Initial Enzyme Activity Research Articles

Catalytic activities of tannase covalently linked to amino-functionalized carbon nanotubes

Immobilized Aspergillus ficuum tannase on amino-functionalized multiwalled carbon nanotubes (A-MWCNT) was assembled through glutaraldehyde-mediated covalent coupling to produce a stable biocatalyst nanocomposite. The effective binding of tannase on the surface of A-MWCNT was evaluated and authenticated by spectroscopic signature signals and morphological differences, before and after enzyme coupling. Both free- and immobilized tannase showed optimal catalytic activities at pH 5.0 and 35 °C, with the immobilized tannase exhibiting enhanced thermostability compared to the soluble enzyme. The immobilized tannase preparation was also found to be reusable up to five cycles, with 66% of initial enzyme activity retained thereafter. The enzyme-carbon nanotube composite preparation allows for bioconversion to be accomplished in a bioreactor with a smaller footprint.

Efficient Biosynthesis of Monacolin J through Enzymatic Hydrolysis Using a Recombinant Lovastatin Hydrolase

Simvastatin is a widely used statin medication that is commonly prescribed to lower cholesterol levels and reduce the risk of cardiovascular events. It is marketed under the brand name Zocor and is known for its effectiveness in treating high cholesterol and managing cardiovascular disease. Monacolin J is an important precursor used to synthesize simvastatin and is mainly produced by chemical methods in industry. Here, monacolin J was synthesized through an enzymatic method under optimized reaction conditions. One recombinant Escherichia coli BL21 (DE3) strain containing lovastatin hydrolase (encoded by CDV55_102090) from Aspergillus turcosus was constructed, which effectively transformed 100 g/L of lovastatin to monacolin J within 3.5 h at pH 8.0 and 30 °C, with a conversion rate of >99.8%. Furthermore, the T5010, the temperature at which the residual activity was half of the initial enzymatic activity after 10 min of heat treatment, was >50 °C, indicating the tremendous potential of this bioprocess for synthesizing monacolin J at an industrial scale.

Synthesis of a Zn(II)-2-aminoimidazole Framework as an Efficient Carbonic Anhydrase Mimic.

Carbonic anhydrase (CA) plays a crucial role in the CO2 capture processes by catalyzing the hydration of CO2. In this study, we synthesized a bioinspired carbonic anhydrase Zn-MOF (metal-organic framework) incorporating 2-aminoimidazole and Zn2+ as initial constituents. The synthesized Zn-MOF exhibited promising potential for efficiently catalyzing the CO2 hydration. Structural analyses such as SEM, XRD, and BET confirmed that the Zn-MOF crystal consisted of stacked grains with an average size of approximately 36 nm, forming a micron-sized spherical structure. Functionally, Zn-MOF exhibited effective catalytic activity toward both CO2 hydration and ester hydrolysis. The introduction of amino groups significantly enhanced the esterase activity of Zn-MOF to 0.28 U/mg at ambient temperature, which was twice that of ZIF-8. Furthermore, the introduction of amino groups resulted in remarkable hydrothermal stability, with the esterase activity reaching 0.72 U/mg after undergoing hydrothermal treatment at 80 °C for 12 h. Additionally, Zn-MOF exhibited enhanced capability in CO2 hydration at a pH value exceeding 8.5. After six repeated uses, ZIF-8 and Zn-MOF retained approximately 68 and 65% of their initial enzyme activity, respectively, underscoring the potential practical applicability of Zn-MOF in industrial CO2 capture processes. This work showcases the development of a novel Zn-MOF crystal as an efficient CA mimic, effectively emulating the active sites of natural CA using 2-aminoimidazole as a coordinating ligand for Zn2+ coordination. These findings not only advance the field of innovative enzyme mimics but also pave the way for further exploration of industrial CO2 capture catalysts.

