| Ginsenoside compound K（CK）was first discovered in 1972 and is currently undetected in ginseng plants.CK is the main metabolite of protopanaxadiol-type ginsenosides（PPDG）and has higher bioavailability,smaller molecular weight,and better membrane permeability than other PPDG.Moreover,CK has shown great potential in anticancer,anti-inflammatory,antioxidant studies,skin protection,neuroprotective,etc.Therefore,CK has been used in functional foods or drugs.The ability to produce large quantities of CK,which does not exist in natural ginseng,is a prerequisite to meeting the increasing market demand for CK.Compared to physical,chemical or microbial transformation methods,enzymatic transformation provides an efficient,green and stable solution for the preparation of CK.Therefore,the exploration of effective catalytic enzymes and the establishment of enzymatic catalytic processes are of great application.In this work,we obtained a strain of Aspergillus tubingensis JE0609 that could transform ginseng extracts into CK.The protopanaxadiol-type ginsenoside hydrolases（PPDGGH）from A.tubingensis JE0609 were heterologously expressed using the Pichia pastoris expression system,and the mechanism of action between the enzyme and the substrates were elucidated.In addition,a combinatorial enzymatic catalytic strategy was used to produce CK,and the effect of low eutectic solvent（DES）on combinatorial enzymatic catalysis was investigated.Based on the above study,a DES-based two-phase aqueous system（ATPS）was constructed for the efficient preparation and green extraction of deglycosylated ginsenosides（DG）.The main results of the study were obtained as follows:（1）The 161β-glucosidase-producing microorganisms were isolated from the inter-rhizosphere soil of ginseng using a plate chromogenic method.Among them,JE0609showed the highest activity in converting ginseng extracts（the total ginsenosides content of 80%）into CK with a yield of 1.22±0.36 g·L-1.The strain was classified as A.tubingensis JE0609.After a series of improvements,10%of JE0609 was inoculated into 20 g solid fermentation substrate（soybean hulls:corn hulls:bran,1:1:1,w/w/w）for 8 d.The amount of ginseng extracts was added to 100 g·L-1,the CK content increased to 3.81±0.36 g·L-1,andβ-glucosidase activity increased to 8.83±0.36U·m L-1.In addition,comparing different CK preparation methods,the highest CK content of 5.22±0.38 g·L-1 was observed in the sample prepared by enzyme catalysis.（2）Combined with transcriptome analysis and peptide mass fingerprinting identification,6 potential PPDGGH（BG05,BG07,BG15,BG17,BG19 and BG23）were rapidly and efficiently digged from A.tubingensis JE0609.The signal peptide sequences,N-glycosylation sites and physicochemical properties of the 6 proteins were analyzed by computational analysis.（3）The PPDGGH BG07,BG19 and BG23 were efficiently expressed in P.pastoris GS115,with enzyme activities of 2.16 U·mg-1(3.00 U·m L-1),45.35 U·mg-1(59.41U·m L-1)and 2354.71 U·mg-1(235.73 U·m L-1),respectively.Among them,BG23 was functionally validated as an efficient PPDGGH.Enzymatic property studies have shown that BG23 is an acidic（p H adaptation range of 4.5-7.0）and mesophilic（thermostable<50°C）enzyme.A one-pot combinatorial enzymatic catalytic strategy based on BG23 and BGA35（β-galactosidase from Aspergillus oryzae）was established to obtain approximately 2.38 g·L-1 CK from 5.00 g·L-1substrates in 6 h at a conversion rate of 396.7 mg·L-1·h-1.In addition,the binding mechanism of BG23 with ginsenosides was analyzed with the help of molecular docking and molecular dynamics.It were showed that Van der Waals force was the main force for the binding of ginsenosides to BG23,and the positive value ofΔEbind for BG23 and Rd indicated that Rd was unfavorable for binding to BG23.（4）The 24 DES were prepared as enzymatic reaction media,in which a buffer containing 10 wt%DES（Bet:EG,2:1）could be used as a booster for the catalysis of the combined enzymes（BG23 and BGA35）.The p H activity and stability of BG23 and BGA35 were improved by the addition of DES.BGA35 had even 231.10%and 230.20%of the initial activity at p H 4.5 and 5.0.After improvements,a variety of DG(0.45 g·L-1 F1,0.39 g·L-1 F2,0.19 g·L-1 20（S）-PPT,and 0.21 g·L-1 CK)could be produced from5 g·L-1 of ginseng extracts in 24 h at an addition of 50 g·L-1 BG23 and 10 g·L-1 BGA35.A DES-based（30 wt%）ATPS（K2HPO4 concentration of 70 wt%）was constructed for the environmentally friendly and rapid enrichment of multiple DG（F1,F2,20（S）-PPT,and CK）within 0.5 h.The analysis of fluorescence spectra and circular dichroism spectra of the proteins showed that DES had a significant effect on the secondary and tertiary structures of BG23 and BGA35. |
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