Isolation,Purification And Functional Analysis Of β-glucosidases From Aspergillus Niger

Ginsenosides are the most important active ingredients of ginseng.Among them,the deglycosylated rare ginsenosides(Rg_2,Rg₃,Compound K,etc.)have stronger pharmacological activity than the other ginsenosides,so there is a huge demand for them in the pharmaceutical market.Therefore,the preparation of rare ginsenosides has received widespread attention.At present,methods for preparing rare ginsenosides mainly include chemical methods（acid and alkali treatment,etc.）and biological methods（microbial and enzymatic catalysis,etc.）.Among them,the enzyme-catalyzed method is more suitable for preparing rare ginsenosides because of its characteristics of high catalytic specificity,mild reaction conditions,few by-products and simple post-processing.In the preliminary studies,an Aspergillus niger strain named NG1306,which can transform ginsenosides,was screened in our laboratory.Through continuous induction screening,this strain can efficiently transform ginsenoside Rb₁ into rare ginsenoside CK.This paper aims to findβ-glucosidases involved in the efficient conversion of ginsenoside Rb₁ to CK from Aspergillus niger NG1306.Through a variety of column chromatography and enzyme-activity tracking methods,two keyβ-glucosidases BGL4Z and BGL8Z were isolated and purified.Combined with protein profiling and transcriptome sequencing data analysis,the nucleic and amino acid sequences of these twoβ-glucosidases were also obtained.We finally cloned and heterologously expressed these two genes,and characterized the catalytic functions of the recombinantβ-glucosidases.The main research results of this study are as follows:（1）A 70 kDa protein was separated and purified from the crude enzyme solution by ammonium sulfate stepwise salting out,dialysis and ultra-filtration.The protein showed both catalytic activity against p-nitrophenyl-β-D-glucopyranoside and Rb₁,and could convert ginsenoside substrate Rb₁ into Rd,F₂ and CK;（2）A protein of 110 kDa was isolated and purified from the crude enzyme solution by gel filtration chromatography and CHT ceramic hydroxyapatite chromatography,which was active against p-nitrophenyl-β-D-glucopyranoside and Rb₁,and could convert ginsenoside substrates Rb₁ into Rd,F₂ and CK;（3）A 100 kDa protein was separated and purified from the crude enzyme solution by ultra-filtration and phenyl sepharose fast flow hydrophobic chromatography and other methods,which was active against p-nitrophenyl-α-D-xylopyranose,p-nitrophenyl-β-D-glucopyranoside and Rb₁,and could convert ginsenoside Rb₁ into Rd;（3）Combined with protein profiling analysis,the types of two protein,100 kDa and 110 kDa,were preliminarily identified.The results showed that the 110 kDa protein was aβ-glucosidase with an actual size of 82kDa;a total of 4 proteins were identified in the 100 kDa protein,i.e.,α-xylosidase,β-glucosidase,catalase andα-L-fucosidase 2,with the actual size of 82 kDa,85 kDa,80 kDa,and 86 kDa,respectively.The actual size is smaller than the expressed protein because of the highly glycosylated modification system in Aspergillus niger.Three of them are ginsenoside hydrolases:twoβ-glucosidases and oneα-xylosidase,named BGL4Z（82 kDa）,BGL8Z（85 kDa）and XYL6Z（82 kDa）,respectively;（4）Combined with transcriptome data,the functions of the three proteins BGL4Z,BGL8Z and XYL6Z were annotated;（5）The total mRNA of Aspergillus niger NG1306 was extracted,and the twoβ-glucosidase genes bgl4z and bgl8z were amplified by RT-PCR,which were heterologously expressed in Escherichia coli BL21（DE3）and Pichia pastoris（GS115）respectively to verify enzyme activity.In this study,three ginsenoside hydrolases BGL4Z,BGL8Z and XYL6Z were isolated,purified and annotated by AKTA purifier automatic chromatograph,protein profiling and transcriptome sequencing.Moreover,β-glucosidases BGL4Z and BGL8Z were also heterologous expressed in E.coli BL21（DE3）and Pichia pastoris（GS115,X33）,and characterized their function.Verification found that the twoβ-glucosidase genes of bgl4z and bgl8z were inactive on natural substrates and synthetic substrates when expressed in E.coli BL21（DE3）;when bgl4z was expressed in Pichia pastoris GS115 and X33.It is inactive when expressed in X33.The protein expressed in GS115 is active on both natural and synthetic substrates and can convert Rb₁ to Rd.Compared with the previously verified active natural protein,it was found that the activity changed,presumably because the prokaryotic expression system lacks the function of post-translational modification of the eukaryotic expression system or there is excessive N-glycosylation modification in the Pichia pastoris expression system.Affect the protein expression and activity.