Reducing residual chlortetracycline in wastewater using a whole-cell biocatalyst

Antibiotic contamination has become an increasingly important environmental problem as a potentially hazardous emergent and recalcitrant pollutant that poses threats to human health. In this study, manganese peroxidase displayed on the outer membrane of Escherichia coli as a whole-cell biocatalyst (E. coli MnP) was expected to degrade antibiotics. The manganese peroxidase activity of the whole-cell biocatalyst was 13.88 ± 0.25 U/L. The typical tetracycline antibiotic chlortetracycline was used to analyze the degradation process. Chlortetracycline at 50 mg/L was effectively transformed via the whole-cell biocatalyst within 18 h. After six repeated batch reactions, the whole-cell biocatalyst retained 87.2 % of the initial activity and retained over 87.46 % of the initial enzyme activity after storage at 25°C for 40 days. Chlortetracycline could be effectively removed from pharmaceutical and livestock wastewater by the whole-cell biocatalyst. Thus, efficient whole-cell biocatalysts are effective alternatives for degrading recalcitrant antibiotics and have potential applications in treating environmental antibiotic contamination.

Superoxide dismutase-contained solid lipid nanoparticles: Formulation development and In-vivo evaluation for second-degree burn wound healing in rats

IntroductionSuperoxide dismutase (SOD), a natural enzyme with high antioxidant activity, reduces injury and accelerates wound healing by scavenging superoxide radicals. This enzyme plays an important role in cellular defense against oxidative stress such as burn injury. The aim of this study was to load SOD into solid lipid nanoparticles for the treatment of rat burn wounds. MethodsSolid lipid nanoparticles were prepared by Solvent Emulsification Diffusion method and evaluated for particle size, enzyme activity and enzyme entrapment efficiency. Twenty-seven rats in 3 different groups were induced with deep second-degree burns and then treated with SOD-loaded solid lipid nanoparticles, solid lipid nanoparticles without enzyme, or SOD solution. After the treatment period, the wounds were evaluated macroscopically for the area of healing and microscopically for indices of re-epithelialization, granulation tissue and angiogenesis. ResultsThe optimized SOD-loaded solid lipid nanoparticles showed a particle size of 35–85 ± 2.41 nm, 78.4 ± 4.31 % entrapment efficiency and 90 % initial enzyme activity. Macroscopic examination showed that the best recovery rate belonged to the solid lipid nanoparticle group. Pathological studies also showed that angiogenesis and granulation tissue were significantly better in this group. Compared to the other two groups, SOD-loaded solid lipid nanoparticles showed a significant improvement in pathological factors, particularly angiogenesis and granulation tissue, as well as a faster reduction in the number of inflammatory cells. ConclusionBased on this study, solid lipid nanoparticles could be used as an effective delivery system for SOD in the treatment of second-degree burns.

Impact of an Ionic Liquid Solution on Horseradish Peroxidase Activity.

Horseradish peroxidase (HRP) is an enzyme that oxidizes pollutants from wastewater. A previous report indicated that peroxidases can have an enhancement in initial enzymatic activity in an aqueous solution of 0.26 M 1-ethyl-3-methylimidazolium ethyl sulfate ([EMIm][EtSO4]) at neutral pH. However, the atomistic details remain elusive. In the enzymatic landscape of HRP, compound II (Cpd II) plays a key role and involves a histidine (H42) residue. Cpd II exists as oxoferryl (2a) or hydroxoferryl (2b(FeIV)) forms, where 2a is the predominantly observed form in experimental studies. Intriguingly, the ferric 2b(FeIII) form seen in synthetic complexes has not been observed in HRP. Here, we have investigated the structure and dynamics of HRP in pure water and aqueous [EMIm][EtSO4] (0.26 M), as well as the reaction mechanism of 2a to 2b conversion using polarizable molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations. When HRP is solvated in aq [EMIm][EtSO4], the catalytic water displaces, and H42 directly orients over the ferryl moiety, allowing a direct proton transfer (PT) with a significant energy barrier reduction. Conversely, in neat water, the reaction of 2a to 2b follows the previously reported mechanism. We further investigated the deprotonated form of H42. Analysis of the electric fields at the active site indicates that the aq [EMIm][EtSO4] medium facilitates the reaction by providing a more favorable environment compared with the system solvated in neat water. Overall, the atomic level supports the previous experimental observations and underscores the importance of favorable electric fields in the active site to promote catalysis.

