Engineering of a Thermostable Biocatalyst for the Synthesis of 2-O-Glucosylglycerol.

2-O-Glucosylglycerol is accumulated by various bacteria and plants in response to environmental stress. It is widely applied as a bioactive moisturising ingredient in skin care products, for which it is manufactured via enzymatic glucosylation of glycerol by the sucrose phosphorylase from Leuconostoc mesenteroides. This industrial process is operated at room temperature due to the mediocre stability of the biocatalyst, often leading to microbial contamination. The highly thermostable sucrose phosphorylase from Bifidobacterium adolescentis could be a better alternative in that regard, but this enzyme is not fit for production of 2-O-glucosylglycerol due to its low regioselectivity and poor affinity for glycerol. In this work, the thermostable phosphorylase was engineered to alleviate these problems. Several engineering approaches were explored, ranging from site-directed mutagenesis to conventional, binary, iterative or combinatorial randomisation of the active site, resulting in the screening of ∼3,900 variants. Variant P134Q displayed a 21-fold increase in catalytic efficiency for glycerol, as well as a threefold improvement in regioselectivity towards the 2-position of the substrate, while retaining its activity for several days at elevated temperatures.

Enhancement of neuroprotection, antioxidant capacity, and water-solubility of crocins by transglucosylation using dextransucrase under high hydrostatic pressure.

Crocin, one of the major carotenoid pigments of Crocus sativus (saffron), is responsible for antioxidant activity, neuroprotection, and the inhibition of tumor cell proliferation. In order to improve the functionality of crocin, α-glucosyl-(1→6)-trans-crocins (C-Gs) were synthesized using sucrose and dextransucrase from Leuconostoc mesenteroides. High hydrostatic pressure (HHP) technique was applied to the synthesis process of C-Gs in order to improve its transglucosylation yield. A 100 MPa HHP condition enhanced the production yield of C-Gs by 1.95 times compared to that of 0.1 MPa atmospheric pressure. Novel C-Gs were purified by HPLC, and their chemical structures were determined using NMR analysis. Novel C-Gs increased water solubility 4.6-5.7 times and antioxidant activity 1.5-2.6 times, respectively, compared to crocin, and their neuroprotections (cell viability 92.5-100.4 %) on HT22 mouse hippocampal neuronal cells were significantly higher than that of crocin (cell viability 84.6 %). This advanced neuroprotection of novel C-Gs could be highly associated with their enhanced antioxidant activity. Thus, the enhanced water solubility and functionality of novel C-Gs can induce better clinical efficacy of neuroprotection than trans-crocin.

Functional Identification of the Dextransucrase Gene of Leuconostoc mesenteroides DRP105.

Leuconostoc mesenteroides DRP105 isolated from Chinese sauerkraut juice is an intensive producer of dextran. We report the complete genome sequence of Leu. mesenteroides DRP105. This strain contains a dextransucrase gene (dsr) involved in the production of dextran, possibly composed of glucose monomers. To explore the dextran synthesis mechanism of Leu. mesenteroides DRP105, we constructed a dsr-deficient strain derived from Leu. mesenteroides DRP105 using the Cre-loxP recombination system. The secondary structure prediction results showed that Leu. mesenteroides DRP105 dextransucrase (Dsr) was coded by dsr and contained 17.07% α-helices, 29.55% ß-sheets, 10.18% ß-turns, and 43.20% random coils. We also analyzed the dextran yield, monosaccharide change, organic acid, and amino-acid content of Leu. mesenteroides DRP105 and Leu. mesenteroides DRP105-Δdsr. The result showed that the lack of dsr changed the Leu. mesenteroides DRP105 sugar metabolism pathway, which in turn affected the production of metabolites.

Engineering Leuconostoc mesenteroides dextransucrase by inserting disulfide bridges for enhanced thermotolerance.

The disulfide bridge is a very important part of the peptide chain and plays an important role in stabilizing the protein structure and maintaining its active function. One hundred and fourteen potential disulfide bridges were determined by Disulfide by Design™, and 4 disulfide bridges were constructed for the purpose of obtaining new enzyme species with high thermotolerance. High thermotolerance is achieved by increasing the number of hydrogen bonds between amino acids. The optimum temperatures of mutant L838C-V887C and A948C-A1013C were improved by 10 °C compared to that of the original enzyme, which was beneficial to reduce the viscosity of the reaction system. Some of the mutations resulted in the alteration of catalytic specificity, and the products D739C-F932C and A948C-A1013C catalyzed synthesis of dextran containing a new α(1-4) glycosidic linkage and α(1-2) glycosidic linkage. This study may provide information valuable for increasing the reaction temperature of recombinant dextransucrase. The molecular docking study presents a plausible explanation for reaction specificity alteration and optimum temperature improvement for the mutants.

