生物技术通报 ›› 2022, Vol. 38 ›› Issue (11): 238-249.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0167
赵宝顶(), 吕佳, 申玉玉, 桂玲, 陈钟秀, 陈杰, 路福平, 黎明()
收稿日期:
2022-02-15
出版日期:
2022-11-26
发布日期:
2022-12-01
作者简介:
赵宝顶,男,硕士研究生,研究方向:微生物与生化药学;E-mail:基金资助:
ZHAO Bao-ding(), LV Jia, SHEN Yu-yu, GUI Ling, CHEN Zhong-xiu, CHEN Jie, LU Fu-ping, LI Ming()
Received:
2022-02-15
Published:
2022-11-26
Online:
2022-12-01
摘要:
以大肠杆菌作为嘧啶核糖核苷水解酶(pyrimidine-specific ribonucleoside hydrolase RihA,RihA)表达宿主,利用生物转化的方法将尿苷高效转化为尿嘧啶。首先构建pET22b-RihA质粒,在大肠杆菌BL21(DE3)中重组表达,研究尿苷的转化情况。采用优化pET22b-RihA质粒信号肽和与分子伴侣质粒共表达两种策略进一步提高大肠杆菌转化尿苷的效率。来源于碱性磷酸酶的PhoA信号肽和分子伴侣GroES-GroEL共表达的菌株F在投底物发酵时转化效果最好,当底物浓度为65 g/L转化15 h时,菌株F几乎将尿苷完全转化,尿苷转化率达到98.9%,而原始菌株A的尿苷转化率仅为80.2%。进一步对菌株F转化尿苷的浓度进行优化,投入一倍体积的尿苷底物后继续培养约53 h尿苷转化完全,得到尿嘧啶产量为73.45 g/L,尿嘧啶产率为98.16%。发酵液上清中RihA酶活最高的为PelB信号肽和分子伴侣GroES-GroEL共表达的菌株C,其RihA酶活是原始菌株A的10.0倍。其中总可溶性RihA酶活最高的为PhoA信号肽和分子伴侣DnaK-DnaJ-GrpE共表达的菌株G,其RihA酶活是原始菌株A的4.45倍。通过优化信号肽和分子伴侣,提高了尿苷的转化效率,并建立了一种高效的利用大肠杆菌全细胞催化生产药物中间体尿嘧啶的方法。与化学合成方法生产相比,避免了繁琐的生产工艺步骤,对环境友好绿色。
赵宝顶, 吕佳, 申玉玉, 桂玲, 陈钟秀, 陈杰, 路福平, 黎明. 基于信号肽和分子伴侣策略促进大肠杆菌高效转化尿苷[J]. 生物技术通报, 2022, 38(11): 238-249.
ZHAO Bao-ding, LV Jia, SHEN Yu-yu, GUI Ling, CHEN Zhong-xiu, CHEN Jie, LU Fu-ping, LI Ming. Efficient Transformation of Uridine by Escherichia coli Based on Signal Peptide and Molecular Chaperone Strategy[J]. Biotechnology Bulletin, 2022, 38(11): 238-249.
菌株和质粒 Strain and plasmid | 相关特性 Related characteristics | 来源 Source | |
---|---|---|---|
菌株 Strain | E. coli BL21 | Wild type E. coli BL21(DE3) | Lab stock |
E. coli DH5α | Cloning host | Lab stock | |
E. coli K12. MG1655 | Wild type | Lab stock | |
SP-1 | BL21 derivate with plasmid pET22b-Fhud-RihA | This work | |
SP-2 | BL21 derivate with plasmid pET22b-OmpA-RihA | This work | |
SP-3 | BL21 derivate with plasmid pET22b-OmpC-RihA | This work | |
SP-4 | BL21 derivate with plasmid pET22b-OmpF-RihA | This work | |
SP-5 | BL21 derivate with plasmid pET22b-OmpT-RihA | This work | |
SP-6 | BL21 derivate with plasmid pET22b-PhoE-RihA | This work | |
BL21-pCDM4-DnaJ | BL21 derivate with plasmid BL21-pCDM4-DnaJ | This work | |
BL21-pCDM4-DnaK | BL21 derivate with plasmid BL21-pCDM4-DnaK | This work | |
BL21-pCDM4-GroEL | BL21 derivate with plasmid BL21-pCDM4-GroEL | This work | |
BL21-pCDM4-GroES | BL21 derivate with plasmid BL21-pCDM4-GroES | This work | |
BL21-pCDM4-GrpE | BL21 derivate with plasmid BL21-pCDM4-GrpE | This work | |
Strain A | BL21 derivate with plasmid pET22b-RihA | This work | |
Strain B | BL21 derivate with plasmid pET22b-PhoA-RihA | This work | |
Strain C | BL21 derivate with plasmid pET22b-RihA and pCDM4-GroES-GroEL | This work | |
Strain D | BL21 derivate with plasmid pET22b-RihA and pCDM4-DnaK-DnaJ-DrpE | This work | |
Strain E | BL21 derivate with plasmid pET22b-RihA and pCDM4-DnaK-DnaJ-GrpE-GroES-GroEL | This work | |
Strain F | BL21 derivate with plasmid pET22b-PhoA-RihA and pCDM4-GroES-GroEL | This work | |
