion compared to the free enzyme. These findings offer valuable insights into optimizing urease immobilization processes and enhancing urease stability and activity, with potential applications in various fields, including biotechnology and biocatalysis.

An In Vitro Hybrid Biocatalytic System Enabled by a Combination of Surface-Displayed, Purified, and Cell-Free Expressed Enzymes.

Enzymatic cascades have become a green and sustainable approach for the synthesis of valuable chemicals and pharmaceuticals. Using sequential enzymes to construct a multienzyme complex is an effective way to enhance the overall performance of biosynthetic routes. Here we report the design of an efficient in vitro hybrid biocatalytic system by assembling three enzymes that can convert styrene to (S)-1-phenyl-1,2-ethanediol. Specifically, we prepared the three enzymes in different ways, which were cell surface-displayed, purified, and cell-free expressed. To assemble them, we fused two orthogonal peptide-protein pairs (i.e., SpyTag/SpyCatcher and SnoopTag/SnoopCatcher) to the three enzymes, allowing their spatial organization by covalent assembly. By doing this, we constructed a multienzyme complex, which could enhance the production of (S)-1-phenyl-1,2-ethanediol by 3 times compared to the free-floating enzyme system without assembly. After optimization of the reaction system, the final product yield reached 234.6 μM with a substrate conversion rate of 46.9% (based on 0.5 mM styrene). Taken together, our strategy integrates the merits of advanced biochemical engineering techniques, including cellular surface display, spatial enzyme organization, and cell-free expression, which offers a new solution for chemical biosynthesis by enzymatic cascade biotransformation. We, therefore, anticipate that our approach will hold great potential for designing and constructing highly efficient systems to synthesize chemicals of agricultural, industrial, and pharmaceutical significance.

Biotransformation of ethylene glycol by engineered Escherichia coli

There has been extensive research on the biological recycling of PET waste to address the issue of plastic waste pollution, with ethylene glycol (EG) being one of the main components recovered from this process. Therefore, finding ways to convert PET monomer EG into high-value products is crucial for effective PET waste recycling. In this study, we successfully engineered Escherichia coli to utilize EG and produce glycolic acid (GA), expecting to facilitate the biological recycling of PET waste. The engineered E. coli, able to utilize 10 g/L EG to produce 1.38 g/L GA within 96 h, was initially constructed. Subsequently, strategies based on overexpression of key enzymes and knock-out of the competing pathways are employed to enhance EG utilization along with GA biosynthesis. An engineered E. coli, characterized by the highest GA production titer and substrate conversion rate, was obtained. The GA titer increased to 5.1 g/L with a yield of 0.75 g/g EG, which is the highest level in the shake flake experiments. Transcriptional level analysis and metabolomic analysis were then conducted, revealing that overexpression of key enzymes and knock-out of the competing pathways improved the metabolic flow in the EG utilization. The improved metabolic flow also leads to accelerated synthesis and metabolism of amino acids.

Structure-guided mining and engineering of nicotinamide riboside kinase for efficient synthesis of nicotinamide mononucleotide

Nicotinamide mononucleotide (NMN) is a bioactive precursor to nicotinamide adenine dinucleotide (NAD+) and has demonstrated anti-aging activity. Nicotinamide riboside kinase (NRK) can convert nicotinamide ribose (NR) into NMN in one step, but NRKs with high catalytic efficiency are rare and are difficult to engineer. In this study, two novel NRKs from Sparassis crispa and Beauveria bassiana were identified. They displayed impressive specific activities of 6.14 U/mg and 6.66 U/mg with high substrate (NR) conversion rates. Furthermore, structural modelling analysis and semi-rational design were used to develop variants of BEANRK. Variant M1 (V172K) exhibited a 1.2-fold of the wild-type BEANRK (WT) in enzyme activity and 91 % of substrate conversion rate with improved thermal stability compared to WT. Moreover, variants generated from site-specific mutagenesis were also computationally analyzed to elucidate which interactions between key amino residues and substrates facilitated an increase in catalytic activity and conversion rate. This study not only identified new NRKs but also provided insights into which amino acid residues were pivotal to the catalytic activity of NRK, paving the way for future NRK engineering to improve the industrial production of NMN.

