Low-calorie d-allulose as a sucrose alternative modulates the physicochemical properties and volatile profile of sponge cake.

d-Allulose, a C-3 epimer of d-fructose, is a rare sugar with ∼70% of the sweetness of sucrose but a caloric content of only 0.4 kcal/g. Due to its low-calorie nature, d-allulose has garnered increasing interest in the food industry. This study was the first attempt to explore the effect of d-allulose as a sucrose replacer on the properties of sponge cake, a widely consumed high-sugar product. Substituting sucrose with d-allulose generated negligible impact on the batter system, while pronounced differences in physicochemical properties of cakes were detected, including specific volume, texture, microstructure, color, and antioxidant activity. In addition, sponge cake containing d-allulose displayed a distinctive aroma volatile profile, with more furans and pyrazines generation. Furthermore, correlations of physicochemical properties across all formulations were depicted, and the potential mechanism behind the property alterations modulated by d-allulose was revealed from the perspectives of starch gelatinization and browning reactions. Overall, this study provides insights into the application potential of d-allulose as a sucrose substitute in bakery product. PRACTICAL APPLICATION: This study elucidates the effect of d-allulose as a low-calorie sugar substitute on sponge cakes. This finding is valuable for the food industry, providing insights into a healthier alternative to traditional sugar in baked goods.

Biosynthesis of nonnutritive monosaccharide d-allulose by metabolically engineered Escherichia coli from nutritive disaccharide sucrose.

Sucrose is a commonly utilized nutritive sweetener in food and beverages due to its abundance in nature and low production costs. However, excessive intake of sucrose increases the risk of metabolic disorders, including diabetes and obesity. Therefore, there is a growing demand for the development of nonnutritive sweeteners with almost no calories. d-Allulose is an ultra-low-calorie, rare six-carbon monosaccharide with high sweetness, making it an ideal alternative to sucrose. In this study, we developed a cell factory for d-allulose production from sucrose using Escherichia coli JM109 (DE3) as a chassis host. The genes cscA, cscB, cscK, alsE, and a6PP were co-expressed for the construction of the synthesis pathway. Then, the introduction of ptsG-F and knockout of ptsG, fruA, ptsI, and ptsH to reprogram sugar transport pathways resulted in an improvement in substrate utilization. Next, the carbon fluxes of the Embden-Meyerhof-Parnas and the pentose phosphate pathways were regulated by the inactivation of pfkA and zwf, achieving an increase in d-allulose titer and yield of 154.2% and 161.1%, respectively. Finally, scaled-up fermentation was performed in a 5 L fermenter. The titer of d-allulose reached 11.15 g/L, with a yield of 0.208 g/g on sucrose.

Metabolic Profiling of Rat Kidney Tissue Following Administration of D-Allulose.

D-Allulose (D-psicose) is a rare sugar and a C-3 epimer of D-fructose. D-Allulose has been reported to have several health benefits via its alteration of both glucose and lipid metabolism. It was previously reported that D-allulose alters the hepatic metabolomic profile. Although the kidneys are crucial organs in metabolic regulation, the effects of D-allulose on renal metabolism have not yet been established. Therefore, this study was designed to capture the overall metabolic response in the kidneys to D-allulose. This was done by providing an AIN-93G diet to Wistar rats, with or without 3 % D-allulose, for four weeks. Renal tissue and blood samples were collected after a 3-hour fasting for evaluation of the renal metabolic profile and their related plasma parameters. D-Allulose increased renal weight without changes in the plasma indices associated with reduced renal function. Metabolic profiling identified a total of 264 peaks. As the contribution rate was too low in the principal component analysis results of the metabolic profiling results, we evaluated the metabolites that were significantly different between two groups and identified 23 up-regulated and 26 down-regulated metabolites in the D-allulose group. D-Allulose also had significant influence on several metabolites involved in glucose metabolism, amino acid metabolism, and purine metabolism. Moreover, the levels of trimethylamine N-oxide and symmetric dimethylarginine, which are associated with several diseases such as chronic kidney disease and cardiovascular disease decreased following D-allulose diets. This study showed that D-allulose affects the renal metabolic profile, and our findings will help elucidate the function of D-allulose.