IMMOBILIZATION OF A MICROBIAL FRUCTOSYLTRANSFERASE ON BIOCHAR AND BIOCHAR FUNCTIONALIZED WITH GLUTARALDEHYDE AIMING AT FRUCTOOLIGOSSACHARIDES PRODUCTION

Fructooligosaccharides (FOS) are a group of carbohydrates that have fructose units, with a glucose molecule at the end. These are structures that can be found in a wide variety of vegetables, being also produced chemically by the enzymatic transfructosylation reaction of sucrose. The present work aimed at the immobilization of the extracellular FTase enzyme from Aspergillus oryzae-IPT-301 on Eucalyptus biochar and Eucalyptus biochar functionalized with glutaraldehyde, for the enzymatic conversion of sucrose into FOS. Immobilization and reuse tests were carried out and immobilization yield and recovered activity calculated. The immobilization yield (IY) and recovered activity (RA) of the FTase immobilized on biochar were, 10.92% and 77.67%, respectively. The IY and RA of the FTase immobilized on the functionalized biochar were, 22.44% and 96.51%, respectively. The enzyme immobilized on functionalized biochar demonstrated transfructosylation activity for more cycles than the enzyme immobilized on biochar. The use of glutaraldehyde allowed 48% of initial enzymatic activity in the third cycle. Analyzes were conducted up to the sixth cycle, however, only 7% of initial enzymatic activity was achieved in the final cycle. These results suggest that functionalized biochar stands out as a support material for FTase immobilization aiming at FOS production catalyzed on heterogeneous biocatalysts.

One‐pot Heterogeneous Biocatalysis under Thermal Decay for Fructose Production from Lactose using Co‐Immobilized Enzymes: Modeling and Simulation

AbstractMulti‐enzyme reaction systems have become an interesting option to avoid transformation and purification steps in productive reactor‐based systems; when carried out in a single vessel, they are termed one‐pot systems. This novel work aims to model and simulate the behavior of a one‐pot heterogeneous catalysis system in a batch‐stirred bioreactor and in a continuous stirred bioreactor with co‐immobilized enzymes to determine the compromise operation temperature that maximizes the volumetric productivity, considering thermal decay of the biocatalyst. The production of fructose from lactose is considered a case study, using co‐immobilized ‐galactosidase ( ‐gal) and glucose‐isomerase (GI) in a spherical particle. A diffusion and reaction mathematical model was used to calculate the effectiveness factor for every reaction species considering the thermal decay of both enzymes. Simulations in both batch and continuous operation were done at temperatures ranging from 40 to 60 °C, for initial enzyme activity load ratios ranging from 0.1 to 0.9 IUGI IU . Resulting effectiveness factors were close to one, indicating no significant limitations on reaction rates due to internal diffusional constraints. In both cases, temperature exerted a more substantial effect than the initial enzyme activity load ratio, showing different scenarios of high volumetric productivity (batch) or amount of produced fructose (continuous).

Expression and characterization of mesophilic GH1 β-glucosidase CdBglA from acidophilic Cuniculiplasma divulgatum

β-glucosidase has important applications in food, pharmaceutics, biomass conversion and other fields, exploring β-glucosidase with strong adaptability and excellent properties thus has received extensive interest. In this study, a novel glucosidase from the GH1 family derived from Cuniculiplasma divulgatum was cloned, expressed, and characterized, aiming to find a better β-glucosidase. The amino acid sequences of GH1 family glucosidase derived from C. divulgatum were obtained from the NCBI database, and a recombinant plasmid pET-30a(+)-CdBglA was constructed. The recombinant protein was induced to express in Escherichia coli BL21(DE3). The enzymatic properties of the purified CdBglA were studied. The molecular weight of the recombinant CdBglA was 56.0 kDa. The optimum pH and temperature were 5.5 and 55 ℃, respectively. The enzyme showed good pH stability, 92.33% of the initial activity could be retained when treated under pH 5.5-11.0 for 1 h. When pNPG was used as a substrate, the kinetic parameters Km, Vmax and Kcat/Km were 0.81 mmol, 291.99 μmol/(mg·min), and 387.50 s-1 mmol-1, respectively. 90.33% of the initial enzyme activity could be retained when CdBglA was placed with various heavy metal ions at a final concentration of 5 mmol/L. The enzyme activity was increased by 28.67% under 15% ethanol solution, remained unchanged under 20% ethanol, and 43.68% of the enzyme activity could still be retained under 30% ethanol. The enzyme has an obvious activation effect at 0-1.5 mol/L NaCl and can tolerate 0.8 mol/L glucose. In conclusion, CdBglA is an acidic and mesophilic enzyme with broad pH stability and strong tolerance to most metal ions, organic solvents, NaCl and glucose. These characteristics may facilitate future theoretical research and industrial production.

