Screening, UV mutagenesis, and production optimization of a strain with high yield of amylase

Abstract:

[Background] It is an important goal for amylase development to improve the stability under variable industrial production conditions. [Objective] Amylase is widely used in food processing, desizing of cloth, brewing, and breeding industry. However, current amylase in industrial production features a few sources available and poor stability in solution. Thus, efforts should be made to expand the sources of amylase and enhance the stability of the enzyme so that it can adapt to the complex and changeable production environment. [Methods] A total of 20 strains with amylase activity were screened out from the surface soil of the Yellow River Wetland in Sanmenxia with a selective medium. The activity of the crude enzyme solution was detected with the cylinder plate method and 3 strains with high amylase activity were preliminarily identified. Among them, the strain with highest amylase activity was mutagenized by ultraviolet (UV) and the lethality rate was determined. The strain with the highest enzyme activity after mutagenesis was selected. Then the culture conditions were optimized, and the activity of amylase after mutation and the ranges of action conditions were determined. 3,5-dinitrosalicylic acid (DNS) method was used to determine the enzyme activity before and after mutation for a comparison. [Results] The 3 strains with high amylase activity were numbered S03, S08, and S17. The 16S rRNA gene sequence alignment, Gram staining, and physiological and biochemical characterization revealed that strains S03, S08, and S17 are members of Aeromonas, Exiguobacterium, and Bacillus, respectively. The optimal NaCl concentration for the amylase was 10%−12%, and the optimal temperature was 45 °C (S03, S17) and 25 °C (S08). Moreover, the optimal pH was 7.0−9.0, and the optimal fermentation time was 36 h (S03 and S17) and 42 h (S08). After UV mutagenesis, the strain with the highest enzyme activity was screened out and marked as S17M. S17M grew well at pH of 8.0 and 40 °C. Mn²⁺, Ti⁴⁺, and Mg²⁺ promoted the growth of S17M, while Cu²⁺, Fe²⁺, Fe³⁺, Na⁺, and organic solvents (particularly formaldehyde and glacial acetic acid) inhibited it. S17M demonstrated high amylase activity in the presence of NaCl at 8%–14%, at pH of 5.0–10.0, and at 15–55 °C, and the inhibitory effect of metal ions and organic solvents was decreased. The maximum enzyme activity was up to 195.62 U/mL, 3.19 folds that of the original strain. [Conclusion] UV mutagenesis can significantly improve enzyme activity and stability, so that the resultant enzyme can adapt to the variable industrial production environments. Thus, it has the potential to be applied to different industrial production conditions.