Elucidation of d-allulose recognition mechanism in ketose 3-epimerase.

d-Allulose is a low-calorie sweetener with multiple nutritional functions that can be produced through d-fructose isomerization by ketose 3-epimerase (KEase). l-Ribulose 3-epimerase from Arthrobacterglobiformis (AgLRE) is one of the most important enzymes that produce d-allulose; however, its substrate recognition mechanism is unknown. In this study, the crystal structures of AgLRE and its complex with d-allulose and d-fructose were determined. Upon substrate binding, the hydrophobic residues around the active-site entrance move toward the bound substrate. A comparison of AgLRE and other KEase structures revealed that the substrate-binding residues are not the main factors responsible for its marked specificity for d-allulose and d-fructose, but the hydrophobicity of the active site pocket influences substrate recognition. Particularly, the two hydrophobic regions at the active site entrance are the regulatory elements that modulate substrate recognition by AgLRE. This study provides useful information for designing AgLRE to increase its affinity for d-allulose and d-fructose.

Effects of Dietary Allitol and D-Allulose on Body Fat Accumulation and Cecal Short-Chain Fatty Acid Production in Rats Fed a High-Fat Diet.

We investigated the effects of a single and simultaneous intake of allitol and d-allulose on body fat accumulation and cecal short-chain fatty acid (SCFA) production and accurately assessed the contribution of rare sugars to body fat in rats fed a high-fat diet that led to obesity. Thirty-two male 3-week-old Wistar rats were randomly divided into four groups: control, allitol, d-allulose, and allitol + d-allulose. The rats were fed experimental diets and water ad libitum for 11 weeks. High doses of allitol or d-allulose can induce diarrhea in rat; hence, each group of rats was acclimated to 1-5% allitol and d-allulose incrementally for the initial 20 days. After the feeding period, all rats were euthanized and collected tissues. Perirenal, mesenteric, and total intra-abdominal adipose tissue weights were significantly reduced by dietary d-allulose, whereas dietary allitol tended to decrease these adipose tissue weights. Both allitol and d-allulose significantly decreased carcass and total body fat mass. We confirmed that both dietary allitol and d-allulose inhibited body fat accumulation; however, d-allulose did not inhibit hepatic lipogenesis and no synergy was observed between dietary allitol and d-allulose in terms of anti-obesity effects. Dietary allitol significantly increased cecal SCFA levels and these effects were more potent than those of dietary d-allulose. The antiobesity effect of allitol may be due to the action of SCFAs, especially butyric acid, produced by the gut microbiota. Many of the effects of allitol as an alternative sweetener remain unknown, and further research is required.

Engineering Escherichia coli for D-allulose biosynthesis from glycerol.

D-allulose, a naturally occurring monosaccharide, is present in small quantities in nature. It is considered a valuable low-calorie sweetener due to its low absorption in the digestive tract and zero energy for growth. Most of the recent efforts to produce D-allulose have focused on in vitro enzyme catalysis. However, microbial fermentation is emerging as a promising alternative that offers the advantage of combining enzyme manufacturing and product synthesis within a single bioreactor. Here, a novel approach was proposed for the efficient biosynthesis of D-allulose from glycerol using metabolically engineered Escherichia coli. FbaA, Fbp, AlsE, and A6PP were used to construct the D-allulose synthesis pathway. Subsequently, PfkA, PfkB, and Pgi were disrupted to block the entry of the intermediate fructose-6-phosphate (F6P) into the Embden-Meyerhof-Parnas (EMP) and pentose phosphate (PP) pathways. Additionally, GalE and FryA were inactivated to reduce D-allulose consumption by the cells. Finally, a fed-batch fermentation process was implemented to optimize the performance of the cell factory. As a result, the titer of D-allulose reached 7.02 g/L with a maximum yield of 0.287 g/g.

Semi-rational engineering of D-allulose 3-epimerase for simultaneously improving the catalytic activity and thermostability based on D-allulose biosensor.