Strain G | BL21 derivate with plasmid pET22b-PhoA-RihA and pCDM4-DnaK-DnaJ-GrpE | This work | |
Strain H | BL21 derivate with plasmid pET22b-RihA and pCDM4-DnaK-DnaJ-GrpE-GroES-GroEL | This work | |
质粒Plasmid | pET22b | T7 promoters,AmpR | Lab stock |
pCDM4 | T7 promoters,SmR | [ | |
pET22b-RihA | pET22b derivate with rihA cloned | This work | |
pET22b-PhoA-RihA | pET22b derivate with PhoA-rihA cloned | This work | |
pET22b-Fhud-RihA | pET22b derivate with Fhud-rihA cloned | This work | |
pET22b-OmpA-RihA | pET22b derivate with OmpA-rihA cloned | This work | |
pET22b-OmpC-RihA | pET22b derivate with OmpC-rihA cloned | This work | |
pET22b-OmpF-RihA | pET22b derivate with OmpF-rihA cloned | This work | |
pET22b-OmpT-RihA | pET22b derivate with OmpT-rihA cloned | This work | |
pET22bpET22b-PhoE-RihA | pET22b derivate with PhoE-rihA cloned | This work | |
pCDM4-DnaJ | pCDM4 derivate with dnaJ cloned | This work | |
pCDM4-DnaK | pCDM4 derivate with dnaK cloned | This work | |
pCDM4-GroEL | pCDM4 derivate with groEL cloned | This work | |
pCDM4-GroES | pCDM4 derivate with groES cloned | This work | |
pCDM4-GrpE | pCDM4 derivate with grpE cloned | This work | |
pCDM4-GroES-GroEL | pCDM4 derivate with groES-groEL cloned | This work | |
pCDM4-DnaK-DnaJ-GrpE | pCDM4 derivate with dnaK-dnaJ-grpE cloned | This work | |
pCDM4-DnaK-DnaJ-GrpE- GroES-GroEL | pCDM4 derivate with dnaK-dnaJ-grpE-groES-groEL cloned | This work |
表1 本研究所用的菌株及质粒
Table 1 Strains and plasmids in this study
菌株和质粒 Strain and plasmid | 相关特性 Related characteristics | 来源 Source | |
---|---|---|---|
菌株 Strain | E. coli BL21 | Wild type E. coli BL21(DE3) | Lab stock |
E. coli DH5α | Cloning host | Lab stock | |
E. coli K12. MG1655 | Wild type | Lab stock | |
SP-1 | BL21 derivate with plasmid pET22b-Fhud-RihA | This work | |
SP-2 | BL21 derivate with plasmid pET22b-OmpA-RihA | This work | |
SP-3 | BL21 derivate with plasmid pET22b-OmpC-RihA | This work | |
SP-4 | BL21 derivate with plasmid pET22b-OmpF-RihA | This work | |
SP-5 | BL21 derivate with plasmid pET22b-OmpT-RihA | This work | |
SP-6 | BL21 derivate with plasmid pET22b-PhoE-RihA | This work | |
BL21-pCDM4-DnaJ | BL21 derivate with plasmid BL21-pCDM4-DnaJ | This work | |
BL21-pCDM4-DnaK | BL21 derivate with plasmid BL21-pCDM4-DnaK | This work | |
BL21-pCDM4-GroEL | BL21 derivate with plasmid BL21-pCDM4-GroEL | This work | |
BL21-pCDM4-GroES | BL21 derivate with plasmid BL21-pCDM4-GroES | This work | |
BL21-pCDM4-GrpE | BL21 derivate with plasmid BL21-pCDM4-GrpE | This work | |
Strain A | BL21 derivate with plasmid pET22b-RihA | This work | |
Strain B | BL21 derivate with plasmid pET22b-PhoA-RihA | This work | |
Strain C | BL21 derivate with plasmid pET22b-RihA and pCDM4-GroES-GroEL | This work | |
Strain D | BL21 derivate with plasmid pET22b-RihA and pCDM4-DnaK-DnaJ-DrpE | This work | |
Strain E | BL21 derivate with plasmid pET22b-RihA and pCDM4-DnaK-DnaJ-GrpE-GroES-GroEL | This work | |
Strain F | BL21 derivate with plasmid pET22b-PhoA-RihA and pCDM4-GroES-GroEL | This work | |
