Semi-rational design based on the interaction between SmFMO and FAD isoalloxazine ring to enhance the enzyme activity.

Flavin monooxygenases (FMOs) have been widely used in the biosynthesis of natural compounds due to their excellent stereoselectivity, regioselectivity and chemoselectivity. Stenotrophomonas maltophilia flavin monooxygenase (SmFMO) has been reported to catalyze the oxidation of various thiols to corresponding sulfoxides, but its activity is relatively low. Herein, we obtained a mutant SmFMOF52G which showed 4.35-fold increase in kcat/Km (4.96 mM-1s-1) and 6.84-fold increase in enzyme activity (81.76 U/g) compared to the SmFMOWT (1.14 mM-1s-1 and 11.95 U/g) through semi-rational design guided by structural analysis and catalytic mechanism combined with high-throughput screening. By forming hydrogen bond with O4 atom of FAD isoalloxazine ring and reducing steric hindrance, the conformation of FAD isoalloxazine ring in SmFMOF52G is more stable, and NADPH and substrate are closer to FAD isoalloxazine ring, shortening the distances of hydrogen transfer and substrate oxygenation, thereby increasing the rate of reduction and oxidation reactions and enhancing enzyme activity. Additionally, the overall structural stability and substrate binding capacity of the SmFMOF52G have significant improved than that of SmFMOWT. The strategy used in this study to improve the enzyme activity of FMOs may have generality, providing important references for the rational and semi-rational engineering of FMOs.

Computer-assisted semi-rational design enhanced the enzymatic activity and protein stability of Proteinase K in calcium-free conditions.

Wild-type Proteinase K binds to two Ca2+ ions, which play an important role in regulating enzymaticactivity and maintaining protein stability. Therefore, a predetermined concentration of Ca2+ must be added during the use of Proteinase K, which increases its commercial cost. Herein, we addressed this challenge using a computational strategy to engineer a Proteinase K mutant that does not require Ca2+ and exhibits high enzymatic activity and protein stability. In the absence of Ca2+, the best mutant, MT24 (S17W-S176N-D260F), displayed an activity approximately 9.2-fold higher than that of wild-type Proteinase K. It also exhibited excellent protein stability, retaining 56.2 % of its enzymatic activity after storage at 4 °C for 5 days. The residual enzymatic activity was 65-fold higher than that of the wild-type Proteinase K under the same storage conditions. Structural analysis and molecular dynamics simulations suggest that the introduction of new hydrogen bond and π-π stacking at the Ca2+ binding sites due to the mutation may be the reasons for the increased enzymatic activity and stability of MT24.

Semi-rational design of nitroarene dioxygenase for catalytic ability toward 2,4-dichloronitrobenzene.

Rieske non-heme dioxygenase family enzymes play an important role in the aerobic biodegradation of nitroaromatic pollutants, but no active dioxygenases are available in nature for initial reactions in the degradation of many refractory pollutants like 2,4-dichloronitrobenzene (24DCNB). Here, we report the engineering of hotspots in 2,3-dichloronitrobenzene dioxygenase from Diaphorobacter sp. strain JS3051, achieved through molecular dynamic simulation analysis and site-directed mutagenesis, with the aim of enhancing its catalytic activity toward 24DCNB. The computationally predicted activity scores were largely consistent with the detected activities in wet experiments. Among them, the two most beneficial mutations (E204M and M248I) were obtained, and the combined mutant reached up to a 62-fold increase in activity toward 24DCNB, generating a single product, 3,5-dichlorocatechol, which is a naturally occurring compound. In silico analysis confirmed that residue 204 affected the substrate preference for meta-substituted nitroarenes, while residue 248 may influence substrate preference by interaction with residue 295. Overall, this study provides a framework for manipulating nitroarene dioxygenases using computational methods to address various nitroarene contamination problems.IMPORTANCEAs a result of human activities, various nitroaromatic pollutants continue to enter the biosphere with poor degradability, and dioxygenation is an important kickoff step to remove toxic nitro-groups and convert them into degradable products. The biodegradation of many nitroarenes has been reported over the decades; however, many others still lack corresponding enzymes to initiate their degradation. Although rieske non-heme dioxygenase family enzymes play extraordinarily important roles in the aerobic biodegradation of various nitroaromatic pollutants, prediction of their substrate specificity is difficult. This work greatly improved the catalytic activity of dioxygenase against 2,4-dichloronitrobenzene by computer-aided semi-rational design, paving a new way for the evolution strategy of nitroarene dioxygenase. This study highlights the potential for using enzyme structure-function information with computational pre-screening methods to rapidly tailor the catalytic functions of enzymes toward poorly biodegradable contaminants.