Use of ultrasound to increase the catalytic activity of α-L-rhamnosidase

α-L-rhamnosidase (Rha) is ubiquitous in nature and has high feasibility in the food and biotechnology industries. A green and environmentally friendly method was used to improve the activity of Rha. Here, we show that the effects of ultrasound treatment on the Rha. Ultrasonic treatment at 80 W for 10 min yielded the highest enzyme activity. Treatment increased enzyme activity by 26.3% and half-life by 124 min. Further, treatment increased the catalytic efficiency of Rha and increased the substrate conversion rate by 33.88%. These results demonstrate that ultrasound increases the catalytic activity and stability of Rha. Thus, ultrasonic treatment of Rha is cost-effective on the industrial scale.

Functional characterization and enzymatic properties of flavonoid glycosyltransferase gene CtUGT49 in Carthamus tinctorius

Carthami Flos, as a traditional blood-activating and stasis-resolving drug, possesses anti-tumor, anti-inflammatory, and immunomodulatory pharmacological activities. Flavonoid glycosides are the main bioactive components in Carthamus tinctorius. Glycosyltransferase deserves to be studied in depth as a downstream modification enzyme in the biosynthesis of active glycoside compounds. This study reported a flavonoid glycosyltransferase CtUGT49 from C. tinctorius based on the transcriptome data, followed by bioinformatic analysis and the investigation of enzymatic properties. The open reading frame(ORF) of the gene was 1 416 bp, encoding 471 amino acid residues with the molecular weight of about 52 kDa. Phylogenetic analysis showed that CtUGT49 belonged to the UGT73 family. According to in vitro enzymatic results, CtUGT49 could catalyze naringenin chalcone to the prunin and choerospondin, and catalyze phloretin to phlorizin and trilobatin, exhibiting good substrate versatility. After the recombinant protein CtUGT49 was obtained by hetero-logous expression and purification, the enzymatic properties of CtUGT49 catalyzing the formation of prunin from naringenin chalcone were investigated. The results showed that the optimal pH value for CtUGT49 catalysis was 7.0, the optimal temperature was 37 ℃, and the highest substrate conversion rate was achieved after 8 h of reaction. The results of enzymatic kinetic parameters showed that the K_m value was 209.90 μmol·L~(-1) and k_(cat) was 48.36 s~(-1) calculated with the method of Michaelis-Menten plot. The discovery of the novel glycosyltransferase CtUGT49 is important for enriching the library of glycosylation tool enzymes and provides a basis for analyzing the glycosylation process of flavonoid glycosides in C. tinctorius.

Installing xylose assimilation and cellodextrin phosphorolysis pathways in obese Yarrowia lipolytica facilitates cost-effective lipid production from lignocellulosic hydrolysates

BackgroundYarrowia lipolytica, one of the most charming chassis cells in synthetic biology, is unable to use xylose and cellodextrins.ResultsHerein, we present work to tackle for the first time the engineering of Y. lipolytica to produce lipids from cellodextrins and xylose by employing rational and combinatorial strategies. This includes constructing a cellodextrin-phosphorolytic Y. lipolytica by overexpressing Neurospora crassa cellodextrin transporter, Clostridium thermocellum cellobiose/cellodextrin phosphorylase and Saccharomyces cerevisiae phosphoglucomutase. The effect of glucose repression on xylose consumption was relieved by installing a xylose uptake facilitator combined with enhanced PPP pathway and increased cytoplasmic NADPH supply. Further enhancing lipid production and interrupting its consumption conferred the obese phenotype to the engineered yeast. The strain is able to co-ferment glucose, xylose and cellodextrins efficiently, achieving a similar μmax of 0.19 h−1, a qs of 0.34 g-s/g-DCW/h and a YX/S of 0.54 DCW-g/g-s on these substrates, and an accumulation of up to 40% of lipids on the sugar mixture and on wheat straw hydrolysate.ConclusionsTherefore, engineering Y. lipolytica capable of assimilating xylose and cellodextrins is a vital step towards a simultaneous saccharification and fermentation (SSF) process of LC biomass, allowing improved substrate conversion rate and reduced production cost due to low demand of external glucosidase.

