High Hydrolytic Activity Research Articles

Comparative analysis of lipolytic enzymes involved in the surface fermentation of dried katsuobushi by xerophilic molds.

Fermented katsuobushi, a traditional Japanese seasoning, is produced from skipjack tuna through smoking, drying and fermentation by xerophilic Aspergillus molds, primarily Aspergillus chevalieri and Aspergillus pseudoglaucus. In this study, we characterized lipolytic enzymes (cLip_1 to cLip_5 and pLip_1 to pLip_3) to clarify their roles in lipid hydrolysis during katsuobushi production under low water activity. The enzymes showed significant diversity in their activity, stability and substrate specificity, and in the hydrolysis profiles of their reactions with fish oil. Phylogenetic analyses revealed that cLip_5 showed a high identity with pLip_2 (94%) and these enzymes formed a phylogenetic cluster with filamentous fungal lipases. Purified recombinant enzymes (rcLip_1, rcLip_2, rcLip_4 and rcLip_5) and wild-type enzymes (cLip_3 and pLip_3) showed varying substrate preferences toward p-nitrophenyl esters. The addition of glycerol to reduce the water activity in the reaction mixture led to increased activities of rcLip_1 and rcLip_4, but it did not affect the activity of the other three enzymes. Among the tested six enzymes, cLip_5 showed the highest hydrolytic activity toward fish oil. The cLip_5 and pLip_2 gene transcript levels were moderately high in strains MK86 and MK88, respectively. cLip_5 and its homolog pLip_2 were identified as the most promising enzymes for katsuobushi fermentation, because of their high hydrolytic activities toward fish oil and adaptability to low water activity conditions. These findings support the selection of optimal Aspergillus strains as starter cultures to potentially shorten the fermentation time and improve the quality and shelf life of katsuobushi. © 2025 Society of Chemical Industry.

Cerium-based nanohydrolase for fast catalytic hydrolysis of β-lactam antibiotics in wastewater effluents

To defuse risks of antibiotic residues in effluent to achieve safe wastewater reuse, direct hydrolysis of the functional group responsible for the antibacterial activity, such as the of β-lactam ring in β-lactam antibiotics, has been recognized as an efficient and cost-effective strategy. However, the instability of natural hydrolases limits their use in treating antibiotic-containing wastewater. Herein, inspired by the active site of natural hydrolase, a Ce-based nanohydrolase was created for rapid hydrolysis of β-lactam antibiotics. The typical β-lactam antibiotic, penicillin G (PG), could be totally removed by the nanohydrolase within 2 min with a hydrolysis efficiency of 44%, and the hydrolysis efficiency could reach 98% within 10 min. It revealed that Ce(IV) in the nanohydrolase adsorbed PG via Lewis acid-Lewis base interaction to activate the β-lactam ring, while the -OH on Ce(III) served as nucleophile to attack the β-lactam ring, thereby promoting the hydrolysis of PG. The Ce-based nanohydrolase also showed good catalytic hydrolysis performance towards other commonly used β-lactam antibiotics and structurally related chemicals, implying its substrate universality. In addition to having high hydrolytic activity similar to that of natural hydrolases, this nanohydrolase exhibited extraordinary reusability and potential for practical applications that natural hydrolases do not possess. This work offers an innovative strategy to eliminate the risks of hydrolysable micropollutants in wastewater effluents and also provides reference for designing better nanohydrolase.

Enhancing aerobic composting of food waste by adding hydrolytically active microorganisms

The biomass of native microorganisms in food waste (FW) suitable for accelerated composting is initially low and requires time for adaptation. Adding of efficient hydrolytic microorganisms should be able to enhance compost-specific microbial activity, adjust microbial community structure, and potentially hasten FW biodegradation. This study aimed to identify bacterial and fungal strains with growth characteristics suitable for accelerating FW composting. Over 7 weeks, FW was composted in a pilot-scale test, either inoculated at the start or on day 28 with three different mixtures of 10 autochthonous Bacillus and Penicillium spp. strains known for their high hydrolytic activity. The effects of inoculation were assessed by measuring the rate of carbon dioxide (CO2) and ammonia (NH3) production and also the increase in temperature due to spontaneous exothermic activity of the enhanced microbial population degrading FW. Inoculation with Bacillus spp., particularly B. amyloliquefaciens and B. subtilis, at the beginning of composting increased CO2 production nearly 3-fold while maintaining stable ammonia production and temperature. The high concentration of Bacillus relative to native FW microorganisms led to dominant fermentation processes even in the presence of oxygen, resulting in moderate heat release and elevated production of volatile organic compounds. Introducing Penicillium spp. at a later stage (day 28) increased CO2 production nearly 2-fold, along with higher NH3 levels and temperature. These findings highlight the significance of inoculation timing and microbial composition in regulating metabolic pathways during FW composting degradation, offering insights for designing effective microbial formulations for composting.

