Purification And Characterization Of Ginsenoside β-glucosidases From Paecilomyces Bainier Sp. 229

Paecilomyces Bainier sp.229 is a fungal strain isolated from the soil of ginseng plantation localities.It shows strongβ-glucosidase activity that can transform ginsenoside Rb1 to CK efficiently.Ginsenoside CK has attracted great interest because of its intriguing anti-tumor activity.It induces tumor cell apoptosis,inhibits tumor metastasis,and restrains tumor invasion.So far,a comprehensive study on ginsenoside hydrolyzingβ-glucosidases of Paecilomyces Bainier sp.229 has not been seen.β-glucosidases are widely used in food and drug industry.To date,only traditional chromatographies for protein purification have been applied to purifyingβ-glucosidases.In this study,a new method for purification ofβ-glucosidases using microcrystalline cellulose(MCC) was developed.The result showed that the MCC chromatography was much more efficient inβ-glucosidase purification than those traditional chromatographies.What's more,the enzyme recovery of MCC method was 63.6%higher than that of traditional method.A total 7 ginsenosideβ-glucosidases were then purified from Paecilomyces Bainier sp.229 by combinations of the MCC chromatography and different traditional protein purification chromatographis.The purified enzymes were named in sequence as ginsenosideβ-glucosidase(1),(2),(3),(4),(5),(6),and(7).Taking crude enzyme as 100%,the activity recovery of eachβ-glucosidase was 6.5%,2.8%, 3.5%,0.7%,1.1%,2.0%and 5.2%respectively;the protein recovery was 0.048%, 0.022%,0.027%,0.004%,0.007%,0.017%and 0.034%respectively.The properties of purified enzyme were studied.The molecular weights(MW) of enzyme subunits and of whole enzymes were determined by SDS-PAGE and gel filtration on a sephacryl S-300 HR column respectively.The MW of whole enzyme of eachβ-glucosidase was 110 KDa,230 KDa,220 KDa,170 KDa,305 KDa,115 KDa,and 120 KDa,respectively.The MW of subunit was 109 KDa,113 KDa,111 KDa,84 KDa,102 KDa,115 KDa,and 117 KDa,respectively.The effects of pH and temperature on enzyme activity and stability were studied.The basic results were:theβ-glucosidases showed their optimal activities within pH 3.0-4.0 and were stable within pH 3.0-6.0.theβ-glucosidases showed their optimal activities within 50-60℃and were stable at temperatures lower than the optimal reaction temperature at least 5℃.The kinetic parameters,K_m and V_max values,of purified enzymes against p-nitrophenyl-β-D-glucoside(NPβG) were determined by typical Lineweaver-Burk double reciprocal plots under pH 3.5 and 45℃.The K_m values of ginsenosideβ-glucosidase(1)-(7) were 0.141 mmol/L,0.150 mmol/L,0.132 mmol/L, 0.119 mmol/L,0.111 mmol/L,0.102 mmol/L,and 0.125 mmol/L,respectively;the V_max values were 0.165 mol/min/mg,0.245 mol/min/mg,0.268 mol/min/mg,0.144 mol/min/mg,0.218 mol/min/mg,0.158 mol/min/mg,and 0.194 mol/min/mg respecitively.The hydrolyzing pathways of protopanaxadiol-type ginsenosides by purifiedβ-glucosidases were further studied.Rb1 was hydrolyzed by the same pathway, Rb1→Rd,by ginsenosideβ-glucosidase(1),(2),and(7).They only could hydrolyze 1,6-β-glucosidic linkage at C-20.The hydrolyzing pathway of Rb1 byβ-glucosidase (3) was Rb1→Rd→F2.Bothβ-glucosidase(4) and(6) could hydrolyze ginsenoside Rb1 to CK.But the pathways were found to be different.The pathway byβ-glucosidase(6) was Rb1→Rd→F2→CK;while the pathway byβ-glucosidase(4) was Rb1→ⅩⅦ→F2→CK.β-glucosidase(5) could hydrolyze ginsenoside Rb1 to Rg3 via Rd.β-glucosidase(4) and(6) are the first reportedβ-glucosidases that can transform Rb1 mainly to CK;β-glucosidase(5) is the first reportedβ-glucosidase that can transform Rb1 mainly to Rg3.After the comprehensive study of hydrolyzing pathways by differentβ-glucosidases,a general flow chart of Rb1 matabolic pathways in Paecilomyces Bainier sp.229 was drawn. The mainstream was Rb1→Rd→F2→CK.The amino acid sequences ofβ-glucosidase(4) and(6),which played the key roles in preparation of ginsenoside CK,were analyzed with the latest PSD-MALDI MS method sulfonated by SPITC.Four peptides ofβ-glucosidase(4) were: A[I/L]S[I/L][I/L]T[I/L]AR,[I/L][I/L]FAEFGDR,TPPNFSSWTR,and ASDY[I/L] FPSG[I/L]NR.Seven peptides ofβ-glucosidase(5) were:VTFP[I/L]TR; A[I/L]MPH[I/L]R;GWHMGGEFR;GWHM(O)GGEFR;Y[I/L]P[I/L]GAYV[I/L] SR;GVQVA[I/L]GPVVGS[I/L]GR,and W[I/L][I/L][Q/K]SGSYNVFVGSSSR. Five peptides ofβ-glucosidase(6) were:DHAS[I/L][I/L]R;S[I/L]VDV[I/L]YGR, GG[I/L]P[I/L]THQER,HY[I/L]GNEQEHFR,and VT[I/L]APGQQ[I/L]QWTAT[I/L] TR.This study on amino acid sequencing is a remarkable contribution to further research on its gene cloning.