BACKGROUND: D-Allulose is one of the most well-known rare sugars widely used in food, cosmetics, and pharmaceutical industries. The most popular method for D-allulose production is the conversion from D-fructose catalyzed by D-allulose 3-epimerase (DAEase). To address the general problem of low catalytic efficiency and poor thermostability of wild-type DAEase, D-allulose biosensor was adopted in this study to develop a convenient and efficient method for high-throughput screening of DAEase variants. RESULTS: The catalytic activity and thermostability of DAEase from Caballeronia insecticola were simultaneously improved by semi-rational molecular modification. Compared with the wild-type enzyme, DAEaseS37N/F157Y variant exhibited 14.7% improvement in the catalytic activity and the half-time value (t1/2) at 65°C increased from 1.60 to 27.56 h by 17.23-fold. To our delight, the conversion rate of D-allulose was 33.6% from 500-g L-1 D-fructose in 1 h by Bacillus subtilis WB800 whole cells expressing this DAEase variant. Furthermore, the practicability of cell immobilization was evaluated and more than 80% relative activity of the immobilized cells was maintained from the second to seventh cycle. CONCLUSION: All these results indicated that the DAEaseS37N/F157Y variant would be a potential candidate for the industrial production of D-allulose.

D-allulose 3-epimerase for low-calorie D-allulose synthesis: microbial production, characterization, and applications.

D-allulose, an epimer of D-fructose at C-3 position, is a low-calorie rare sugar with favorable physiochemical properties and special physiological functions, which displays promising perspectives in the food and pharmaceutical industries. Currently, D-allulose is extremely sparse in nature and is predominantly biosynthesized through the isomerization of D-fructose by D-allulose 3-epimerase (DAEase). In recent years, D-allulose 3-epimerase as the key biocatalyst for D-allulose production has received increasing interest. The current review begins by providing a summary of D-allulose regarding its characteristics and applications, as well as different synthesis pathways dominated by biotransformation. Then, the research advances of D-allulose 3-epimerase are systematically reviewed, focusing on heterologous expression and biochemical characterization, crystal structure and molecular modification, and application in D-allulose production. Concerning the constraint of low yield of DAEase for industrial application, this review addresses the various attempts made to promote the production of DAEase in different expression systems. Also, various strategies have been adopted to improve its thermotolerance and catalytic activity, which is mainly based on the structure-function relationship of DAEase. The application of DAEase in D-allulose biosynthesis from D-fructose or low-cost feedstocks through single- or multi-enzymatic cascade reaction has been discussed. Finally, the prospects for related research of D-allulose 3-epimerase are also proposed, facilitating the industrialization of DAEase and more efficient and economical bioproduction of D-allulose.

Enhanced Biosynthesis of d-Allulose from a d-Xylose-Methanol Mixture and Its Self-Inductive Detoxification by Using Antisense RNAs in Escherichia coli.

d-Allulose, a C-3 epimer of d-fructose, has great market potential in food, healthcare, and medicine due to its excellent biochemical and physiological properties. Microbial fermentation for d-allulose production is being developed, which contributes to cost savings and environmental protection. A novel metabolic pathway for the biosynthesis of d-allulose from a d-xylose-methanol mixture has shown potential for industrial application. In this study, an artificial antisense RNA (asRNA) was introduced into engineered Escherichia coli to diminish the flow of pentose phosphate (PP) pathway, while the UDP-glucose-4-epimerase (GalE) was knocked out to prevent the synthesis of byproducts. As a result, the d-allulose yield on d-xylose was increased by 35.1%. Then, we designed a d-xylose-sensitive translation control system to regulate the expression of the formaldehyde detoxification operon (FrmRAB), achieving self-inductive detoxification by cells. Finally, fed-batch fermentation was carried out to improve the productivity of the cell factory. The d-allulose titer reached 98.6 mM, with a yield of 0.615 mM/mM on d-xylose and a productivity of 0.969 mM/h.

The Formation of D-Allulose 3-Epimerase Hybrid Nanoflowers and Co-Immobilization on Resins for Improved Enzyme Activity, Stability, and Processability.

As a low-calorie sugar, D-allulose is produced from D-fructose catalyzed by D-allulose 3-epimerase (DAE). Here, to improve the catalytic activity, stability, and processability of DAE, we reported a novel method by forming organic-inorganic hybrid nanoflowers (NF-DAEs) and co-immobilizing them on resins to form composites (Re-NF-DAEs). NF-DAEs were prepared by combining DAE with metal ions (Co2+, Cu2+, Zn2+, Ca2+, Ni2+, Fe2+, and Fe3+) in PBS buffer, and were analyzed by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, and X-ray diffraction. All of the NF-DAEs showed higher catalytic activities than free DAE, and the NF-DAE with Ni2+ (NF-DAE-Ni) reached the highest relative activity of 218%. The NF-DAEs improved the thermal stability of DAE, and the longest half-life reached 228 min for NF-DAE-Co compared with 105 min for the free DAE at 55 °C. To further improve the recycling performance of the NF-DAEs in practical applications, we combined resins and NF-DAEs to form Re-NF-DAEs. Resins and NF-DAEs co-effected the performance of the composites, and ReA (LXTE-606 neutral hydrophobic epoxy-based polypropylene macroreticular resins)-based composites (ReA-NF-DAEs) exhibited outstanding relative activities, thermal stabilities, storage stabilities, and processabilities. The ReA-NF-DAEs were able to be reused to catalyze the conversion from D-fructose to D-allulose, and kept more than 60% of their activities after eight cycles.