On the donor substrate dependence of group-transfer reactions by hydrolytic enzymes: Insight from kinetic analysis of sucrose phosphorylase-catalyzed transglycosylation.

Chemical group-transfer reactions by hydrolytic enzymes have considerable importance in biocatalytic synthesis and are exploited broadly in commercial-scale chemical production. Mechanistically, these reactions have in common the involvement of a covalent enzyme intermediate which is formed upon enzyme reaction with the donor substrate and is subsequently intercepted by a suitable acceptor. Here, we studied the glycosylation of glycerol from sucrose by sucrose phosphorylase (SucP) to clarify a peculiar, yet generally important characteristic of this reaction: partitioning between glycosylation of glycerol and hydrolysis depends on the type and the concentration of the donor substrate used (here: sucrose, α-d-glucose 1-phosphate (G1P)). We develop a kinetic framework to analyze the effect and provide evidence that, when G1P is used as donor substrate, hydrolysis occurs not only from the ß-glucosyl-enzyme intermediate (E-Glc), but additionally from a noncovalent complex of E-Glc and substrate which unlike E-Glc is unreactive to glycerol. Depending on the relative rates of hydrolysis of free and substrate-bound E-Glc, inhibition (Leuconostoc mesenteroides SucP) or apparent activation (Bifidobacterium adolescentis SucP) is observed at high donor substrate concentration. At a G1P concentration that excludes the substrate-bound E-Glc, the transfer/hydrolysis ratio changes to a value consistent with reaction exclusively through E-Glc, independent of the donor substrate used. Collectively, these results give explanation for a kinetic behavior of SucP not previously accounted for, provide essential basis for design and optimization of the synthetic reaction, and establish a theoretical framework for the analysis of kinetically analogous group-transfer reactions by hydrolytic enzymes.

Enzymatic synthesis and biological characterization of a novel mangiferin glucoside.

Mangiferin, a major constituent of Mangifera indica L., has attracted substantial attention due to its anti-oxidant, anti-diabetic, anti-inflammatory, and anti-microbial activities. However, its poor solubility in water limits its use in food and pharmaceutical industries. In this study, novel mangiferin-(1→6)-α-d-glucopyranoside (Mg-G1) was enzymatically synthesized from mangiferin and sucrose using glucansucrase from Leuconostoc mesenteroides B-512F/KM, and optimized using response surface methodology. The water solubility of Mg-G1 was found to be 824.7 mM, which is more than 2300-fold higher than that of mangiferin. Mg-G1 also showed DPPH radical scavenging activity and superoxide dismutase (SOD)-like scavenging activity, which were 4.77- and 3.71-fold higher than that of mangiferin, respectively. Mg-G1 displayed inhibitory activity against human intestinal maltase and COX-2. Thus, the novel glucosylated mangiferin may be used as an ingredient in functional food and pharmaceutical application.

Enzymatic synthesis of non-digestible oligosaccharide catalyzed by dextransucrase and dextranase from maltose acceptor reaction.

Non-digestible oligosaccharides have wide food industrial applications as dietary fibers and prebiotics. The aim of this study is to realize the effective biosynthesis of isomalto-oligosaccharides (IMOs) and reduce the production of by-product dextran. In the presence of acceptors improved the dextransucrase reaction shifting to oligosaccharides formation but a number of by-products dextran appeared. Maltose acceptor performed stronger inhibition behaviors in dextran synthesis than lactose and glucose acceptor due to its higher efficiencies. Acceptors had no influence on the structure of by-product dextran which mainly composed of α-(1,6)-glycosidic linkages and low α-(1,3)-glycosidic branch. In addition, the Mw and contents of IMOs and oligodextrans synthesized by dual-enzyme were hard to control. Addition of maltose acceptor in the dual-enzyme reaction, the adequate dextranase preferentially degraded dextran than the acceptor products to yield the IMOs. Results indicated that the combined use of the dual-enzyme and the maltose acceptor is a simple and effective method to promote the high-quality of functional IMOs.

