Enhancement of the thermostability of Aspergillus niger α-l-rhamnosidase based on PoPMuSiC algorithm.

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.

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