Effects of D-Allulose with Sucrose Beverage on Glucose Tolerance and Insulin Levels among Thai Healthy Volunteers.

D-Allulose has blood glucose suppression effects in both animal and clinical studies. The mechanism mediating glucose suppression in animals is controlled by several actions including the inhibition of sucrase. To investigate the dose-response effects of D-allulose with a sucrose beverage on glucose tolerance and insulin levels using Thai volunteers. This was a prospective, randomized, double-blinded, crossover study. Subjects had five oral sucrose tolerance tests (OSTT) with escalating doses of D-allulose (0, 2.5, 5, 7.5 or 10 g) with a 50 g sucrose beverage in a random order once a week for five consecutive weeks. The five drinks were consumed in a random order; the order being blinded for both subjects and investigators. Blood samples were drawn immediately before consumption and at 30, 60, 90 and 120 min after consumption of the study product for measurement of plasma glucose and insulin levels. Thirty healthy subjects (11 men and 19 women) completed the study. The peak postprandial glucose (PePPG) and insulin levels (PePPI) were lower when D-allulose was added in a dose-dependent manner. The lowest plasma glucose and insulin levels occurred at 120 min after OSTT in all five products and they were raised when D-allulose was added in a dose-dependent manner. D-Allulose has a suppression response on glucose and insulin shown by the decrease in postprandial plasma glucose and insulin levels following the addition of D-allulose to sucrose in a dose-dependent manner. The more D-allulose added, the less marked the glucose and insulin response occurred.

Designable immobilization of D-allulose 3-epimerase on bimetallic organic frameworks based on metal ion compatibility for enhanced D-allulose production.

D-allulose, a low-calorie rare sugar catalyzed by D-allulose 3-epimerase (DAE), is highly sought after for its potential health benefits. However, poor reusability and stability of DAE limited its popularization in industrial applications. Although metal-organic frameworks (MOFs) offer a promising enzyme platform for enzyme immobilization, developing customized strategies for MOF immobilization of enzymes remains challenging. In this study, we introduce a designable strategy involving the construction of bimetal-organic frameworks (ZnCo-MOF) based on metal ions compatibility. The DAE@MOFs materials were prepared and characterized, and the immobilization of DAE and the enzymatic characteristics of the MOF-immobilized DAE were subsequently evaluated. Remarkably, DAE@ZnCo-MOF exhibited superior recyclability which could maintain 95 % relative activity after 8 consecutive cycles. The storage stability is significantly improved compared to the free form, with a relative activity of 116 % remaining after 30 days. Molecular docking was also employed to investigate the interaction between DAE and the components of MOFs synthesis. The results demonstrate that the DAE@ZnCo-MOF exhibited enhanced catalytic efficiency and increased stability. This study introduces a viable and adaptable MOF-based immobilization strategy for enzymes, which holds the potential to expand the implementation of enzyme biocatalysts in a multitude of disciplines.

Enhancing the stability of a novel D-allulose 3-epimerase from Ruminococcus sp. CAG55 by interface interaction engineering and terminally attached a self-assembling peptide.

D-allulose, a highly desirable sugar substitute, is primarily produced using the D-allulose 3-epimerase (DAE). However, the availability of usable DAE enzymes is limited. In this study, we discovered and engineered a novel DAE Rum55, derived from a human gut bacterium Ruminococcus sp. CAG55. The activity of Rum55 was strictly dependent on the presence of Co2+, and it exhibited an equilibrium conversion rate of 30.6 % and a half-life of 4.5 h at 50 °C. To enhance its performance, we engineered the interface interaction of Rum55 to stabilize its tetramer structure, and the best variant E268R was then attached with a self-assembling peptide to form active enzyme aggregates as carrier-free immobilization. The half-life of the best variant E268R-EKL16 at 50 °C was dramatically increased 30-fold to 135.3 h, and it maintained 90 % of its activity after 13 consecutive reaction cycles. Additionally, we identified that metal ions played a key role in stabilizing the tetramer structure of Rum55, and the dependence on metal ions for E268R-EKL16 was significantly reduced. This study provides a useful route for improving the thermostability of DAEs, opening up new possibilities for the industrial production of D-allulose.

