| Maltooligosyl trehalose trehalohydrolase（MTHase,EC3.2.1.141）specifically recognizes the trehalose moiety of the maltooligosyl trehalose and generates trehalose by hydrolysis.The product trehalose has been used in biomedicine,food and cosmetic industries due to its moisturizing properties such as sun protection and low sweetness.The MTHase derived from Sulfolobus acidocaldarius ATCC 33909 has the excellent performance of producing trehalose,but during the recombination preparation,intracellular precipitate are easily formed,which affects the soluble expression.In this study,the S.acidocaldarius MTHase was modified:based on multiple sequence alignments,21 amino acid sites were selected and replaced by hydrophobic amino acids by mutation to increase the soluble expression of MTHase.The mutant L202P/L218D/Y323G plasmid was used as a template to obtain effective mutants by random mutation.Saturation mutations and superimposed mutations were performed on the effective mutants,and the best mutant was optimized for 3 L bioreactor fermentation based on the optimal mutant shake flask fermentation.The main conclusions are as follows:（1）Perform multiple sequence alignments on S.acidocaldarius MTHase,select 21hydrophobic amino acids located on the surface of the protein in the non-conserved region,and replace the hydrophobic amino acids with hydrophilic amino acids from other sources at the same site（except 202 sites）.The amino acid substitutions were hydrophilic amino acids to study protein soluble effects.It was found that the mutants Y21Q,L77E,L202P,L218D and Y323G had 1.2,1.4,1.3,1.3 and 1.4 times higher activity,respectively.The mutations were superimposed,and the mutant L202P/L218D/Y323G had the highest activity,reaching 59.4U·mL-1.The enzyme activity of the mutant L202P/L218D/Y323G was increased by 1.9 times.（2）The enzymatic properties of the mutant L202P/L218D/Y323G were studied.The results showed that the amino acid substitution had little effect on the enzymatic properties of MTHase compared to the wild-type enzyme.The increase in enzyme activity of this mutant is due to an increase in the amount of protein expression.（3）Using the plasmid of the above mutant as a template,a mutant library was constructed by random mutation and combined with a high-throughput rapid screening technique to obtain two mutants with improved enzyme activity.Plasmids were extracted and sequenced to confirm the mutation sites.Saturation mutations and superimposed mutations wereperformedontheeffectivemutants.Themutant M140L/L202P/L218D/Y323G/F338S/I404T（MTHasem）was finally obtained.The enzyme activity was 2.4 times higher than that of the wild type.（4）The enzymatic properties of the above mutants were studied and the results showed that the mutants had similar enzymatic properties as the wild type.The mutant was produced with maltooligosyl trehalose synthase（MTSase,EC5.4.99.15）of the same origin stored in the lab to produce trehalose with a maltodextrin（DE 5-7）.The trehalose conversion rate was not greatly affected.（5）The mutant MTHasem was optimized for 3 L bioreactor fermentation.The optimum fermentation conditions were as follows:in the process of bacterial fermentation,the temperature was maintained at 37℃ in the early stage.When the OD600 reached 50,the temperature decreased to 30℃,lactose was fed at a constant rate(flow rate 0.1 g·L-1·h-1)for induction.At 27 h after induction,the activity of the mutant reached 444.8 U·mL-1,approximately 5.9 times than that of the shake flask fermentation. |