Substrate access tunnel engineering of a Fe-type nitrile hydratase from Pseudomonas fluorescens ZJUT001 for substrate preference adjustment and catalytic performance enhancement.

Substrate access tunnel engineering is a useful strategy for enzyme modification. In this study, we improved the catalytic performance of Fe-type Nitrile hydratase (Fe-type NHase) from Pseudomonas fluorescens ZJUT001 (PfNHase) by mutating residue Q86 at the entrance of the substrate access tunnel. The catalytic activity of the mutant PfNHase-αQ86W towards benzonitrile, 2-cyanopyridine, 3-cyanopyridine, and 4-hydroxybenzonitrile was enhanced by 9.35-, 3.30-, 6.55-, and 2.71-fold, respectively, compared to that of the wild-type PfNHase (PfNHase-WT). In addition, the mutant PfNHase-αQ86W showed a catalytic efficiency (kcat/Km) towards benzonitrile 17.32-fold higher than the PfNHase-WT. Interestingly, the substrate preference of PfNHase-αQ86W shifted from aliphatic nitriles to aromatic nitrile substrates. Our analysis delved into the structural changes that led to this altered substrate preference, highlighting an expanded entrance tunnel region, theenlarged substrate-binding pocket, and the increased hydrophobic interactions between the substrate and enzyme. Molecular dynamic simulations and dynamic cross-correlation Matrix (DCCM) further supported these findings, providing a comprehensive explanation for the enhanced catalytic activity towards aromatic nitrile substrates.

Enhancing the imidase activity of BpIH toward 3-isobutyl glutarimide via semi-rational design.

(R)-3-Isobutylglutarate monoamide (R-IBM) is a key intermediate in the synthesis of the analgesic drug pregabalin. Recently, the imidase BpIH derived from Burkholderia phytofirmans was identified as a promising catalyst for the industrial production of R-IBM. Notably, this catalyst has the distinct advantage of achieving a 100% theoretical yield from 3-isobutyl glutarimide (IBI). In this study, homology modeling and structure alignment techniques were used to determine the substrate binding pocket of BpIH. Semi-rational design was used to analyze the amino acid residue conservation in the binding pocket region of BpIH. Interestingly, mutations of several low-conserved amino acid located 6-9 Å from the substrate significantly enhanced the catalytic activity of BpIH. Among them, the triple mutant Y37FH133NS226I (YHS-I) showed approximately a fivefold increase in enzyme activity and a significantly improved catalytic efficiency (kcat/Km). Under the same reaction time and conditions, YHS-I successfully converted IBI into R-IBM with a conversion rate of 88.87%, with an enantiomeric excess (ee) of the product exceeding 99.9%. In comparison, wild-type BpIH had a conversion rate of only 38.15%. Molecular dynamics and docking results indicated that YHS-I had higher rigidity around the mutation sites. The synergistic substitutions of Y37F, H133N, and S226I altered the interaction network within the mutation site, enhancing the protein's affinity for the substrate and improving catalytic efficiency. KEY POINTS: • 100% theoretical yield of R-IBM by BpIH compared with 50% by resolution • Semi-rational design of BpIH based on conservativity with homologous enzymes • Mutant with enzyme activity of sixfold and product ee value of 99.9.

Chemoenzymatic Synthesis of Selegiline: An Imine Reductase-Catalyzed Approach.

(R)-Homobenzylic amines are key structural motifs present in (R)-selegiline, a drug indicated for the treatment of early-stage Parkinson's disease. Herein, we report a new short chemoenzymatic approach (in 2 steps) towards the synthesis of (R)-selegiline via stereoselective biocatalytic reductive amination as the key step. The imine reductase IR36-M5 mutant showed high conversion (97%) and stereoselectivity (97%) toward the phenylacetone and propargyl amine substrates, offering valuable biocatalysts for synthesizing alkylated homobenzylic amines.

Efficient 2,3-Butanediol Production from Ethanol by a Modified Four-Enzyme Synthetic Biosystem.