Bioinformatics-assisted mining and design of novel pullulanase suitable for starch cold hydrolysis.

Cold-active pullulanases with good catalytic performance possess promising applications in cold hydrolysis of starch. Adopting bioinformatics-assisted mining strategies, 7 candidate cold-active pullulanases were initially screened out from IMG/MER database. Among the candidates, PulBs exhibited good thermostability and the highest specific activity of 147.4 U/mg. The half-life of PulBs was about 200 h at 35 °C. Employing PulBs as the initial enzyme, the active-site design of FuncLib was implemented to enhance the activity. The design PulBs-20 exhibited an enhanced specific activity of 209.9 U/mg, which was 1.4 times that of PulBs. Furthermore, the thermostability of PulBs-20 was augmented, with a half-life of 250 h at 35 °C. When applied in the cold hydrolysis of starch, PulBs-20 can effectively enhance the hydrolysis effect of raw starch. Supplemented with the raw starch-hydrolyzing α-amylase AmyZ1 and PulBs-20, the hydrolysis rate of raw corn starch increased to 53.5 %, which was 1.3 times that of using AmyZ1 alone. Due to its high hydrolysis activity and good thermostability, PulBs-20 can serve as an efficient accessory enzyme in starch cold hydrolysis.

Functional Analysis of Two Carboxylesterase Genes Involved in Beta-Cypermethrin and Phoxim Resistance in Plutella xylostella (L.)

Enhanced expression of carboxylesterase (CarE) genes is an important mechanism of insecticide resistance in pests. However, their roles in multi-insecticide resistance have rarely been reported. Herein, two CarE genes (PxαE6 and PxαE9) were identified; their relative expression levels in three multi-insecticide-resistant populations of P. xylostella (HN, GD-2017 and GD-2019) were 2.69- to 15.32-fold higher than those in the sensitive population, and they were considerably overexpressed at the larval stage and in the midgut of the 4th instar. PxαE6 and PxαE9 knockdown increased the susceptibility of GD-2019 larvae to phoxim or/and beta-cypermethrin. The recombinant PxαE6 and PxαE9 expressed in Escherichia coli exhibited high hydrolysis activity towards α-NA. GC–MS and LC–MS/MS assays revealed that PxαE9 could metabolize beta-cypermethrin and phoxim with efficiency determinations of 51.6% and 21.1%, respectively, while PxαE6 could metabolize phoxim with an efficiency of 12.0%. Homology modelling, molecular docking and molecular-dynamics simulation analyses demonstrated that beta-cypermethrin or/and phoxim could fit well into the active pocket and stably bind to PxαE6 or PxαE9. These results show that PxαE6 and PxαE9 overexpression were involved in resistance to beta-cypermethrin or/and phoxim in multi-insecticide-resistant P. xylostella populations, a finding which sheds light on the molecular mechanisms of multi-insecticide resistance in insect pests.

The functional role of N-link glycosylation in a novel cellobiohydrolase II (LsCel6A) from a white-rot fungus Lentinus sp. WR2

White-rot fungi produce a wide spectrum of lignocellulose-degradation enzymes, which can be used in bioenergy, bioremediation, and other industrial applications. This study identified a cellobiohydrolase II (Cel6A, GH6 cellobiohydrolase, EC 3.2.1.91) with high hydrolytic activity toward crystalline cellulose from a white-rot fungus Lentinus sp. WR2. Both native (nLsCel6A) and recombinant (rLsCel6A) enzymes expressed in Pichia pastoris were purified and characterized. Three N-glycosylation sites at Asn102, Asn145, and Asn392 containing high-mannose glycans, were confirmed by mass spectrometry. To elucidate the functional role of N-linked glycans, three deglycosylated mutants of rLsCel6A, i.e., N102A, N145A, and N392A, were created and characterized for their biochemical and kinetic properties. While no discernible changes in the secondary structure of the three mutants were determined by circular dichroism spectrometry, deterioration of thermostability was revealed in N392A but not in N102A and N145A. Structure modeling and molecular dynamics analyses revealed that the N-linked glycan on Asn392 may restrict the flexibility of the C-terminal loop in LsCel6A, affecting the protein integrity and appropriate dynamics for the enzymatic function. In summary, this study identified a novel LsCel6A enzyme with high catalytic activity against insoluble forms of cellulose and demonstrated the role of N-linked glycosylation in the thermostability of the enzyme.