Surface activation of hierarchically porous diatomite modified with polyethyleneimine for immobilizing d-allulose 3-epimerase

d-Allulose, a low-calorie functional sweetener popular among the obese population, can be biosynthesized using d-allulose 3-epimerase (DAE). However, there are currently no commercially viable immobilized DAE enzymes on the market for the large-scale production of d-Allulose. In this study, immobilizing the DAE enzyme was developed based on polymer-activated diatomite with surface enhancement using crosslinkers. Specifically, the amino groups of polyethyleneimine were successfully carried on the porous diatomite and then activated by glutaraldehyde to form a biopolymer coating. The obtained carrier was firmly covalently linked to the DAE enzyme via the Schiff-base reaction. The constructed immobilized DAE with a “core-shell” structure showed a high specific activity of 2088.93 ± 9.85 U/g and an excellent conversion rate of 34.4%. This biocatalyst was stable within a wide temperature and pH range. It also showed outstanding thermal stability, the half-lives of the immobilized DAE were 109 and 124 times that of the free DAE at 55 °C and 60 °C, respectively. Moreover, the immobilized DAE remained at about 81.33% of the initial enzyme activity after 8 recycles. The results of this study may contribute to the cost reduction of d-Allulose industrial production and provide a potential immobilized enzyme strategy for future large-scale manufacturing of d-Allulose.

Efficient secreted expression of natural intracellular β-galactosidase from Bacillus aryabhattai via non-classical protein secretion pathway in Bacillus subtilis

In this study, the natural intracellular β-galactosidase (lacZBa) from Bacillus aryabhattai was expressed extracellularly in Bacillus subtilis. Sec and Tat signal peptides from different secretion pathways were incorporated to facilitate extracellular secretion of lacZBa, resulting in a yield of only 0.54 U/mL. Interestingly, it was discovered that lacZBa could be efficiently expressed and secreted in B. subtilis via a non-classical secretory pathway without the need for a signal peptide. The extracellular activity and secretion ratio were 5.3 U/mL and 65 %, respectively. Compared to E. coli, the expression of lacZBa in B. subtilis resulted in increased acid resistance and higher pH stability and thermostability, with a 1.7-fold increase in half-life at 50 °C and pH 6.0. Additionally, we combined single-factor experiments and response surface methodology to enhance extracellular expression of β-galactosidase in shake-flasks. The resulting optimal medium contained 4.46 % glucose, 1.47 % corn steep liquor, 1.5 % beef extract, 0.82 % CaCl2, and 0.1 % MgSO4. Under optimal conditions, the yield of extracellularly secreted β-galactosidase at the shake flask level was 17.41 U/mL, representing a 32.2-fold increase in initial extracellular enzyme activity. This study represents the first successful report of natural intracellular β-galactosidase being expressed through the non-classical secretory pathway in B. subtilis.

Properties and application of pectinase obtained from Galactomyces candidum using pectin extracted from mango peels as a carbon source