Bioconversion performances of black soldier fly larvae (<em>Hermetia illucens</em> L. Diptera: Stratiomyidae) on selected organic substrates

Black Soldier Fly Larvae (BSFL; Hermetia illucens L.) is a bioconversion agent of organic substrates and its proven ability to degrade and biotransform of waste strengthens organic waste management and waste recycling strategies. BSFL have the advantages of rapid development and very efficient conversion of various organic wastes into body biomass. This process reduces the disposal costs of organic waste as well as the use of energy providing a sustainable solution to the treatment of organic waste. In addition, it provides valuable products and fertilizers for agriculture, potentially reducing crop production costs for farmers. The current study investigated the potential live biomass production of BSFL using different organic substrates available locally while understanding their decomposition ability. Seven treatments were compared (T1- equal proportion of poultry manure and rice bran, T2- equal proportion of goat manure and rice bran, T3- rabbit droppings, T4- soybean meal, T5- coconut scrapings, T6- equal proportion of vegetable and fruit waste and T7- cow dung) and the experiment was set up as completely randomized design with three replicates. The study results examined the ability of BSFL to reduce the weight of the end-substrate and promote the degradation of organic waste materials. The live biomass production of larvae, changes in live biomass production, substrate consumption rate, the weight of the end-substrate and substrate conversion efficiency were significantly different among treatments. The greatest live biomass production of BSFL was recorded in coconut scrapings and vegetables & fruit waste mixture while the lowest value was observed in cow dung. Coconut scrapings reduced most of their weight at the end of the study indicating the highest substrate consumption rate and the total weight of the end-substrate consisted of < 2mm particle in size. However, the highest substrate conversion rate was observed in cow dung. Aqueous solutions prepared from the end-substrates varied widely in pH, electrical conductivity, total dissolved solids, and salinity. In addition, significant differences were found among treatments for the colour and texture of the end-substrate. However, the appearance and odour did not differ significantly. According to the results of the present study, it can be concluded that the most efficient media for BSFL was coconut scraping though; it cannot be used as organic fertilizer as it exceeded the pH value of the SLS recommendation for compost. Overall, BSFL can be recognized as an effective and environmentally friendly tool to use in the biotransformation process of locally available organic waste materials.

Rational design of polyphosphate kinase dual-substrate channel cavity for efficient production of glutathione by cell free catalysis

ATP is an important cofactor involved in many biocatalytic reactions that require energy input. Polyphosphate kinases (PPK) can provide energy for ATP-consuming reactions due to their cheap and readily available substrate polyphosphate. We selected ChPPK from Cytophaga hutchinsonii for substrate profiling and tolerance analysis. By molecular docking and site-directed mutagenesis, we rationally engineered the dual-substrate channel cavity of polyphosphate kinase to improve the catalytic activity of PPK. Compared with the wild type, the relative enzyme activity of the screened mutant ChPPKK81H-K103V increased by 326.7%. Meanwhile, the double mutation expanded the substrate utilization range and tolerance of ChPPK, and improved its heat and alkali resistance. Subsequently, we coupled the glutathione bifunctional enzyme GshAB and ChPPKK81H-K103V based on this ATP regeneration system, and glutathione was produced by cell-free catalysis upon disruption of cells. This system produced (25.4±1.9) mmol/L glutathione in 6 h upon addition of 5 mmol/L ATP. Compared with the system before mutation, glutathione production was increased by 41.9%. After optimizing the buffer, bacterial mass and feeding time of this system, (45.2±1.8) mmol/L glutathione was produced in 6 h and the conversion rate of the substrate l-cysteine was 90.4%. Increasing the ability of ChPPK enzyme to produce ATP can effectively enhance the conversion rate of substrate and reduce the catalytic cost, achieving high yield, high conversion rate and high economic value for glutathione production by cell-free catalysis. This study provides a green and efficient ATP regeneration system that may further power the ATP-consuming biocatalytic reaction platform.

A high-throughput dual system to screen polyphosphate kinase mutants for efficient ATP regeneration in L-theanine biocatalysis

