【作者】 严钦；

【导师】 周珮；

【作者基本信息】 复旦大学 ， 药物化学， 2008， 博士

【摘要】 拟青霉菌sp.229是本实验室从人参种植区土壤里分离筛选得到的菌株,经菌种诱变、筛选,它具有很强的人参皂苷β-葡萄糖苷酶活性,能将人参皂苷Rb1高效转化为CK。人参皂苷CK由于其良好的抗肿瘤活性而受到关注,但是目前还未见对该拟青霉菌中人参皂苷转化酶进行系统的研究。β-葡萄糖苷酶（β-glucosidases）是在食品工业和医药工业中均有广泛应用的酶类。但是目前对该类酶的分离纯化过程仍局限一般的蛋白纯化柱层析的组合应用。本研究用微晶纤维素（microcrystalline cellulose）作为亲和层析材料对β-葡萄糖苷酶进行分离纯化,结果显示,微晶纤维素柱层析法在纯化β-葡萄糖苷酶时效率远高于常规的蛋白纯化方法,并且酶的回收率比常规方法高63.6%。通过新建立的微晶纤维素柱层析方法和一系列蛋白常规纯化方法,从拟青霉菌sp.229中共分离纯化得到7个电泳纯的β-葡萄糖苷酶,分别命名为人参皂苷β-葡萄糖苷酶（1）、（2）、（3）、（4）、（5）、（6）和（7）。以粗酶液为参照,各个酶的酶活力回收率依次为6.5%,2.8%,3.5%,0.7%,1.1%,2.0%和5.2%;各个酶的蛋白回收率为0.048%,0.022%,0.027%,0.004%,0.007%,0.017%和0.034%。本研究接着对这7个人参皂苷β-葡萄糖苷酶进行酶学性质的研究。通过对各个酶的sephacryl S-300 HR凝胶分子筛过滤层析和SDS-PAGE实验,分别得出全酶的分子量和亚基分子量。七个酶的全酶分子量依次为:110 KDa、230 KDa、220 KDa、170 KDa、305 KDa、115 KDa和120 KDa;亚基分子量分别为109 KDa、113 KDa、111 KDa、84 KDa、102 KDa、115 KDa和117 KDa。对各个酶进行最适反应pH/温度和稳定pH/温度范围进行研究后,得出如下基本结论:最适反应pH值都在3-4范围内,在pH 3-6范围内都保持相对的稳定;最适反应温度在50-60℃范围内,在低于最适反应温度5℃以上各个酶保持相对的稳定。通过Lineweaver-Burk双倒数曲线,计算出各个酶的酶动力学参数。在pH 3和45℃条件下,以p-硝基苯酚-β-D-葡萄糖苷（NPβG）为底物,酶（1）—（7）的米氏常数（K_m）依次为:0.141 mmol/L、0.150 mmol/L、0.132mmol/L、0.119 mmol/L、0.111 mmol/L、0.102 mmol/L和0.125 mmol/L:最大反应速度(V_max)依次为:0.165 mol/min/mg、0.245 mol/min/mg、0.268mol/min/mg、0.144 mol/min╱mg、0.218 mol/min/mg、0.158 mol/min╱mg、0.194mol/min/mg。本研究进一步深入研究各人参皂苷β-葡萄糖苷酶对二醇型人参皂苷的催化方式。酶（1）、（2）和（7）对人参皂苷Rb1的催化途径均为Rb1→Rd,它们只催化酶解Rb1在20位上的1,6-β-葡萄糖苷,而对其它的糖苷键都不起作用。酶（3）可催化酶解Rb1分别在20位上的1,6-β-葡萄糖苷和3位上的1,2-β-葡萄糖苷,将Rb1转化为F2,其催化途径为Rb1→Rd→F2。酶（4）和（6）都能催化酶解二醇型人参皂苷在20位上的1,6-β-葡萄糖苷和在3位上的两个葡萄糖苷,将Rb1直接转化为CK。但是经过深入研究,证明它们的催化途径不尽相同,酶（6）对Rb1的催化途径为:Rb1→Rd→F2→CK,而酶（4）的催化途径为:Rb1→ⅩⅦ→F2→CK。酶（5）能酶解Rb1在20位上的两个葡萄糖苷,将Rb1主要转化为Rg3。该酶还能酶解二醇型人参皂苷3位上与达玛烷骨架直接相连的葡萄糖苷,将F2转化为CK,然后再进一步将CK酶解为二醇型人参皂苷苷元（PPD）。酶（4）、（6）是首次发现的能将Rb1主要转化为CK的β-葡萄糖苷酶;酶（5）是首次发现的能将Rb1主要转化为Rg3的β-葡萄糖苷酶。对各个人参皂苷β-葡萄糖苷酶的作用方式进行研究后,推断出人参皂苷Rb1在拟青霉菌sp.229中可能总的转化代谢途径,其主要途径为:Rb1→Rd→F2→CK,同时包括其它一些支路,这为代谢调控提供理论依据。本研究最后研究在人参皂苷CK合成过程起重要作用的酶（4）、（5）和（6）的氨基酸序列,通过磺基异硫氰酸苯酯（SPITC）修饰的PSD-MALDI-MS（带有源后衰变的基质辅助激光解吸电离飞行时间质谱）方法对它们进行从头（de novo）氨基酸序列分析,结果显示酶（4）的4个肽段的氨基酸序列分别为:A[I/L]S[I/L][I/L]T[I/L]AR;[I/L][I/L]FAEFGDR;TPPNFSSWTR;ASDY[I/L]FPSG[I/L]NR。酶（5）的7个肽段的氨基酸序列分别为: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:W[I/L][I/L][Q/K]SGSYNVFVGSSSR。酶（6）的5个肽段序列分别为:DHAS[I/L][I/L]R;S[I/L]VDV[I/L]YGR;GG[I/L]P[I/L]THQER;HY[I/L]GNEQEHFR;VT[I/L]APGQQ[I/L]QWTAT[I/L]TR。这些研究为进一步从拟青霉菌sp.229中提取这些关键酶的基因及此后的基因工程研究打下基础。更多还原

【Abstract】 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.更多还原