2,3-butanediol (2,3-BD) is a versatile bio-based platform chemical. An artificial four-enzyme synthetic biosystem composed of ethanol dehydrogenase, NADH oxidase, formolase and 2,3-butanediol dehydrogenase was designed for upgrading ethanol to 2,3-BD in our previous study. However, a key challenge in developing in vitro enzymatic systems for 2,3-BD synthesis is the relatively sluggish catalytic efficiency of formolase, which catalyzes the rate-limiting step in such systems. Herein, this study reports how engineering the tunnel and substrate binding pocket of FLS improved its catalytic performance. A series of single-point and combinatorial variants were successfully obtained which displayed both higher catalytic efficiency and better substrate tolerance than wild-type FLS. Subsequently, a cell-free biosystem based on the FLS:I28V/L482E enzyme was implemented for upgrading ethanol to 2,3-BD. Ultimately, this system achieved efficient production of 2,3-BD from ethanol by the fed-batch method, reaching a concentration of 1.39 M (124.83 g/L) of the product and providing both excellent productivity and yield values of 5.94 g/L/h and 92.7%, respectively. Taken together, this modified enzymatic catalysis system provides a highly promising alternative approach for sustainable and cost-competitive production of 2,3-BD.

Engineering Glucose Dehydrogenase to Favor Totally Synthetic Biomimetic Cofactors Containing Carboxyl Group.

The utilization of unnatural nicotinamide cofactors for reactions catalyzed by oxidoreductases has gained increasing interest. Totally synthetic nicotinamide cofactor biomimetics (NCBs) are cost-effective and convenient to synthesize. Thus, it has become increasingly important to develop enzymes that accept NCBs. Here, we have engineered SsGDH to favor a newly synthesized unnatural cofactor 3-carbamoyl-1-(4-carboxybenzyl) pyridin-1-ium (BANA+ ). Using in situ ligand minimization tool, sites 44 and 114 were identified as hotspots for mutagenesis. All the double mutants demonstrated 2.7-7.7-fold improvements in catalytic activity, and the best double mutant E44D/E114 L exhibited 10.6-fold increased catalytic efficiency toward BANA+ . These results provide valuable information for the rational engineering of oxidoreductases with versatile NCBs-dependency, as well as the design of novel biomimetic cofactors.

Tailoring Galactose Oxidase for Self-Powered Benzyl Alcohol Sensing.

Benzyl alcohol (BnOH) is a widely-used preservative in a variety of cosmetics, but the excess addition (≥1.0 %) may cause strong symptoms such as nausea, gastrointestinal irritation, convulsion, even death, making it crucial to monitor and control the addition quantity. Herein, we have developed a test-strip-like BnOH detection method via tailoring a galactose oxidase (GOase) towards BnOH oxidation and preparing a self-powered electrochromic strip for BnOH concentration visualization. A double-substituted GOase variant (Y329S/R330F), on the basis of the reported GOase M1 , has been obtained by semi-rational design with a 24.6-fold improved activity towards BnOH compared to GOase M1 . The GOase Y329S/R330F electrode has a response to BnOH with a linear range of 0.04 to 3.25 mM (R2 =0.9985), a sensitivity of 122.78 µA mM-1 cm-2 , and a detection limit of 0.03 mM (S/N=3). Coupling an electrochromic Prussian blue (PB) cathode helps the successful sensing visualization without any further power supply. The present sensing is more convenient and user-friendly than the generally used gas chromatography (GC) and high performance liquid chromatography (HPLC), and brings a more accessible solution to the field of quality controlling.

A high-throughput expression and screening platform for applications-driven PETase engineering.

The environmental consequences of plastic waste have impacted all kingdoms of life in terrestrial and aquatic ecosystems. However, as the burden of plastic pollution has increased, microbes have evolved to utilize anthropogenic polymers as nutrient sources. Of depolymerase enzymes, the best characterized is PETase, which hydrolyzes aromatic polyesters. PETase engineering has made impressive progress in recent years; however, further optimization of engineered PETase toward industrial application has been limited by lower throughput techniques used in protein purification and activity detection. Here, we address these deficiencies through development of a higher-throughput PETase engineering platform. Secretory expression via YebF tagging eliminates lysis and purification steps, facilitating production of large mutant libraries. Fluorescent detection of degradation products permits rapid screening of depolymerase activity in microplates as opposed to serial chromatographic methods. This approach enabled development of more stable PETase, semi-rational (SR) PETase variant containing previously unpublished mutations. SR-PETase releases 1.9-fold more degradation products and has up to 7.4-fold higher activity than wild-type PETase over 10 days at 40°C. These methods can be adapted to a variety of chemical environments, enabling screening of PETase mutants in applications-relevant conditions. Overall, this work promises to facilitate advancements in PETase engineering toward industrial depolymerization of plastic waste.