Characterization of a novel ribose-5-phosphate isomerase B from Curtobacterium flaccumfaciens ZXL1 for D-allose production.

Enzymatic preparation of rare sugars as an alternative to traditional sweeteners is an effective strategy to achieve a low-calorie healthy diet. Ribose-5-phosphate isomerase B (RpiB) is a key enzyme in the non-oxidative branch of the catalytic pentose phosphate pathway. Here, we investigated the potential of Curtobacterium flaccumfaciens ZXL1 (C. flaccumfaciens ZXL1) derived RpiB (CfRpiB) in D-allose preparation. The optimal reaction conditions for recombinant CfRpiB were found experimentally to be pH 7.0, 55 °C, and no metal ions. The kinetic parameters Km, kcat, and catalytic efficiency kcat/Km were 320 mM, 4769 s-1, and 14.9 mM-1 s-1 respectively. The conversion of D-allulose by purified enzyme (1 g L-1 ) to D-allose was 13% within 1 h. In addition, homology modeling and molecular docking were used to predict the active site residues: Asp13, Asp14, Cys72, Gly73, Thr74, Gly77, Asn106, and Lys144.

Identification of hyperthermophilic D-allulose 3-epimerase from Thermotoga sp. and its application as a high-performance biocatalyst for D-allulose synthesis.

D-Allulose 3-epimerase (DAE) is a vital biocatalyst for the industrial synthesis of D-allulose, an ultra-low calorie rare sugar. However, limited thermostability of DAEs hinders their use at high-temperature production. In this research, hyperthermophilic TI-DAE (Tm = 98.4 ± 0.7 ℃) from Thermotoga sp. was identified via in silico screening. A comparative study of the structure and function of site-directed saturation mutagenesis mutants pinpointed the residue I100 as pivotal in maintaining the high-temperature activity and thermostability of TI-DAE. Employing TI-DAE as a biocatalyst, D-allulose was produced from D-fructose with a conversion rate of 32.5%. Moreover, TI-DAE demonstrated excellent catalytic synergy with glucose isomerase CAGI, enabling the one-step conversion of D-glucose to D-allulose with a conversion rate of 21.6%. This study offers a promising resource for the enzyme engineering of DAEs and a high-performance biocatalyst for industrial D-allulose production.

Rational design improves both thermostability and activity of a new D-tagatose 3-epimerase from Kroppenstedtia eburnean to produce D-allulose.

D-allulose is a naturally occurring rare sugar and beneficial to human health. However, the efficient biosynthesis of D-allulose remains a challenge. Here, we mined a new D-tagatose 3-epimerase from Kroppenstedtia eburnean (KeDt3e) with high catalytic efficiency. Initially, crucial factors contributing to the low conversion of KeDt3e were identified through crystal structure analysis, density functional theory calculations (DFT), and molecular dynamics (MD) simulations. Subsequently, based on the mechanism, combining restructuring the flexible region, proline substitution based onconsensus sequence analysis, introducing disulfide bonds, and grafting properties, and reshaping the active center, the optimal mutant M5 of KeDt3e was obtained with enhanced thermostability and activity. The optimal mutant M5 exhibited an enzyme activity of 130.8 U/mg, representing a 1.2-fold increase; Tm value increased from 52.7 °C to 71.2 °C; and half-life at 55 °C extended to 273.7 min, representing a 58.2-fold improvement, and the detailed mechanism of performance improvement was analyzed. Finally, by screening the ribosome-binding site (RBS) of the optimal mutant M5 recombinant bacterium (G01), the engineered strain G08 with higher expression levels was obtained. The engineered strain G08 catalyzed 500 g/L D-fructose to produce 172.4 g/L D-allulose, with a conversion of 34.4% in 0.5 h and productivity of 344.8 g/L/h on a 1 L scale. This study presents a promising approach for industrial-scale production of D-allulose.

D-Allulose Reduces Hypertrophy and Endoplasmic Reticulum Stress Induced by Palmitic Acid in Murine 3T3-L1 Adipocytes.