Fabricating cholyglycine-glucose-6-phosphate dehydrogenase conjugates for cholyglycine detection.

To establish cholyglycine (CG) detection via enzyme-multiplied immunoassay technique (EMIT), glucose-6-phosphate dehydrogenase (G6PD) was used as a reporter enzyme to prepare hapten-enzyme conjugate. Gel electrophoresis and UV scanning demonstrated that G6PD was successfully labeled with cholyglycine, and CG-G6PD conjugate was obtained. Furthermore, the effects of various parameters on the preparation of CG-G6PD conjugates were investigated. Consequently, CG amount, nicotinamide adenine dinucleotide, d-glucose-6-phosphate (G6P), phosphate buffer and the pH, and ionic strength of solution had important effects on the residual activity of CG-G6PD. Moreover, CG amount, the pH, and G6P played important roles in changing CG labeling location on G6PD. Using the CG-G6PD conjugate as test kit, the cholyglycine-EMIT calibration curve was established, which could be employed in clinical detection of cholyglycine. This study provides some valuable information for preparing hapten-G6PD conjugates.

Synthesis and characterization of stevioside having low degree polymerized glucosides using dextransucrase and dextranase.

Transglycosylation is one of enzymatic methods to improve the physical and biochemical properties of various functional compounds. In this study, stevioside glucosides were synthesized using sucrose as a substrate, stevioside (Ste) as an acceptor, and dextransucrase from Leuconostoc mesenteroides B-512 F/KM. The highest Ste conversion yield of 98% was obtained with 50 mg/mL Ste, 800 mM sucrose, and dextransucrase 4 U/mL at 28 °C for 6 h. The concentration of Ste was unchanged while of Ste-G1 was increased from 7.7 mM to 9.1 mM as the Ste acceptor reaction digest was treated with dextranase from Lipomyces starkeyi. Ste-G1 (13-O-ß-sophorosyl-19-O-ß-isomaltosyl-steviol), Ste-G2 (13-O-(ß-(1→6) glucosyl)-ß-glucosylsophorosyl-19-O-ß-isomaltosyl-steviol), and Ste-G2' (13-O-ß-sophorosyl-19-O-ß-isomaltotriosyl-steviol) were determined by NMR. These glucosylated Ste showed increased stabilities at pH 2, 60 °C for 48 h as compared to Ste. Ste-G1, Ste-G2, and Ste-G2' inhibited the insoluble glucan synthesis from sucrose by mutansucrase from Streptococcus muntans by the transfer of the glucosyl group of sucrose to Ste-G1, Ste-G2, and Ste-G2'. The relative water solubility of curcumin, pterostilbene or idebenone was increased by Ste or Ste glucosides treatment. Ste and Ste-G1 restored cell viability in RAW264.7 cells at concentrations up to 8 mg/mL and inhibited nitric oxide production in LPS-induced RAW264.7 cells with IC50 of 3.29 and 1.87 mg/mL.

Alginate-pectin co-encapsulation of dextransucrase and dextranase for oligosaccharide production from sucrose feedstocks.

The genes for dextransucrase and dextranase were cloned from the genomic regions of Leuconostoc mesenteroides MTCC 10508 and Streptococcus mutans MTCC 497, respectively. Heterologous expression of genes was performed in Escherichia coli. The purified enzyme fractions were entrapped in the alginate-pectin beads. A high immobilization yield of dextransucrase (~ 96%), and dextranase (~ 85%) was achieved. Alginate-pectin immobilization did not affect the optimum temperature and pH of the enzymes; rather, the thermal tolerance and storage stability of the enzymes was improved. The repetitive batch experiments suggested substantially good operational stability of the co-immobilized enzyme system. The synergistic catalytic reactions of alginate-pectin co-entrapped enzyme system were able to produce 7-10 g L-1 oligosaccharides of a high degree of polymerization (DP 3-9) from sucrose (~ 20 g L-1) containing feedstocks, e.g., table sugar and cane molasses. The alginate-pectin-based co-immobilized enzyme system is a useful catalytic tool to bioprocess the agro-industrial bio-resource for the production of prebiotic biomolecules.

Determination of glucansucrase encoding gene in Leuconostoc mesenteroides.