Enzyme instability, poor levels of activity, low substrate specificity, and cost, limit the use of pectinase in the juice industry as well as in galacturonic acid production. Therefore, this research explored the properties of low-cost Galactomyces candidum pectinase with high heat and pH stability capable of degrading mango pectin. Pectinase was produced from Galactomyces candidum on the second day in a submerged fermentation broth supplemented with beef-extract, yeast-extract, and mango pectin at a pH of 5.0. The crude enzyme had a specific activity of 31.34 U/mg. After gel filtration, the specific activity and molecular weight of the enzyme were 39.96U/mg and 26 kDa, respectively. The enzyme's optimal pH and temperature were 5.5 and 50 °C, respectively. The maximum velocity (Vmax) and Michaelis constant (KM) of the enzyme were 73.53 μmol/min and 1.49 mg/ml. The activation effect of some metal ions on the enzyme was in the following order Mn2+˃Ca2+˃K+˃Fe2+> Co2+> Na+˃Zn2+> Mg2+˃Cu2+. Galactomyces candidum pectinase retained more than 70% of its original activity after incubation for 120 min at all the pH tested. Also, over 80% of the enzyme's original activity was retained after incubation for 120 min at 50 and 60 °C, while at 70 °C, 50% of its initial activity was retained. In addition, over 70% of the enzyme's initial activity was lost at 80 °C after incubation for 120 min. Spectral studies on the clarification of mango fruit juice showed that both crude and purified Galactomyces candidum pectinase were able to clarify mango juice, suggesting the enzyme's potential in the beverage industry.

Biochemical characterization and elucidation of the action mode of a GH16 family κ-carrageenase for efficient preparation of carrageenan oligosaccharides.

κ-Carrageenan oligosaccharides have a variety of biological activities. Degradation of κ-carrageenan by κ-carrageenase leads to degradation products with different degrees of polymerization (DPs). A novel gene (CecgkA) encoding a new κ-carrageenase was cloned from Colwellia echini and heterologously expressed in Escherichia coli BL21 (DE3). The enzyme is 1104bp in length, encodes 367 amino acid residues and has a molecular weight of 41.30kDa. Multiple alignment analysis showed that CeCgkA belongs to the glycoside hydrolase (GH16) family and has the highest homology with the κ-carrageenase of Rhodopirellula maiorica SM1, with 58% homology. The CeCgkA showed maximum activity (453.15 U/mg) at pH 8.0 and 35°C. Determination of biochemical properties showed that CeCgkA was a thermal recovery enzyme, and 51.6% of the initial enzyme activity was recovered by immediately placing the sample at 35°C for 60min after enzymatic inactivation by boiling for 10min. K+, Na+, and EDTA had an activating effect on the enzyme activity, while Ni2+, Cu2+, and Zn2+ inhibited the activity of the enzyme. In addition, TLC and ESI-MS analysis showed that the maximum recognition unit of CecgkA was decasaccharide and that the main degradation products were disaccharides, tetrasaccharides and hexasaccharides, indicating that the enzyme is an endo-type carrageenase.

Preparation of magnetic polyacrylamide hydrogel with chitosan for immobilization of glutamate decarboxylase to produce γ-aminobutyric acid

Gamma-aminobutyric acid (GABA) is an vital neurotransmitter, and the reaction to obtain GABA through biocatalysis requires coenzymes, which are therefore limited in the production of GABA. In this study, polyacrylamide hydrogels doped with chitosan and waste toner were synthesized for glutamate decarboxylase (GAD) and coenzyme co-immobilization to realize the production of GABA and the recovery of coenzymes. Enzymatic properties of immobilized GAD were discussed. The immobilized enzymes have significantly improved pH and temperature tolerance compared to free enzymes. In terms of reusability, after 10 repeated reuses of the immobilized GAD, the residual enzyme activity of immobilized GAD still retains 100% of the initial enzyme activity, and the immobilized coenzyme can also be kept at about 32%, with better stability and reusability. And under the control of no exogenous pH, immobilized GAD showed good performance in producing GABA. Therefore, in many ways, the new composite hydrogel provides another way for the utilization of waste toner and promises the possibility of industrial production of GABA.

3D printed polylactide scaffolding for laccase immobilization to improve enzyme stability and estrogen removal from wastewater

This study reports a biocatalytic system of immobilized laccase and 3D printed open-structure biopolymer scaffoldings. The scaffoldings were computer-designed and 3D printed using polylactide (PLA) filament. The immobilization of laccase onto the 3D printed PLA scaffolds were optimized with regard to pH, enzyme concentration, and immobilization time. Laccase immobilization resulted in a small reduction in reactivity (in terms of Michaelis constant and maximum reaction rate) but led to significant improvement in chemical and thermal stability. After 20 days of storage, the immobilized and free laccase showed 80% and 35% retention of the initial enzymatic activity, respectively. The immobilized laccase on 3D printed PLA scaffolds achieved 10% improvement in the removal of estrogens from real wastewater as compared to free laccase and showed the significant reusability potential. Results here are promising but also highlight the need for further study to improve enzymatic activity and reusability.