ATP, an important cofactor, is involved in many biocatalytic reactions that require energy. Polyphosphate kinases (PPK) can provide energy for ATP-consuming reactions due to their cheap and readily available substrate polyphosphate. We determined the catalytic properties of PPK from different sources and found that PPK from Cytophaga hutchinsonii (ChPPK) had the best catalytic activity for the substrates ADP and polyP6. An extracellular–intracellular dual system was constructed to high-throughput screen for better catalytic activity of ChPPK mutants. Finally, the specific activity of ChPPKD82N-K103E mutant was increased by 4.3 times. Therefore, we focused on the production of L-theanine catalyzed by GMAS as a model of ATP regeneration. Supplying 150 mM ATP, GMAS enzyme could produce 16.8 ± 1.3 g/L L-theanine from 100 mM glutamate. When 5 mM ATP and 5 U/mL ChPPKD82N-K103E were added, the yield of L-theanine was 16.6 ± 0.79 g/L with the conversion rate of 95.6 ± 4.5% at 4 h. Subsequently, this system was scaled up to 200 mM and 400 mM glutamate, resulting in the yields of L-theanine for 32.3 ± 1.6 g/L and 62.7 ± 1.1 g/L, with the conversion rate of 92.8 ± 4.6% and 90.1 ± 1.6%, respectively. In addition, we also constructed an efficient ATP regeneration system from glutamate to glutamine, and 13.8 ± 0.2 g/L glutamine was obtained with the conversion rate of 94.4 ± 1.4% in 4 h after adding 6 U/ mL GS enzyme and 5 U/ mL ChPPKD82N-K103E, which further laid the foundation from glutamine to L-theanine catalyzed by GGT enzyme. This proved that giving the reaction an efficient ATP supply driven by the mutant enzyme enhanced the conversion rate of substrate to product and maximized the substrate value. This is a positively combination of high yield, high conversion rate and high economic value of enzyme catalysis. The mutant enzyme will further power the ATP-consuming biocatalytic reaction platform sustainably.

Production of hydroxytyrosol through whole-cell bioconversion from L-DOPA using engineered Escherichia coli

Hydroxytyrosol (HT), a polyphenolic molecule of high value, is used in the nutraceutical, cosmetic, food, and livestock nutrition industries. As a natural product, HT is chemically manufactured or extracted from olives; nevertheless, the increasing demand mandates the exploration and development of alternative sources, such as heterologous production by recombinant bacteria. In order to achieve this purpose, we have molecularly modified Escherichia coli to carry two plasmids. For conversion of L-DOPA (Levodopa) into HT efficiently, it is necessary to enhance the expression of DODC (DOPA decarboxylase), ADH (alcohol dehydrogenases), MAO (Monoamine oxidase) and GDH (glucose dehydrogenases). The step that significantly affects the rate of ht biosynthesis is likely to be associated with the reaction facilitated by DODC enzymatic activity, as suggested by the result of in vitro catalytic experiment and HPLC. Then Pseudomonas putida, Sus scrofa, Homo sapiens and Levilactobacillus brevis DODC were taken into comparsion. The DODC from H. sapiens is superior to that of P. putida, S. scrofa or L. brevis for HT production. Seven promoters were introduced to increase the expression levels of catalase (CAT) to remove the byproduct H2O2 and optimized coexpression strains were obtained after screening. After the 10-hour operation, the optimized whole-cell biocatalyst produced HT at a maximum titer of 4.84 g/L with over 77.5% molar substrate conversion rate.

Remediation of petroleum contaminated soil by persulfate oxidation coupled with microbial degradation

This study was conducted on soil polluted with high concentrations of crude oil (12,835 ± 572.76 mg/kg) with different dosages of persulfate (PS) coupled with hydrocarbon-degrading mixed bacteria (including Enterobacteriaceae, Stenotrophomonas, Pseudomonas, Acinetobacter, and Achromobacter) obtained from long-term oil-contaminated soil. The interaction among total petroleum hydrocarbons (TPH), soil parameters, and microbial communities during a 187-d combined remediation process was evaluated. The result showed that the TPH removal of 1% PS oxidation combined with bioremediation was 80.05%; this was 4.88% and 20.94% higher than that of bioremediation and 1% PS oxidation combined with natural attenuation. This is a result of 1%PS stimulated enzyme secretion and dissolved organic carbon content, especially fulvic acid, improving TPH mobility and bioavailability in soil. Furthermore, introducing mixed bacteria after oxidation further increased enzyme activities and TPH-degrading ability, thus promoting carbon substrate conversion and biodegradation rate. Conversely, the excessive oxidation of 3% and 5% PS had a negative impact on the soil environment and microbial diversity resulting in lower TPH degradation (36%-57%). High-throughput sequencing revealed that the abundance and diversity of soil bacteria decreased in all remediation groups compared to those in the pristine soil. Redundancy analysis showed that the main factors affecting 1% PS oxidation combined with bioremediation during subsequent microbial remediation were pH, cation exchange capacity, and lipase activity, which may indirectly influence TPH biodegradation by increasing the abundance of genus Immundisolibacteraceae, Comamonadaceae, and Acinetobacter. This study provides data on in situ chemical oxidation integrated with the bioremediation of petroleum hydrocarbon-contaminated soils.