Regio- and stereo-selective 1ß-hydroxylation of lithocholic acid by cytochrome P450 BM3 mutants.

Regio- and stereo-selective hydroxylation of bile acids is a valuable reaction but often lacks suitable catalysts. In the research, semi-rational design in protein engineering techniques had been applied on cytochrome P450 monooxygenase CYP102A1 (P450 BM3) from Bacillus megaterium, and a mutation library had been set up for the 1ß-hydroxylation of lithocholic acid (LCA) to produce 1ß-OH-LCA. After four rounds of mutagenesis, a key residue at W72 was identified to regulate the regio- and stereo-selectivity at C1 of LCA. A quadruple variant (G87A/W72T/A74L/L181M) was identified to reach 99.4% selectivity of 1ß-hydroxylation and substrate conversion of 68.1% resulting in a 21.5-fold higher level of 1ß-OH-LCA production than the template LG-23. Molecular docking indicated that introducing hydrogen bonds at W72 was responsible for enhancing selectivity and catalytic activity, which gave some insights into the structure-based understanding of Csp3 -H activation by the developed P450 BM3 mutants.

Modification of the 4-Hydroxyphenylacetate-3-hydroxylase Substrate Pocket to Increase Activity towards Resveratrol.

4-Hydroxyphenylacetate-3-hydroxylase (4HPA3H; EC 1.14.14.9) is a heterodimeric flavin-dependent monooxygenase complex that catalyzes the ortho-hydroxylation of resveratrol to produce piceatannol. Piceatannol has various health benefits and valuable applications in food, medicine, and cosmetics. Enhancing the catalytic activity of 4HPA3H toward resveratrol has the potential to benefit piceatannol production. In this study, the critical amino acid residues in the substrate pocket of 4HPA3H that affect its activity toward resveratrol were identified using semi-rational engineering. Two key amino acid sites (I157 and A211) were discovered and the simultaneous "best" mutant I157L/A211D enabled catalytic efficiency (Kcat/Km-resveratrol) to increase by a factor of 4.7-fold. Molecular dynamics simulations indicated that the increased flexibility of the 4HPA3H substrate pocket has the potential to improve the catalytic activity of the enzyme toward resveratrol. On this basis, we produced 3.78 mM piceatannol by using the mutant I157L/A211D whole cells. In this study, we successfully developed a highly active 4HPA3H variant for the hydroxylation of resveratrol to piceatannol.

Efficient synthesis of 2'-deoxyguanosine in one-pot cascade by employing an engineered purine nucleoside phosphorylase from Brevibacterium acetylicum.

2'-deoxyguanosine is a key medicinal intermediate that could be used to synthesize anti-cancer drug and biomarker in type 2 diabetes. In this study, an enzymatic cascade using thymidine phosphorylase from Escherichia coli (EcTP) and purine nucleoside phosphorylase from Brevibacterium acetylicum (BaPNP) in a one-pot whole cell catalysis was proposed for the efficient synthesis of 2'-deoxyguanosine. BaPNP was semi-rationally designed to improve its activity, yielding the best triple variant BaPNP-Mu3 (E57A/T189S/L243I), with a 5.6-fold higher production of 2'-deoxyguanosine than that of wild-type BaPNP (BaPNP-Mu0). Molecular dynamics simulation revealed that the engineering of BaPNP-Mu3 resulted in a larger and more flexible substrate entrance channel, which might contribute to its catalytic efficiency. Furthermore, by coordinating the expression of BaPNP-Mu3 and EcTP, a robust whole cell catalyst W05 was created, capable of producing 14.8 mM 2'-deoxyguanosine (74.0% conversion rate) with a high time-space yield (1.32 g/L/h) and therefore being very competitive for industrial applications.

Regulating substrate preference of phosphatase by reshaping the "cap domain" for multi-enzymatic biosynthesis of high-purity monosaccharides.