Immobilized KDN Lipase on Macroporous Resin for Isopropyl Myristate Synthesis

Free enzymes often face economic problems because of their non-repeatability and variability, which limit their application in industrial production. In this study, KDN lipase was immobilized with the macroporous resin LXTE-1000 and glutaraldehyde. The optimal conditions of enzyme immobilization were defined by a single factor experiment and response surface methodology (RSM). The concentration of the cross-linking agent glutaraldehyde was 0.46% (v/v), the cross-linking temperature was 25.0 °C, and the cross-linking time was 157 min. The enzyme activity of the immobilized KDN lipase after adsorption/cross-linking was 291.36 U/g, and the recovery of the enzyme activity was 9.90%. The optimal conditions for the synthesis of isopropyl myristate were catalyzed by the immobilized KDN lipase in a solvent-free system: immobilized enzyme 53 mg, reaction temperature 36.1 °C, myristic acid 228.4 mg, isopropanol 114 µL, and reaction time 18 h. The yield of isopropyl myristate was 66.62%. After ten cycles, the activity of the immobilized KDN lipase preserved more than 46.87% of its initial enzyme activity, and it demonstrated high tolerance to solvents compared to free KDN lipase.

Immobilization of the crude enzyme extracted from Stenotrophomonas sp. GYH within modified zeolitic imidazolate framework (ZIF-8-NH2) and its application in trichloromethane removal

Enzyme immobilization, due to its higher effective density in a limited micro space, can effectively improve the removal efficiency for some recalcitrant compounds. Metal–organic frameworks (MOFs) have been identified as having attractive properties for the immobilization of enzymes, such as high surface area, large internal pore volumes and easily adjustable pore size. In the present study, a new immobilization carrier was synthesized through the modification of zeolitic imidazole framework-8 (ZIF-8) by 2-aminobenzimidazole, which was employed for enzymes immobilization for the first time. The immobilized bio-enzyme was extracted from trichloromethane (TCM) degrader Stenotrophomonas sp. GYH, and identified through label-free quantitative proteomics. Based on the metabolites detection and molecular docking, a proper degradation pathway for TCM was proposed, in which some key enzymes were tagged with the specific role. XRD, BET, and FTIR analyses proved that ZIF-8-NH2 was proper as the immobilization carrier. The bio-enzyme@ZIF-8-NH2 prepared under the best immobilized conditions had a similar relative enzyme activity to that of free enzyme (the Michaelis kinetic constant Km was 3.86 and 3.78 ​min−1), but it had better pH and temperature adaptability (pH 5−9 and temperature 10−50 ​°C), better storage stability (83% and 40% of the initial enzyme activity at 30 ​d) and so on. The density functional theory (DFT) results show that the ZIF-8-NH2 carrier has a better TCM and CO2 adsorption energy, which is consistent with the fact that the bio-enzyme@ZIF-8-NH2 had better TCM degradation efficiency and less CO2 emission to the surroundings.

Cloning, Expression and Characterization of an Alginate Lyase in Bacillus subtilis WB600

The aim of this study was to further broaden the heterologous expression of alginate lyase from Vibrio alginolyticus in a Bacillus subtilis expression vector. A B. subtilis WB600/pP43NMK-alg62 strain was constructed. (NH4)2SO4 precipitation and Ni-affinity chromatography were performed to purify the enzyme. We then characterized the enzyme. Its molecular weight was 57.64 kDa, and it worked optimally at 30 °C with a pH of 8.0. Ca2+ markedly enhanced the enzymatic activity of Alg62 while Cu2+ and Ni2+ inhibited its activity. Alg62 had a wide range of substrate specificity, showing high activity toward sodium alginate and polyG. Following optimization of the fermentation process, the optimal conditions for the recombinant expression of Alg62 were as follows: temperature of 37 °C, pH of 7.0, medium consisting of glycerol 15 g/L, yeast powder 25 g/L and K+ 1.5 mmol/L. At these optimal conditions, enzyme activity reached 318.21 U/mL, which was 1.54 times higher than the initial enzyme activity.