Salidroside synthesis via glycosylation by β-D-glucosidase immobilized on chitosan microspheres in deep eutectic solvents

This study aimed to establish a novel method for catalysing salidroside synthesis by immobilized β-D-glucosidase with deep eutectic solvents (DESs) as a new green non-aqueous medium. To this end, the catalytic properties of β-D-glucosidase (i.e. activity, stability, and enzymatic properties) were analysed within sixteen different DESs systems. Although most DESs significantly improved enzyme stability, they also reduced enzymatic activity. This was verified at the molecular level by employing fluorescence spectroscopy to evaluate conformational changes in β-D-glucosidase molecules. Subsequently, chitosan microspheres were used to immobilize β-D-glucosidase; the structures of the microspheres and immobilized enzyme were observed by scanning electron microscopy (SEM). Through single factor experiments and application of a response surface design, the optimal conditions for production of salidroside by the reaction of tyrosol with immobilized β-D-glucosidase were established. These conditions comprised 80 vol% ChCl/G (1:2), 50 °C reaction temperature, 5.8 pH, 100 h reaction time, 45 U/mL enzyme dosage, and a tyrosol/D-glucose molar ratio of 10. Under these conditions, the average conversion rate of the tyrosol substrate was 31.6%. Moreover, a salidroside crude product was obtained with a purity >70% (g/g) and a substrate conversion rate of immobilized β-D-glucosidase >50% of its initial value after five repeated uses. Collectively, this study establishes a novel green reaction system for the efficient preparation of salidroside and other glycoside compounds, while also providing a theoretical foundation for the analysis of β-D-glucosidase catalytic reactions in DESs systems.

Mussel-Inspired Electro-oxidation-Modified Three-Dimensional Printed Carriers for a Versatile Enzyme Immobilization Approach

Conventional enzyme immobilization approaches can only immobilize certain specific enzymes with poor generality. Attempts to improve the universality of enzyme types tend to impart them with more enzymatic catalysis applications. Here, inspired by mussel adhesive proteins, we present a novel eco-friendly surface carrier that was 3D printed and modified by electro-oxidation for enzyme immobilization. The carrier was fabricated through 3D printing by transforming acrylonitrile butadiene styrene (ABS) material into a suitable structure (3DABS). Then, electro-oxidative modification was performed on the surface to form a polydopamine (PDA) coating (3DABS-PDA). The desired structures for the enzyme immobilization carriers were obtained through 3D printing technology, while electro-oxidation modification of the surface provided numerous and firmly covalent binding sites. Based on these features, we have demonstrated that 3D printed and electro-oxidation-modified carriers could be applied to immobilize different types of enzymes. The loading capacity of all immobilized enzymes (galV, EG5C-1, XynLK9, and kdcA) exceeded 25 mg·g–1 (37.7 mg·g–1 for galV), and after 10 reuse cycles, the substrate conversion rate of 3DABS-PDA@galV was still over 85%. The carriers can be reused after simple processing. These results indicate that 3DABS-PDA provides an efficient, sustainable, and versatile approach for enzyme immobilization and exhibits excellent value in various enzymatic catalysis applications.

Construction of engineered bifunctional enzymes to improve the production of kaempferol in an in vitro synthetic biosystem

Kaempferol, an important flavonol, has numerous health-beneficial bioactivities and possesses a great potential for application in medicine, food, and cosmetics industries. To improve the production of kaempferol in an in vitro synthetic biosystem, we designed and constructed a panel of bifunctional enzymes by fusing the flavanone 3-hydroxylase (AtF3H) and the flavonol synthase (AtFLS1) of Arabidopsis thaliana with different orientation and different peptide linker type and length. By comparing the output of kaempferol, we obtained a highly active bifunctional enzyme AtF3H-(GGGGS)2-AtFLS1 with a Km value of 0.129 ± 0.016 mM. After optimization of a series of reaction parameters, kaempferol was produced at 100.54 ± 0.54 mg/L, the currently highest kaempferol output in an in vitro synthetic biosystem, and the substrate conversion rate was 68.26% ± 0.05%. In addition, we observed substrate inhibition for the AtFLS1, which eventually limited the production of kaempferol. This study provides a highly active biocatalyst for production of kaempferol and an insight into biosynthesis of other valuable molecules.