High-level Cell-surface Display Of α-L-rhamnosidase From Aspergillus Terreus In Pichia Pastoris And The Primary Application In Selective Trimming Of Rutin

α-L-rhamnosidase(α-L-rhamnosidase(EC 3.2.1.40))can hydrolyze a variety of natural products such as naringin,rutin,quercetin,hesperidin,terpene glycosides and containingα-L-rhamnose at the end of glycosides.The α-L-rhamnosidase are widely used in citrus debittering,beverage flavor enhancement,and biotransformation fields such as flavonoid glycoside conversion.The α-L-rhamnosidase At Rha derived from Aspergillus terreus CCF3059 is considered to be a heat-resistant and alkali-resistant glycoside hydrolase that can specifically catalyze the hydrolysis of rutin to isoquercitrin.The Pichia pastoris surface display system is one of the microbial surface display systems.The use of Pichia pastoris surface display technology to immobilize enzymes can effectively improve the stability and recycling efficiency of the enzymes,and reduce production and purification costs.This project uses GPI-type anchoring protein to display At Rha on the surface of Pichia pastoris,and successfully constructs three recombinant engineered strains strains.On this basis,the effects of different expression strategies on the expression of At Rha of recombinant strains were explored.Finally,it explores the enzymatic properties of the enzyme as a whole-cell catalyst and the preliminary application of catalyzing the hydrolysis of rutin to isoquercitrin,which provides a theoretical basis for industrial large-scale biotransformation.The main research contents and results of this article are as follows:(1)Using GPI-type cell wall proteins Gcw21,Gcw51,Gcw61 and rhamnosidase At Rha encoding gene fusion expression to construct a recombinant surface display engineering strains.The results of immunofluorescence analysis showed that At Rha was successfully displayed on the surface of Pichia pastoris.The growth and enzyme production status of different anchored proteins displaying At Rha in shake flasks were determined.The growth of the recombinant strain expressing At Rha fusion with Gcw21 was slightly impaired,while the growth of the recombinant strain expressing At Rha fusion with Gcw51 and Gcw61 was not affected.The enzyme activity analysis using p NPR as the substrate showed that the recombinant strain expressing At Rha fusion with Gcw61 had the highest enzyme activity.The enzyme activity reached 72.56 ± 4.54 U/m L in shake flask fermentation for 120 h,and the specific enzyme activity was 7961 ± 415 U/g,which is the highest reported enzyme activity for immobilizing rhamnosidase.It shows that At Rha can be efficiently displayed on the surface of Pichia pastoris cells by fusion expression with the anchoring protein Gcw61.(2)Different expression strategies were used to induce the expression of the display enzyme At Rha.The results showed that the overexpression regulators Fhl1 p and Hac1 could promote the expression of the display enzyme,which was increased by 25.05 % and 24.21 %compared with the original strain,reaching 13494 ± 831 U/g,13403 ± 576 U/g.However,replacing the promoter,overexpression of P180,and increasing the gene dosage can not promote the expression of the display enzyme,on the contrary,the specific enzyme activity has decreased.In addition,when the enzyme production temperature was controlled to 25 ℃,the enzyme activity of the recombinant strain increased by 15.08 %.The experimental results of optimizing the p H of enzyme production show that the recombinant strain has good stability of enzyme production in the range of p H 5.0-7.0,which is conducive to industrial large-scale fermentation production.(3)Using the recombinant surface display engineering strains as a whole-cell catalyst,the optimal temperature,optimal p H,thermal stability and p H stability of the display enzyme are not significantly different than those of the free enzyme.In the bioconversion system that catalyzes the hydrolysis of rutin to isoquercitrin,the enzyme can specifically catalyze the hydrolysis of rutin.When the total cell catalyst is added at 20 U/m L,100-200 g/L rutin can be completely converted into isoquercetin within 20 hours,and the final conversion rate of 250g/L rutin for 20 hours is 88.06 % The final conversion rate of 300 g/L rutin for 20 h was74.73 %.In addition,after 5 rounds of repeated use,the whole-cell catalyst maintained74.84 % of its catalytic activity.It shows that the whole-cell catalyst has the potential for industrial large-scale biotransformation applications.