Purification, fibrinolytic activity and substrate binding of nattokinase from Bacillus subtilis: A rapid and sensitive detection for fibrinolytic activity of nattokinase

Nattokinase (NK, EC 3.4.21.62) is an alkaline serine protease secreted by B. subtilis, which has a strong fibrinolytic activity in vitro and in vivo. Here, we fermented NK using a B. subtilis strain and purified the protein, designed a peptide segment derived from fibrin as a substrate of NK. Based on fluorescence resonance energy transfer (FRET), we found that NK exhibits a high hydrolytic activity towards the peptide, with Km of 9.33 ± 1.28 μM, kcat of 0.20 ± 0.01s-1, and kcat/Km of 21,436.23s-1M-1, demonstrating that the peptide is a substrate for NK. Furthermore, we investigated the binding of the substrate with NK by tryptophan fluorescence quenching, molecular docking and dynamics simulation. Fluorescence quenching showed that the substrate binds to NK mainly through hydrogen bonding and van der Waals forces, with dissociation constants KD of 13.73 and 21.24μM at 25 and 35°C, respectively. Molecular docking and dynamics analysis revealed that the substrate recognizes the active site of NK, and provides new information on the characteristics of the binding of the substrate with NK. Our study demonstrated a rapid and sensitive method for the quantitative measurement of fibrinolytic activity of NK based on the substrate, which is very reproducible and rapid with virtually complete results in approximately 20min.

Mechanism of maltogenic α-amylase modification on barley granular starches spanning the full range of amylose

Amylopectin (AP)-only (APBS), normal (NBS), and amylose (AM) only (AOBS) barley starches were selected here to investigate catalysis pattern of maltogenic α-amylase (MA) on hydrolyzing AP and AM granular starches. MA shortened starch side chains with degree of polymerization (DP) 11-30. MA-treated APBS exhibited porous granular structures and dramatically increased degree of branching (DB, 17-20%), and reduced ordered degrees, suggesting high hydrolysis and transglycosylation activities of MA. MA-treated NBS showed less pronounced porous structures and slightly increased DB (2-4%), indicating high hydrolysis but low transglycosylation activities. AOBS displayed minimal changes in DB (0.2-0.3%) and starch structures, implying low hydrolysis and transglycosylation activities. Therefore, MA preferred to attack the AP molecules with abundant glucan substrates with DP 11-30, while AM restricted MA activity likely by creating ineffective binding sites and undergoing rapid reorganization. These findings deepened the understanding of the mechanisms of MA in modifying granular starches with varying AM content.

Catalytic Hydrolysis of Paraoxon by Immobilized Copper(II) Complexes of 1,4,7-Triazacyclononane Derivatives.

Organophosphate neuroactive agents represent severe security threats in various scenarios, including military conflicts, terrorist activities and industrial accidents. Addressing these threats necessitates effective protective measures, with a focus on decontamination strategies. Adsorbents such as bentonite have been explored as a preliminary method for chemical warfare agent immobilization, albeit lacking chemical destruction capabilities. Chemical decontamination, on the other hand, involves converting these agents into non-toxic or less toxic forms. In this study, we investigated the hydrolytic activity of a Cu(II) complex, previously studied for phosphate ester hydrolysis, as a potential agent for chemical warfare decontamination. Specifically, we focused on a ligand featuring a thiophene anchor bound through an aliphatic spacer, which exhibited high hydrolytic activity in its Cu(II) complex form in our previous studies. Paraoxon, an efficient insecticide, was selected as a model substrate for hydrolytic studies due to its structural resemblance to specific chemical warfare agents and due to the presence of a chromogenic 4-nitrophenolate moiety. Our findings clearly show the hydrolytic activity of the studied Cu(II) complexes. Additionally, we demonstrate the immobilization of the studied complex onto a solid substrate of Amberlite XAD4 via copolymerization of its thiophene side group with dithiophene. The hydrolytic activity of the resultant material towards paraoxon was studied, indicating its potential utilization in organophosphate neuroactive agent decontamination under mild conditions and the key importance of surface adsorption of paraoxon on the polymer surface.