High Molecular Weight α-Galactosidase from the Novel Strain Aspergillus sp. D-23 and Its Hydrolysis Performance

Aspergillus sp. D-23 was obtained by ultraviolet-diethyl sulfate (UV-DES) compound mutagenesis from Aspergillus sp. C18 that the α-galactosidase was purified from. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and non-denaturing polyacrylamide gel electrophoresis (Native-PAGE), the purified enzyme demonstrated apparent homogeneity. The monomeric α-galactosidase’s native molecular weight was 125 kDa. The optimal temperature of α-galactosidase was 65 °C, and 75% of the initial enzyme activity could be maintained between 45 and 55 °C. Its optimal pH was 5.0 with good pH stability. After incubating for 2 h at pH 3.0–8.0, it could retain more than 80% of its original activity. Different concentrations of metal ions had different effects on the α-galactosidase activity. High concentrations of Cu2+ could strongly inhibit enzyme activity and low concentrations of Fe2+ could promote enzyme activity. Additionally, as shown by thin layer chromatography and high-performance liquid chromatography, the enzyme also had good hydrolysis ability, which could efficiently hydrolyze melibiose and raffinose by more than 95%. Therefore, these excellent characteristics could make α-galactosidase a good candidate for the food and feed industries.

Preparation and catalytic properties of catalase-inorganic hybrid nanoflowers

Catalase is widely used in the food, medical, and textile industries. It possesses exceptional properties including high catalytic efficiency, high specificity, and environmental friendliness. Free catalase cannot be recycled and reused in industry, resulting in a costly industrial biotransformation process if catalase is used as a core ingredient. Developing a simple, mild, cost-effective, and environmentally friendly approach to immobilize catalase is anticipated to improve its utilization efficiency and enzymatic performance. In this study, the catalase KatA derived from Bacillus subtilis 168 was expressed in Escherichia coli. Following separation and purification, the purified enzyme was prepared as an immobilized enzyme in the form of enzyme-inorganic hybrid nanoflowers, and the enzymatic properties were investigated. The results indicated that the purified KatA was obtained through a three-step procedure that included ethanol precipitation, DEAE anion exchange chromatography, and hydrophobic chromatography. Then, by optimizing the process parameters, a novel KatA/Ca3(PO4)2 hybrid nanoflower was developed. The optimum reaction temperature of the free KatA was determined to be 35 ℃, the optimum reaction temperature of KatA/Ca3(PO4)2 hybrid nanoflowers was 30-35 ℃, and the optimum reaction pH of both was 11.0. The free KatA and KatA/Ca3(PO4)2 hybrid nanoflowers exhibited excellent stability at pH 4.0-11.0 and 25-50 ℃. The KatA/Ca3(PO4)2 hybrid nanoflowers demonstrated increased storage stability than that of the free KatA, maintaining 82% of the original enzymatic activity after 14 d of storage at 4 ℃, whereas the free KatA has only 50% of the original enzymatic activity. In addition, after 5 catalytic reactions, the nanoflower still maintained 55% of its initial enzymatic activity, indicating that it has good operational stability. The Km of the free KatA to the substrate hydrogen peroxide was (8.80±0.42) mmol/L, and the kcat/Km was (13 151.53± 299.19) L/(mmol·s). The Km of the KatA/Ca3(PO4)2 hybrid nanoflowers was (32.75±2.96) mmol/L, and the kcat/Km was (4 550.67±107.51) L/(mmol·s). Compared to the free KatA, the affinity of KatA/Ca3(PO4)2 hybrid nanoflowers to the substrate hydrogen peroxide was decreased, and the catalytic efficiency was also decreased. In summary, this study developed KatA/Ca3(PO4)2 hybrid nanoflowers using Ca2+ as a self-assembly inducer, which enhanced the enzymatic properties and will facilitate the environmentally friendly preparation and widespread application of immobilized catalase.