A glucansucrase encoding gene was cloned into pET-28a(+) vector and expression in Escherichia coli BL21(DE3). An about 160 kDa recombinant glucansucrase was purified with a yield of 50.73% and a 4.02-fold increase in activity. The 1464 amino acid residue enzyme belongs to the GH70 subfamily and shares 90% similarity with Leuconostoc sp. glucansucrase. The optimal temperature and pH were 30 °C and pH 5.5, and 80% of activity was retained after incubation at 10-30 °C and pH 5-7. Enzyme activity was strongly activated by Ca2+ and Mn2+ and inhibited by various metal ions and chemical agents, and a high affinity for sucrose (Km = 11.6 mM, Vmax = 8.1 mmol/(mL·min)). Circular dichroism (CD) and Raman spectra collectively indicated a high proportion of random coil structure.

Transglycosylation Improved Caffeic Acid Phenethyl Ester Anti-Inflammatory Activity and Water Solubility by Leuconostoc mesenteroides Dextransucrase.

Bioglycosylation is an efficient strategy to improve the biological activity and physicochemical properties of natural compounds for therapeutic drug development. In this study, two caffeic acid phenethyl ester (CAPE) glucosides (G-CAPE and 2G-CAPE) were synthesized by transglycosylation with dextransucrase from Leuconostoc mesenteroides 0326 with CAPE as an acceptor and sucrose as a donor. The products were purified and the structures were characterized. The physicochemical properties, anti-inflammatory activity, and cytotoxicity of the two CAPE glucosides were measured. The water solubility of G-CAPE and 2G-CAPE is 35 and 90 times higher, respectively, than that of CAPE. Compared to CAPE, the monoglycoside product showed superior anti-inflammatory effects, and its inhibition rate of NO, IF-6, and TNF-α is 93.4%, 76.81%, and 56.58% in RAW 264.7 macrophages, respectively, at 20 µM. Also, the cytotoxicity of both products was significantly improved. These glycosylation-modified CAPEs circumvent some of the flaws in CAPE application in anti-inflammatory drugs.

Catalytic biosynthesis of levan and short-chain fructooligosaccharides from sucrose-containing feedstocks by employing the levansucrase from Leuconostoc mesenteroides MTCC10508.

Levansucrase gene (LmLEVS) was cloned from Leuconostoc mesenteroides MTCC 10508. The heterologous expression and purification of the truncated (TrLmLEVS) gene, lacking the N-terminal signal peptide, was performed in Escherichia coli. The recombinant enzyme (TrLmLEVS) was physico-kinetically characterized using sucrose as substrate. TrLmLEVS exhibited the maximum activity at pH 6 and temperature 30 °C. Thin layer chromatography and high performance liquid chromatography analyses unveiled the biosynthesis of fructooligosaccharides and levan by TrLmLEVS using sucrose as substrate. The catalytically synthesized polymer was characterized by Fourier-Transform Infrared Spectroscopy and Nuclear Magnetic Resonance analyses, confirming it as levan. TrLmLEVS was capable of catalyzing the transformation of raffinose-derived molecules, besides sucrose, into fructans. Further, TrLmLEVS was employed for the genesis of non-digestible fructans from sucrose-containing feedstocks like table sugar, jaggery, cane molasses, and sweet sorghum juice. The results suggest that Leu. mesenteroides MTCC 10508 levansucrase is a potential candidate for the production of levan-type biomolecules in plant-based food products.

The first identification and characterization of a histidine-specific amino acid racemase, histidine racemase from a lactic acid bacterium, Leuconostoc mesenteroides subsp. sake NBRC 102480.

We expressed a histidine racemase from Leuconostoc mesenteroides subsp. sake NBRC 102480 (Lm-HisR) successively in a soluble fraction of Escherichia coli BL21 (DE3) and then highly purified it from the cell-free extract. Lm-HisR showed amino acid racemase activity on histidine specifically. This is the first example of an amino acid racemase specifically acting on histidine. Phylogenetic analysis of Lm-HisR showed that Lm-HisR was located far from the cluster of alanine racemases reported thus far and only in lactic acid bacteria of the genus Leuconostoc. Alignment of the primary structure of Lm-HisR with those of lysine and alanine racemases and alanine racemase homologs previously reported revealed that the PLP-binding lysine and catalytic tyrosine were completely conserved, and some residues that are unique to the phylogenetic branch of Lm-HisR, Phe44, Ser45, Thr174, Thr206, His286, Ser287, Phe292, Gly312, Val357, and Ala358 were identified. We determined the crystal structure of Lm-HisR complexed with PLP at a 2.1-Å resolution. The crystal structure contained four molecules (two dimers) in the asymmetric unit. When comparing the 3D structure of Lm-HisR with those of racemases from Geobacillus stearothermophilus and Oenococcus oeni, Met315 was completely conserved, but Val357 was not. In addition, two significant differences were observed between Lm-HisR and G. stearothermophilus alanine racemase. Phe44 and His286 in Lm-HisR corresponded to Tyr43 and Tyr284 in G. stearothermophilus alanine racemase, respectively. Based on the structural analysis, comparison with alanine racemase, and docking simulation, three significant residues, Phe44, His286, and Val357, were identified that may control the substrate specificity of Lm-HisR.