Multienzyme Immobilization on PVDF Membrane via One-Step Mussel-Inspired Method: Enhancing Fouling Resistance and Self-Cleaning Efficiency.

Immobilizing different enzymes on membranes can result in biocatalytic active membranes with a self-cleaning capacity toward a complex mixture of foulants. The membrane modification can reduce fouling and enhance filtration performance. Protease, lipase, and amylase were immobilized on poly(vinylidene fluoride) (PVDF) microfiltration membranes using a polydopamine coating in a one-step method. The concentrations of polydopamine precursor and enzymes were optimized during the immobilization. The higher hydrolytic activities were obtained using 0.2 mg/mL of dopamine hydrochloride and 4 mg/mL of enzymes: 0.90 mgstarch/min·cm2 for amylase, 10.16 nmoltyrosine/min·cm2 for protease, and 20.48 µmolp-nitrophenol/min·cm2 for lipase. Filtration tests using a protein, lipid, and carbohydrate mixture showed that the modified membrane retained 41%, 29%, and 28% of its initial water permeance (1808 ± 39 L/m2·h·bar) after three consecutive filtration cycles, respectively. In contrast, the pristine membrane (initial water permeance of 2016 ± 40 L/m2·h·bar) retained only 23%, 12%, and 8%. Filtrations of milk powder solution were also performed to simulate dairy industry wastewater: the modified membrane maintained 28%, 26%, and 26% of its initial water permeance after three consecutive filtration cycles, respectively, and the pristine membrane retained 34%, 21%, and 7%. The modified membrane showed increased fouling resistance against a mixture of foulants and presented a similar water permeance after three cycles of simulated dairy wastewater filtration. Membrane fouling is reduced by the immobilized enzymes through two mechanisms: increased membrane hydrophilicity (evidenced by the reduced water contact angle after modification) and the enzymatic hydrolysis of foulants as they accumulate on the membrane surface.

Redox interference-free bimodal paraoxon sensing enabled by an aggregation-induced emission nanozyme catalytically hydrolyzing phosphoesters specifically

In view of the current serious situation of organophosphorus pesticides (OPs) residue contamination, developing rapid and accurate OPs sensors is a matter of urgency. Redox-nanozyme based colorimetric sensors have been widely researched and utilized in OPs residue determination, but overcoming the interference of external redox substances and the effect of single-signal modes on detection performance is still a challenge. Here we fabricated a Zr-based metal-organic framework (MOF) featuring specific phosphatase-like activity and strong aggregation-induced emission (AIE) fluorescence for redox interference-free bimodal pesticide sensing. In the MOF, the activity-tunable Zr4+ node offered high hydrolytic activity and affinity toward P-O containing substrates, and the rigid framework structure effectively enhanced the fluorescence emission of the ligand 1,1,2,2-tetra(4-carboxylphenyl)ethylene. The developed AIEzyme could efficiently catalyze the hydrolysis of paraoxon to yellow p-nitrophenol, which further reduced the intrinsic AIE fluorescence of AIEzyme through internal filtration effect. Thereby, a natural enzyme-free dual-mode colorimetric/fluorescence approach was established for paraoxon detection with no interference from redox substances, and a smartphone-assisted portable platform was further developed to enable the facile, rapid, and high-performance sensing of the pesticide in complex practical matrices.

Single-Atom Ce-Doped Metal Hydrides with High Phosphatase-like Activity Amplify Oxidative Stress-Induced Tumor Apoptosis.

Phosphates within tumors function as key biomolecules, playing a significant role in sustaining the viability of tumors. To disturb the homeostasis of cancer cells, regulating phosphate within the organism proves to be an effective strategy. Herein, we report single-atom Ce-doped Pt hydrides (Ce/Pt-H) with high phosphatase-like activity for phosphate hydrolysis. The resultant Ce/Pt-H exhibits a 26.90- and 6.25-fold increase in phosphatase-like activity in comparison to Ce/Pt and Pt-H, respectively. Mechanism investigations elucidate that the Ce Lewis acid site facilitates the coordination with phosphate groups, while the surface hydrides enhance the electron density of Pt for promoting catalytic ability in H2O cleavage and subsequent nucleophilic attack of hydroxyl groups. Finally, by leveraging its phosphatase-like activity, Ce/Pt-H can effectively regulate intracellular phosphates to disrupt redox homeostasis and amplify oxidative stress within cancer cells, ultimately leading to tumor apoptosis. This work provides fresh insights into noble-metal-based phosphatase mimics for inducing tumor apoptosis.

