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In this study, we found the addition of sorbitol could improve the thermostability of α‐l‐rhamnosidase from Aspergillus niger. When α‐l‐rhamnosidase with sorbitol was heat‐treated at 60°C, 65°C, and 70°C, the half‐life t1/2 increased by 28‐, 18‐, and 9‐fold, respectively. Inactivation thermodynamic analysis showed that both Ea and ΔG≠ of α‐l‐rhamnosidase increased. Through the response surface methodology (RSM) analysis, the higher hesperidin conversion (63.26%) by α‐l‐rhamnosidase was attained with 0.7 M sorbitol at 60°C and pH 4.5 for 10 min. Furthermore, hesperidin could be completely hydrolyzed after 10 hr of reaction. Overall, the results indicated that the addition of sorbitol improved the thermostability of α‐l‐rhamnosidase and increased the enzymatic conversion of hesperidin to hesperetin‐7‐O‐glucoside (HMG). It also provided a simple and efficient way to increase enzymatic conversion of other valuable flavonoid monomers due to the broad substrate specificities of α‐l‐rhamnosidase from A. niger. Practical applications: Hesperetin‐7‐O‐glucoside (HMG), a derhamnosylation product of hesperidin, is considered as a synthetic precursor for novel and efficient sweeteners and is important in food, functional food, and nutraceutical industries. Compared to chemical hydrolysis methods, the enzymatic conversion of hesperidin is milder and has the advantages of high specificity. Adding sorbitol can improve the thermostability of α‐l‐rhamnosidase and increase the enzyme efficacy against hesperidin. This study gave more evidence that adding sorbitol could improve the thermostability of enzymes and provide a better choice for improving biotransformation potency of enzymes. [ABSTRACT FROM AUTHOR]

Catalytic efficiency and thermostability are the two most important characteristics of enzymes. However, it is always tough to improve both catalytic efficiency and thermostability of enzymes simultaneously. In the present study, a computational strategy with double‐screening steps was proposed to simultaneously improve both catalysis efficiency and thermostability of enzymes; and a fungal α‐l‐rhamnosidase was used to validate the strategy. As the result, by molecular docking and sequence alignment analysis within the binding pocket, seven mutant candidates were predicted with better catalytic efficiency. By energy variety analysis, A355N, S356Y, and D525N among the seven mutant candidates were predicted with better thermostability. The expression and characterization results showed the mutant D525N had significant improvements in both enzyme activity and thermostability. Molecular dynamics simulations indicated that the mutations located within the 5 Å range of the catalytic domain, which could improve root mean squared deviation, electrostatic, Van der Waal interaction, and polar salvation values, and formed water bridge between the substrate and the enzyme. The study indicated that the computational strategy based on the binding energy, conservation degree and mutation energy analyses was effective to develop enzymes with better catalysis and thermostability, providing practical approach for developing industrial enzymes. [ABSTRACT FROM AUTHOR]

α‐l‐Rhamnosidase is a biotechnologically important enzyme in food industry and in the preparation of drugs and drug precursors. To expand the functionality of our previously cloned α‐l‐rhamnosidase from Aspergillus niger JMU‐TS528, 14 mutants were constructed based on the changes of the folding free energy (ΔΔG), predicted by the PoPMuSiC algorithm. Among them, six single‐site mutants displayed higher thermal stability than wild type (WT). The combinational mutant K573V–E631F displayed even higher thermostability than six single‐site mutants. The spectra analyses displayed that the WT and K573V–E631F had almost similar secondary and tertiary structure profiles. The simulated protein structure‐based interaction analysis and molecular dynamics calculation were further implemented to assess the conformational preferences of the K573V–E631F. The improved thermostability of mutant K573V–E631F may be attributed to the introduction of new cation–π and hydrophobic interactions, and the overall improvement of the enzyme conformation. Practical applications: The stability of enzymes, particularly with regards to thermal stability remains a critical issue in industrial biotechnology and industrial processing generally tends to higher ambient temperature to inhibit microbial growth. Most of the α‐l‐rhamnosidases are usually active at temperature from 30 to 60°C, which are apt to denature at temperatures over 60°C. To expand the functionality of our previously cloned α‐l‐rhamnosidase from Aspergillus niger JMU‐TS528, we used protein engineering methods to increase the thermal stability of the α‐l‐rhamnosidase. Practically, conducting reactions at high temperatures enhances the solubility of substrates and products, increases the reaction rate thus reducing the reaction time, and inhibits the growth of contaminating microorganisms. Thus, the improvement on the thermostability of α‐l‐rhamnosidase on the one hand can increase enzyme efficacy; on the other hand, the high ambient temperature would enhance the solubility of natural substrates of α‐l‐rhamnosidase, such as naringin, rutin, and hesperidin, which are poorly dissolved in water at room temperature. Protein thermal resistance is an important issue beyond its obvious industrial importance. The current study also helps in the structure–function relationship study of α‐l‐rhamnosidase. [ABSTRACT FROM AUTHOR]