High Levels of CO2 Induce Spoilage by Leuconostoc mesenteroides by Upregulating Dextran Synthesis Genes.

During nonventilated storage of carrots, CO2 gradually accumulates to high levels and causes modifications in the carrot's microbiome toward dominance of Lactobacillales and Enterobacteriales The lactic acid bacterium Leuconostoc mesenteroides secretes a slimy exudate over the surface of the carrots. The objective of this study was to characterize the slime components and the potential cause for its secretion under high CO2 levels. A proteomic analysis of the exudate revealed bacterial glucosyltransferases as the main proteins, specifically, dextransucrase. A chemical analysis of the exudate revealed high levels of dextran and several simple sugars. The exudate volume and dextran amount were significantly higher when L. mesenteroides was incubated under high CO2 levels than when incubated in an aerated environment. The treatment of carrot medium plates with commercial dextransucrase or exudate protein extract resulted in similar sugar profiles and dextran production. Transcriptome analysis demonstrated that dextran production is related to the upregulation of the L. mesenteroides dextransucrase-encoding genes dsrD and dsrT during the first 4 to 8 h of exposure to high CO2 levels compared to aerated conditions. A phylogenetic analysis of L. mesenteroides YL48 dsrD revealed a high similarity to other dsr genes harbored by different Leuconostoc species. The ecological benefit of dextran production under elevated CO2 requires further investigation. However, this study implies an overlooked role of CO2 in the physiology and fitness of L. mesenteroides in stored carrots, and perhaps in other food items, during storage under nonventilated conditions.IMPORTANCE The bacterium Leuconostoc mesenteroides is known to cause spoilage of different types of foods by secreting a slimy fluid that damages the quality and appearance of the produce. Here, we identified a potential mechanism by which high levels of CO2 affect the spoilage caused by this bacterium by upregulating dextran synthesis genes. These results have broader implications for the study of the physiology, degradation ability, and potential biotechnological applications of Leuconostoc.

Catalytic, Computational, and Evolutionary Analysis of the d-Lactate Dehydrogenases Responsible for d-Lactic Acid Production in Lactic Acid Bacteria.

d-Lactate dehydrogenase (d-LDH) catalyzes the reversible reaction pyruvate + NADH + H+ ↔ lactate + NAD+, which is a principal step in the production of d-lactate in lactic acid bacteria. In this study, we identified and characterized the major d-LDH (d-LDH1) from three d-LDHs in Leuconostoc mesenteroides, which has been extensively used in food processing. A molecular simulation study of d-LDH1 showed that the conformation changes during substrate binding. During catalysis, Tyr101 and Arg235 bind the substrates by hydrogen bonds and His296 acts as a general acid/base for proton transfer. These residues are also highly conserved and have coevolved. Point mutations proved that the substrate binding sites and catalytic site are crucial for enzyme activity. Network and phylogenetic analyses indicated that d-LDH1 and the homologues are widely distributed but are most abundant in bacteria and fungi. This study expands the understanding of the functions, catalytic mechanism, and evolution of d-LDH.

Purification and Characterization of Lipase Produced by Leuconostoc mesenteroides Subsp. mesenteroides ATCC 8293 Using an Aqueous Two-Phase System (ATPS) Composed of Triton X-100 and Maltitol.