A novel Al/BiCl3/γ-Al2O3 composite for small-sized fuel cell: Fabrication, performance and mechanisms

A novel Al/BiCl3/γ-Al2O3 composite for small-sized fuel cell: Fabrication, performance and mechanisms

Fabrication of an In‐situ Synthesized Porous Silica‐Ruthenium‐Nickel Composite Catalyst for Hydrolysis of Ammonia Borane

AbstractThis work investigated in‐situ synthesis of porous silica‐nickel‐ruthenium composite catalysts for hydrolysis of ammonia borane. The precursors were prepared with an anionic surfactant, sodium dodecyl sulfate (SDS), to form porous structure in the precursor particles, and the precursors was treated in aqueous solution including sodium borohydride and ammonia borane for in‐situ process to parallelly activate and use the catalysts for hydrolysis of ammonia borane. The result of TEM and nitrogen sorption measurements suggested that the primary particle size of the in‐situ synthesized catalysts decrease with increasing the amount of SDS and sodium chloride as the additive for controlling the primary particle size probably because of the effect of sodium ions to inhibit nuclei growth of the primary particles. From the EDS analyses, the composition of active nickel and ruthenium contents increased with increasing the amount of SDS and sodium chloride except for the highest amount of SDS probably because of adsorption of the metal cations with the excess amount of the surfactant anion to decrease the amount of the metal cations including the catalyst. The in‐situ synthesized catalyst including small size primary particles and high nickel and ruthenium content tended to exhibit high activity for hydrolysis of ammonia borane.

Deciphering the Dual Roles of an Alginate-Based Biodegradable Flocculant in Anaerobic Fermentation of Waste Activated Sludge: Dewaterability and Degradability.

Biodegradable flocculants are rarely used in waste activated sludge (WAS) fermentation. This study introduces an alginate-based biodegradable flocculant (ABF) to enhance both the dewatering and degradation of WAS during its fermentation. Alginate was identified in structural extracellular polymeric substances (St-EPS) of WAS, with alginate-producing bacteria comprising ∼4.2% of the total bacterial population in WAS. Owing to its larger floc size, higher contact angle, and lower free energy resulting from the Lewis acid-base interaction, the addition of the prepared ABF with a network structure significantly improved the dewaterability of WAS and reduced capillary suction time (CST) by 72%. The utilization of ABF by an enriched alginate-degrading consortium (ADC) resulted in a 35.5% increase in the WAS methane yield owing to its higher hydrolytic activity on both ABF and St-EPS. Additionally, after a 30 day fermentation, CST decreased by 62% owing to the enhanced degradation of St-EPS (74.4%) and lower viscosity in the WAS + ABF + ADC group. The genus Bacteroides, comprising 12% of ADC, used alginate lyase (EC 4.2.2.3) and pectate lyase (EC 4.2.2.2 and EC 4.2.2.9) to degrade alginate and polygalacturonate in St-EPS, respectively. Therefore, this study introduces a new flocculant and elucidates its dual roles in enhancing both the dewaterability and degradability of WAS. These advancements improve WAS fermentation, resulting in higher methane production and lower CSTs.

Coupling Peptide-Based Encapsulation of Enzymes with Bacteria for Paraoxon Bioremediation.

The catalytic efficiency of enzymes can be harnessed as an environmentally friendly solution for decontaminating various xenobiotics and toxins. However, for some xenobiotics, several enzymatic steps are needed to obtain nontoxic products. Another challenge is the low durability and stability of many native enzymes in their purified form. Herein, we coupled peptide-based encapsulation of bacterial phosphotriesterase with soil-originated bacteria, Arthrobacter sp. 4Hβ as an efficient system capable of biodegradation of paraoxon, a neurotoxin pesticide. Specifically, recombinantly expressed and purified methyl parathion hydrolase (MPH), with high hydrolytic activity toward paraoxon, was encapsulated within peptide nanofibrils, resulting in increased shelf life and retaining ∼50% activity after 132 days since purification. Next, the addition of Arthrobacter sp. 4Hβ, capable of degrading para-nitrophenol (PNP), the hydrolysis product of paraoxon, which is still toxic, resulted in nondetectable levels of PNP. These results present an efficient one-pot system that can be further developed as an environmentally friendly solution, coupling purified enzymes and native bacteria, for pesticide bioremediation. We further suggest that this system can be tailored for different xenobiotics by encapsulating the rate-limiting key enzymes followed by their combination with environmental bacteria that can use the enzymatic step products for full degradation without the need to engineer synthetic bacteria.