To improve the thermostability of α-L-rhamnosidase (r-Rha1), an enzyme previously identified from Aspergillus niger JMU-TS528 , multiple arginine (Arg) residues were introduced into the r-Rha1 sequence to replace several lysine (Lys) residues that located on the surface of the folded r-Rha1. Hinted by in silico analysis, five surface Lys residues (K134, K228, K406, K440, K573) were targeted to produce a list of 5 single-residue mutants and 4 multiple-residue mutants using site-directed mutagenesis. Among these mutants, a double Lys to Arg mutant, i.e. K406R/K573R, showed the best thermostability improvement. The half-life of this mutant's enzyme activity increased 3 h at 60 °C, 23 min at 65 °C, and 3.5 min at 70 °C, when compared with the wild type. The simulated protein structure based interaction analysis and molecular dynamics calculation indicate that the thermostability improvement of the mutant K406R-K573R was possibly due to the extra hydrogen bonds, the additional cation-π interactions, and the relatively compact conformation. With the enhanced thermostability, the α-L-rhamnosidase mutant, K406R-K573R, has potentially broadened the r-Rha1 applications in food processing industry. [ABSTRACT FROM AUTHOR]

The glycoside hydrolase, α- l -rhamnosidase, could remove the bitter taste of naringin from citrus juices. However, most α- l -rhamnosidases are easily deactivated at high temperatures, limiting the practice in debittering citrus juices. The V529A mutant of the α- l -rhamnosidase r-Rha1 from Aspergillus niger JMU-TS528 was developed with improved thermostability using directed evolution technology and site-directed mutagenesis. The enzyme mutant had a half-live of thermal inactivation T (1/2) of 1.92 h, 25.00 min, and 2 min at 60, 65, and 70 °C, respectively. In addition, it had improved substrate affinity and better resistance to the inhibition of glucose. The improved substrate affinity was related to its lowered binding energy. Most significantly, the naringin content was reduced to below the bitter taste threshold by treatment with 75 U/mL of the mutant during the preheating process of orange juice production. The comprehensive results indicate that thermostability improvement could promote the practical value of α- l -rhamnosidase in citrus juice processing. [ABSTRACT FROM AUTHOR]

This study aimed to improve the thermostability of arylsulfatase from Pseudoalteromonas carrageenovora . A total of 10 single-site mutants were chosen using the PoPMuSiC program, and two mutants of K253N and P314T showed enhanced thermal stability. By saturation mutagenesis and thermostability analysis, K253H and P314T were the best mutants at the two sites. Combinational mutations of K253H, P314T and H260L were subsequently introduced, and the best mutant of K253H/H260L was selected. Thermal inactivation analysis showed the half-life ( t 1/2 ) value at 55 °C for K253H/H260L was 7.7-fold that of the wild-type enzyme (WT), meanwhile this mutant maintained the specific enzyme activity. Structure modeling demonstrated that the additional hydrogen bonds, optimization of surface charge–charge interactions, and increasing of hydrophobic interaction could account for the improved thermostability imparted by K253H/H260L. [ABSTRACT FROM AUTHOR]