Purification of lipase produced by L. mesenteroides subsp. mesenteroides ATCC 8293 was conducted for the first time using a novel aqueous two-phase system (ATPS) composed of Triton X-100 and maltitol. The partitioning of lipase was optimized according to several parameters including pH, temperature, and crude load. Results showed that lipase preferentially migrated to the Triton X-100 rich phase and optimum lipase partitioning was achieved in ATPS at TLL of 46.4% and crude load of 20% at 30 °C and pH 8, resulting in high lipase purification factor of 17.28 and yield of 94.7%. The purified lipase showed a prominent band on SDS-PAGE with an estimated molecular weight of 50 kDa. The lipase was stable at the temperature range of 30⁻60 °C and pH range of 6⁻11, however, it revealed its optimum activity at the temperature of 37 °C and pH 8. Moreover, lipase exhibited enhanced activity in the presence of non-ionic surfactants with increased activity up to 40%. Furthermore, results exhibited that metals ions such as Na⁺, Mg2+, K⁺ and Ca2+ stimulated lipase activity. This study demonstrated that this novel system could be potentially used as an alternative to traditional ATPS for the purification and recovery of enzymes since the purified lipase still possesses good process characteristics after undergoing the purification process.

Identification and Characterization of l-Malate Dehydrogenases and the l-Lactate-Biosynthetic Pathway in Leuconostoc mesenteroides ATCC 8293.

One putative l-lactate dehydrogenase gene (l- ldh) and three putative d- ldh genes from Leuconostoc mesenteroides ATCC 8293 were overexpressed, and their enzymatic properties were investigated. Only one gene showed d-LDH activity, catalyzing pyruvate and d-lactate interconversion, whereas the other genes displayed l- and d-malate dehydrogenase (MDH) activity, catalyzing oxaloacetate and l- and d-malate interconversion, suggesting that strain ATCC 8293 may not harbor an l- ldh gene. Putative phosphoenolpyruvate carboxylase (PEPC)- and malolactic enzyme (MLE)-encoding genes were identified from strain ATCC 8293, and sequence analysis showed that they could exhibit PEPC and MLE activities, respectively. l-Lactate production and transcriptional expression of the mle gene in this strain were highly increased in the presence of l-malate. We propose that in strain ATCC 8293, which lacks an l- ldh gene, l-lactate is produced through sequential enzymatic conversions from phosphoenolpyruvate to oxaloacetate, then l-malate, and finally l-lactate by PEPC, l-MDH, and MLE, respectively.

Optimization and purification of glucansucrase produced by Leuconostoc mesenteroides DRP2-19 isolated from Chinese Sauerkraut.

Strain DRP2-19 was detected to produce high yield of glucansucrase in MRS broth, which was identified to be Leuconostoc mesenteroides. In order for industrial glucansucrase production of L. mesenteroides DRP2-19, a one-factor test was conducted, then response surface method was applied to optimize its yield and discover the best production condition. Based on Plackett-Burman (PB) experiment, sucrose, Ca2+, and initial pH were found to be the most significant factors for glucansucrase production. Afterwards, effects of the three main factors on glucansucrase activity were further investigated by central composite design and the optimum composition was sucrose 35.87 g/L, Ca2+ 0.21 mmol/L, and initial pH 5.56. Optimum results showed that glucansucrase activity was increased to 3.94 ± 0.43 U/mL in 24 hr fermentation, 2.66-fold higher than before. In addition, the crude enzyme was purified using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of glucansucrase was determined as approximately 170 kDa by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified 15.77-fold and showed a final specific activity of 338.56 U/mg protein.

A Synthetic Light-Driven Substrate Channeling System for Precise Regulation of Enzyme Cascade Activity Based on DNA Origami.

Substrate channeling, in which a metabolic intermediate is directly passed from one enzyme to the next enzyme in an enzyme cascade, accelerates the processing of metabolites and improves substrate selectivity. Synthetic design and precise control of channeling outside the cellular environment are of significance in areas such as synthetic biology, synthetic chemistry, and biomedicine. In particular, the precise control of synthetic substrate channeling in response to light is highly important, but remains a major challenge. Herein, we develop a photoresponsive molecule-based synthetic substrate channeling system on DNA origami to regulate enzyme cascade activity. The photoresponsive azobenzene molecules introduced into DNA strands enable reversible switching of the position of substrate channeling to selectively activate or inhibit the enzyme cascade activity. Moreover, DNA origami allows precise control of interenzyme distance and swinging range of the swing arm to optimize the regulation efficiency. By combining the accurate and addressable assembly ability of DNA origami and the clean, rapid, and reversible regulation of photoresponsive molecules, this light-driven substrate channeling system is expected to find important applications in synthetic biology and biomedicine.