Phosphatases are a class of enzymes catalyzing the cleavage of monophosphate ester bonds from the phosphorylated substrates. They have important applications in construction of in vitro multi-enzymatic system for monosaccharides. However, the enzymes generally show substrate ambiguity, which has become a bottleneck for efficient biosynthesis of target products with high purity. In this study, semirational design was performed on phosphatase from Thermosipho atlanticus (Ta-PST). The hotspot amino acid residues forming a "cap domain" were identified and selected for saturation mutagenesis. The mutant F179T and F179M showed improved substrate preference toward fructose-6-phosphate and mannose-6-phosphate, respectively. Coupling with other enzymes involved in the multi-enzymatic system under optimized conditions, the application of F179T led to fructose yield of 80% from 10 g/L maltodextrin and the ratio between the target product and by-product glucose was increased from 2:1 to 19:1. On the other hand, the application of F179M led to mannose yield of 59% with ratio of mannose to the by-products glucose and fructose increased from 1:1:1 to 14:2:1. Moreover, the molecular understanding of the beneficial substitution was gained by structural analysis and molecular dynamic simulations, giving important guidance to regulate the enzyme's substrate preference.

Thermostability engineering of industrial enzymes through structure modification.

Thermostability is an essential requirement of enzymes in the industrial processes to catalyze the reactions at high temperatures; thus, enzyme engineering through directed evolution, semi-rational design and rational design are commonly employed to construct desired thermostable mutants. Several strategies are implemented to fulfill enzymes' thermostability demand including decreasing the entropy of the unfolded state through substitutions Gly → Xxx or Xxx → Pro, hydrogen bond, salt bridge, introducing two different simultaneous interactions through single mutant, hydrophobic interaction, filling the hydrophobic cavity core, decreasing surface hydrophobicity, truncating loop, aromatic-aromatic interaction and introducing positively charged residues to enzyme surface. In the current review, horizons about compatibility between secondary structures and substitutions at preferable structural positions to generate the most desirable thermostability in industrial enzymes are broadened. KEY POINTS: • Protein engineering is a powerful tool for generating thermostable industrial enzymes. • Directed evolution and rational design are practical approaches in enzyme engineering. • Substitutions in preferable structural positions can increase thermostability.

Enantioselectivity and key residue of Herbaspirillum huttiense monooxygenase in asymmetric epoxidation of styrenes.

Styrene monooxygenases (SMOs) are powerful enzymes for the synthesis of enantiopure epoxides, but these SMOs have narrow substrate spectra, and the residues in controlling enantioselectivity of SMOs remains unclear. A monooxygenase from Herbaspirillum huttiense (HhMO) was found to have excellent enantioselectivities and diastereoselectivities in the epoxidation of unconjugated terminal alkenes. Here we found that HhMO could also transfer styrene into styrene epoxide with 75% ee, and it could also catalyze the epoxidation of styrene derivatives into the corresponding epoxides with enantioselectivities up to 99% ee. Meanwhile, site 199 in the substrate access channel of HhMO was found to play an important role in the controlling enantioselectivity of the epoxidation. The E199L variant catalyzed the epoxidation of styrene with > 99% ee. The identification of critical residue that affects the enantioselectivity of SMOs would thus be valuable for creating efficient monooxygenases for the preparation of essential enantiopure epoxides. KEY POINTS: • Bioexpoxidation of both conjugated and unconjugated alkenes by HhMO with excellent enantioselectivities. • Gating residue 199 played an essential role in controlling the enantioselectivity of SMO. • HhMO E199L catalyzed the epoxidation of styrenes with up to > 99% ee.

Enzyme engineering and its industrial applications.

Recently, there has been an increase in the demand for enzymes with modified activity, specificity, and stability. Enzyme engineering is an important tool to meet the demand for enzymes adjusted to different industrial processes. Knowledge of the structure and function of enzymes guides the choice of the best strategy for engineering enzymes. Each enzyme engineering strategy, such as rational design, directed evolution, and semi-rational design, has specific applications, as well as limitations, which must be considered when choosing a suitable strategy. Engineered enzymes can be optimized for different industrial applications by choosing the appropriate strategy. This review features engineered enzymes that have been applied in food, animal feed, pharmaceuticals, medical applications, bioremediation, biofuels, and detergents.