Natural rare sugars are an alternative category of sweeteners with positive physiologic and metabolic effects both in in vitro and animal models. D-allulose is a D-fructose epimer that combines 70% sucrose sweetness with the advantage of an extremely low energy content. However, there are no data about the effect of D-allulose against adipose dysfunction; thus, it remains to be confirmed whether D-allulose is useful in the prevention and in treatment of adipose tissue alterations. With this aim, we evaluated D-allulose's preventive effects on lipid accumulation in 3T3-L1 murine adipocytes exposed to palmitic acid (PA), a trigger for hypertrophic adipocytes. D-allulose in place of glucose prevented adipocyte hypertrophy and the activation of adipogenic markers C/EBP-ß and PPARγ induced by high PA concentrations. Additionally, D-allulose pretreatment inhibited the NF-κB pathway and endoplasmic reticulum stress caused by PA, through activation of the Nrf2 pathway. Interestingly, these effects were also observed as D-allulose post PA treatment. Although our data need to be confirmed through in vivo models, our findings suggest that incorporating D-allulose as a glucose substitute in the diet might have a protective role in adipocyte function and support a unique mechanism of action in this sugar as a preventive or therapeutic compound against PA lipotoxicity through the modulation of pathways connected to lipid transport and metabolism.

Enhancement of the d-Allulose 3-Epimerase Expression in Bacillus subtilis through Both Transcriptional and Translational Regulations.

d-Allulose, a functional bulk sweetener, has recently attracted increasing attention because of its low-caloric-ness properties and diverse health effects. d-Allulose is industrially produced by the enzymatic epimerization of d-fructose, which is catalyzed by ketose 3-epimerase (KEase). In this study, the food-grade expression of KEase was studied using Bacillus subtills as the host. Clostridium sp. d-allulose 3-epimerase (Clsp-DAEase) was screened from nine d-allulose-producing KEases, showing better potential for expression in B. subtills WB600. Promoter-based transcriptional regulation and N-terminal coding sequence (NCS)-based translational regulation were studied to enhance the DAEase expression level. In addition, the synergistic effect of promoter and NCS on the Clsp-DAEase expression was studied. Finally, the strain with the combination of a PHapII promoter and gln A-Up NCS was selected as the best Clsp-DAEase-producing strain. It efficiently produced Clsp-DAEase with a total activity of 333.2 and 1860.6 U/mL by shake-flask and fed-batch cultivations, respectively.

Safety evaluation of the food enzyme preparation D-psicose 3-epimerase from the non-genetically modified Microbacterium foliorum strain SYG27B.

This assessment addresses a food enzyme preparation consisting of the immobilised non-viable cells of the non-genetically modified bacterium identified by the applicant (Samyang Corporation) as Microbacterium foliorum strain SYG27B. This strain produces the enzyme D-psicose 3-epimerase (EC 5.1.3.30). The food enzyme preparation is used for the isomerisation of fructose to produce the speciality carbohydrate D-allulose (synonym D-psicose). Since the hazard identification and characterisation could not be made and the identity of the production organism could not be established, the Panel was unable to complete the assessment of this food enzyme preparation containing D-psicose 3-epimerase.

Simultaneous Enhancement of the Thermostability and Catalytic Activity of D-Allulose 3-Epimerase from Clostridium bolteae ATTC BAA-613 Based on the "Back to Consensus Mutations" Hypothesis.

D-Allulose is a high value rare sugar with multiple physiological functions and commercial potential that can be enzymatically synthesized from D-fructose by D-allulose 3-epimerase (DAEase). Poor catalytic activity and thermostability of DAEase prevent the industrial production of D-allulose. In this work, rational design was applied to a previously identified DAEase from Clostridium bolteae ATCC BAA-613 based on the "back to consensus mutations" hypothesis, and the catalytic activity of the Cb-I265 V variant was enhanced 2.5-fold. Furthermore, the Cb-I265 V/E268D double-site variant displayed 2.0-fold higher specific catalytic activity and 1.4-fold higher thermostability than the wild-type enzyme. Molecular docking and kinetic simulation results indicated increased hydrogen bonds between the active pocket and substrate, possibly contributing to the improved thermal stability and catalytic activity of the double-site mutant. The findings outlined a feasible approach for the rational design of multiple preset functions of target enzymes simultaneously.