The Improved Antineoplastic Activity of Thermophilic L-Asparaginase Tli10209 via Site-Directed Mutagenesis.

Amino acid deprivation therapy (AADT) is a novel anticancer therapy, considered nontoxic and selective. Thermophilic L-asparaginase enzymes display high stability and activity at elevated temperatures. However, they are of limited use in clinical applications because of their low substrate affinity and reduced activity under physiological conditions, which may necessitate an improved dosage, leading to side effects and greater costs. Thus, in an attempt to improve the activity of L-Asn at 37 °C, with the use of a semi-rational design, eight active-site mutants of Thermococcus litoralis DSM 5473 L-asparaginase Tli10209 were developed. T70A exhibited a 5.11-fold increase compared with the wild enzyme in physiological conditions. Double-mutant enzymes were created by combining mutants with higher hydrolysis activity. T70A/F36Y, T70A/K48L, and T70A/D50G were enhanced by 5.59-, 6.38-, and 5.58-fold. The immobilized enzyme applied in MCF-7 breast cancer cells only required one-seventh of the dose of the free enzyme to achieve the same inhibition rate under near-infrared irradiation. This provides a proof of concept that it is possible to reduce the consumption of L-Asn by improving its activity, thus providing a method to manage side effects.

In situ hydrogen production from Al–BiCl3 hydrolysis for small-sized fuel cell

In situ hydrogen production from Al–BiCl3 hydrolysis for small-sized fuel cell

Characterization of a novel aspartic protease from Trichoderma asperellum for the preparation of duck blood peptides

A novel aspartic protease gene (TaproA1) from Trichoderma asperellum was successfully expressed in Komagataella phaffii (Pichia pastoris). TaproA1 showed 52.8% amino acid sequence identity with the aspartic protease PEP3 from Coccidioides posadasii C735. TaproA1 was efficiently produced in a 5 L fermenter with a protease activity of 4092 U/mL. It exhibited optimal reaction conditions at pH 3.0 and 50 °C and was stable within pH 3.0–6.0 and at temperatures up to 45 °C. The protease exhibited broad substrate specificity with high hydrolysis activity towards myoglobin and hemoglobin. Furthermore, duck blood proteins (hemoglobin and plasma protein) were hydrolyzed by TaproA1 to prepare bioactive peptides with high ACE inhibitory activity. The IC50 values of hemoglobin and plasma protein hydrolysates from duck blood proteins were 0.105 mg/mL and 0.091 mg/mL, respectively. Thus, the high yield and excellent biochemical characterization of TaproA1 presented here make it a potential candidate for the preparation of duck blood peptides.Key points• An aspartic protease (TaproA1) from Trichoderma asperellum was expressed in Komagataella phaffii.• TaproA1 exhibited broad substrate specificity and the highest activity towards myoglobin and hemoglobin.• TaproA1 has great potential for the preparation of bioactive peptides from duck blood proteins.

Sucrose phosphorylase from Alteromonas mediterranea: Structural insight into the regioselective α-glucosylation of (+)-catechin

Sucrose phosphorylases, through transglycosylation reactions, are interesting enzymes that can transfer regioselectively glucose from sucrose, the donor substrate, onto acceptors like flavonoids to form glycoconjugates and hence modulate their solubility and bioactivity. Here, we report for the first time the structure of sucrose phosphorylase from the marine bacteria Alteromonas mediterranea (AmSP) and its enzymatic properties. Kinetics of sucrose hydrolysis and transglucosylation capacities on (+)-catechin were investigated. Wild-type enzyme (AmSP-WT) displayed high hydrolytic activity on sucrose and was devoid of transglucosylation activity on (+)-catechin. Two variants, AmSP-Q353F and AmSP-P140D catalysed the regiospecific transglucosylation of (+)-catechin: 89% of a novel compound (+)-catechin-4'-O-α-d-glucopyranoside (CAT-4') for AmSP-P140D and 92% of (+)-catechin-3'-O-α-d-glucopyranoside (CAT-3') for AmSP-Q353F. The compound CAT-4' was fully characterized by NMR and mass spectrometry. An explanation for this difference in regiospecificity was provided at atomic level by molecular docking simulations: AmSP-P140D was found to preferentially bind (+)-catechin in a mode that favours glucosylation on its hydroxyl group in position 4' while the binding mode in AmSP-Q353F favoured glucosylation on its hydroxyl group in position 3'.