Substrate access tunnel engineering for improving the catalytic activity of a thermophilic nitrile hydratase toward pyridine and pyrazine nitriles.

Nitrile hydratase (NHase) is able to bio-transform nitriles into amides. As nitrile hydration being an exothermic reaction, a NHase with high activity and stability is needed for amide production. However, the widespread use of NHase for amide bio-production is limited by an activity-stability trade-off. In this study, through the combination of substrate access tunnel calculation, residue conservative analysis and site-saturation mutagenesis, a residue located at the substrate access tunnel entrance of the thermophilic NHase from extremophile Caldalkalibacillus thermarum TA2. A1, ßLeu48, was semi-rationally identified as a potential gating residue that directs the enzymatic activity toward various pyridine and pyrazine nitriles. The specific activity of the corresponding mutant ßL48H towards 3-cyanopyridine, 2-cyanopyridine and cyanopyrazine were 2.4-fold, 2.8-fold and 3.1-fold higher than that of its parent enzyme, showing a great potential in the industrial production of high-value pyridine and pyrazine carboxamides. Further structural analysis demonstrated that the ßHis48 could form a long-lasting hydrogen bond with αGlu166, which contributes to the expansion of the entrance of substrate access tunnel and accelerate substrate migration.

Semi-rational design of L-amino acid deaminase for production of pyruvate and D-alanine by Escherichia coli whole-cell biocatalyst.

In our previous study, one-step pyruvate and D-alanine production from D,L-alanine by a whole-cell biocatalyst Escherichia coli expressing L-amino acid deaminase (Pm1) derived from Proteus mirabilis was investigated. However, due to the low catalytic efficiency of Pm1, the pyruvate titer was relatively low. Here, semi-rational design based on site-directed saturation mutagenesis was carried out to improve the catalytic efficiency of Pm1. A novel high-throughput screening (HTS) method for pyruvate based on 2,4-dinitrophenylhydrazine indicator was then established. The catalytic efficiency (kcat/Km) of the mutant V437I screened out by this method was 1.88 times higher than wild type. Next, to improve the growth of the engineered strain BLK07, the genes encoding for Xpk and Fbp were integrated into its genome to construct non-oxidative glycolysis (NOG) pathway. Finally, the CRISPR/Cas9 system was used to integrate the N6-pm1-V437I gene into the genome of BLK07. Pyruvic acid titer of the plasmid-free strain reached 42.20 g/L with an L-alanine conversion rate of 77.62% and a D-alanine resolution of 82.4%. This work would accelerate the industrial production of pyruvate and D-alanine by biocatalysis, and the HTS method established here could be used to screen other Pm1 mutants with high pyruvate titers.

Development of thermostable sucrose phosphorylase by semi-rational design for efficient biosynthesis of alpha-D-glucosylglycerol.

Sucrose phosphorylase (SPase) can specifically catalyze transglycosylation reactions and can be used to enzymatically synthesize α-D-glycosides. However, the low thermostability of SPase has been a bottleneck for its industrial application. In this study, a SPase gene from Leuconostoc mesenteroides ATCC 12,291 (LmSPase) was synthesized with optimized codons and overexpressed successfully in Escherichia coli. A semi-rational design strategy that combined the FireProt (a web server designing thermostable proteins), structure-function analysis, and molecular dynamic simulations was used to improve the thermostability of LmSPase. Finally, one single-point mutation T219L and a combination mutation I31F/T219L/T263L/S360A (Mut4) with improved thermostability were obtained. The half-lives at 50 °C of T219L and Mut4 both increased approximately two-fold compared to that of wild-type LmSPase (WT). Furthermore, the two variants T219L and Mut4 were used to produce α-D-glucosylglycerol (αGG) from sucrose and glycerol by incubating with 40 U/mL crude extracts at 37 °C for 60 h and achieved the product concentration of 193.2 ± 12.9 g/L and 195.8 ± 13.1 g/L, respectively, which were approximately 1.3-fold higher than that of WT (150.4 ± 10.0 g/L). This study provides an effective strategy for improving the thermostability of an industrial enzyme. KEY POINTS: • Predicted potential hotspot residues directing the thermostability of LmSPase by semi-rational design • Screened two positive variants with higher thermostability and higher activity • Synthesized α-D-glucosylglycerol to a high level by two screened positive variants.