Study On Protopanaxadiol Type Saponins Hydrolases From Aspergillus Niger NG1306 Strain

Ginsenoside is the main pharmacological active component of Panaxnoto ginseng,which has pharmacological activities such as anti-inflammatory,immune regulation,tissue damage repair and metabolic homeostasis regulation.It has been confirmed by human and animal metabolomics studies that naturally existing ginsenoside,due to poor membrane permeability,is difficult to be absorbed by the human intestine.Only through the intestinal flora metabolic transformation of deglycosylation can products-rare ginsenosides be absorbed into the blood and have a medical effect.The cost of chemical synthesis of rare ginsenosides is very high,and the selectivity of preparing rare ginsenosides with chemical degradation method is low;Owing to good specificity,high efficiency and controllability,the enzymatic hydrolysis method is an excellent alternative.Rare ginsenosides can be prepared directionally by the action of different properties of glycoside hydrolase.The glycosidic bonds in ginsenosides of different configurations and compositions can be used to prepare rare ginsenosides.Therefore,the discovery of glycoside hydrolases that can efficiently convert ginsenosides is a key issue to be solved urgently.The research strain Aspergillus niger NG1306 was isolated from the stem and leaf tissue of Panax notoginseng.Its crude enzyme solution has the function to convert a variety of ginsenosides.Aspergillus niger NG1306 was cultured using ginsenosides as the carbon source.Through transcriptome sequencing,annotation and analysis,13glycoside hydrolase genes were inferred to have the potential to transform ginsenosides.These genes were heterologously expressed and recombinant enzymes were verified in Escherichia coli and Pichia pastoris.The experimental results showed that glycoside hydrolase expressed by glycoside hydrolase genes anglu ZL1,anglu ZL2,anglu ZL3,anglu ZL656,anglu ZL9-1 and anglu ZLGYN can hydrolyze the glucose outside the C3 position of ginsenoside Rb1 to produce Rd,and the conversion pathway is Rb₁→Rd.Theβ-glucosidase Anglu ZL3 can hydrolyze two glucoses at the C3 position and the glucose outside the C20 position of ginsenosides Rb₁,Rb₂,Rb₃,Rc.It can convert various ginsenosides into rare ginsenosides.The conversion pathways are:Rb₁→Gyp VXII/Rd→Gyp LXXV/F₂→C-K;Rb₂→C-O→C-Y;Rb₃→C-Mx₁→C-Mx;Rc→C-Mc₁→C-Mc.The recombinantβ-xylosidase gene anxyl from Aspergillus niger NG1306 was recombinantly expressed in Pichia pastoris KM71H,and the enzymatic properties of the recombinant enzyme Anxyl were characterized.Anxyl can hydrolyze the xylose in the outer C20 position of ginsenoside Rb₃ and C-Mx to generate Rd and C-K,respectively.Anxyl has no catalytic activity on other ginsenosides without xylan side chain.Using ginsenoside Rb₃as the ligand of Anxyl,molecular docking model analysis was carried out,and the main catalytically active residues were inferred,which provided a reference for the subsequent directed evolutionary transformation of Anxyl.Through in-depth exploration of glycoside hydrolase genes from multi-species origin and targeted evolutionary modification of known glycoside hydrolase genes,we will provide an efficient enzyme source for the bioprocessing engineering of rare ginsenosides,and promote the in-depth development of medicinal plant resources such as Panax ginseng and Panax notoginseng.