Strain G | BL21 derivate with plasmid pET22b-PhoA-RihA and pCDM4-DnaK-DnaJ-GrpE | This work | |
Strain H | BL21 derivate with plasmid pET22b-RihA and pCDM4-DnaK-DnaJ-GrpE-GroES-GroEL | This work | |
质粒Plasmid | pET22b | T7 promoters,AmpR | Lab stock |
pCDM4 | T7 promoters,SmR | [ | |
pET22b-RihA | pET22b derivate with rihA cloned | This work | |
pET22b-PhoA-RihA | pET22b derivate with PhoA-rihA cloned | This work | |
pET22b-Fhud-RihA | pET22b derivate with Fhud-rihA cloned | This work | |
pET22b-OmpA-RihA | pET22b derivate with OmpA-rihA cloned | This work | |
pET22b-OmpC-RihA | pET22b derivate with OmpC-rihA cloned | This work | |
pET22b-OmpF-RihA | pET22b derivate with OmpF-rihA cloned | This work | |
pET22b-OmpT-RihA | pET22b derivate with OmpT-rihA cloned | This work | |
pET22bpET22b-PhoE-RihA | pET22b derivate with PhoE-rihA cloned | This work | |
pCDM4-DnaJ | pCDM4 derivate with dnaJ cloned | This work | |
pCDM4-DnaK | pCDM4 derivate with dnaK cloned | This work | |
pCDM4-GroEL | pCDM4 derivate with groEL cloned | This work | |
pCDM4-GroES | pCDM4 derivate with groES cloned | This work | |
pCDM4-GrpE | pCDM4 derivate with grpE cloned | This work | |
pCDM4-GroES-GroEL | pCDM4 derivate with groES-groEL cloned | This work | |
pCDM4-DnaK-DnaJ-GrpE | pCDM4 derivate with dnaK-dnaJ-grpE cloned | This work | |
pCDM4-DnaK-DnaJ-GrpE- GroES-GroEL | pCDM4 derivate with dnaK-dnaJ-grpE-groES-groEL cloned | This work |
名称 Name | 引物序列Primer sequence(5'-3') |
---|---|
pET22bRihAF | cctcgctgcccagccggcgatggccatgGCACTGCCAATTCTGTTAGATTG |
pET22bRihAR | tgtcgacggagctcgaattcggatccttaAGCGTAAAATTTCAGACGATCAGCC |
FhuD-RihA F1 | gatgaataccgcccacgcggcggctattgatcccaatatgGCACTGCCAATTCTGTTAGA |
FhuD F1 | gactgttaacggcgatggcgctttctccgttgttatggcagatgaataccgcccacgcg |
FhuD F2 | taagaaggagatatacatatgagcggcttacctcttatttcgcgccgtcgactgttaacggcgatggcg |
OmpA-RihA F1 | gcactggctggtttcgctaccgtagcgcaggccatgGCACTGCCAATTCTGTTAG |
OmpA F1 | taagaaggagatatacatatgaaaaagacagctatcgcgattgcagtggcactggctggtttcgc |
OmpC F1 | taagaaggagatatacatatgaaagttaaagtactgtccctcctggtcccagctctgctggtagcag |
OmpC-RihA F1 | ccagctctgctggtagcaggcgcagcaaacgctatgGCACTGCCAATTCTGTTAGA |
OmpF F1 | ttaagaaggagatatacatatgatgaagcgcaatattctggcagtgatcgtccctgctctgttagtagcaggt |
OmpF-RihA F1 | ccctgctctgttagtagcaggtactgcaaacgctatgGCACTGCCAATTCTGTTAG |
OmpT F1 | tttaagaaggagatatacatatgcgggcgaaacttctgggaatagtcctgacaacccctattgcga |
OmpT-RihA F1 | cctgacaacccctattgcgatcagctcttttgctatgGCACTGCCAATTCTGTTAG |
PhoA F1 | tttaagaaggagatatacatatgaaacaaagcactattgcactggcactcttaccgttactgtttacccctgt |
PhoA-RihA F1 | cttaccgttactgtttacccctgtgacaaaagccatgGCACTGCCAATTCTGTT |
PhoE F1 | tttaagaaggagatatacatatgaaaaagagcactctggcattagtggtgatgggcattgtggcatct |
PhoE-RihA F1 | gatgggcattgtggcatctgcatctgtacaggctatgGCACTGCCAATTCTGTTAG |
dnaJ F1 | GGAATTCCATatggctaagcaagattattacgaga |
dnaJ R1 | GGACTAGTttagcgggtcaggtcgtca |
dnaK F1 | GGAATTCCATatgggtaaaataattggtatcgacctgg |
dnaK R1 | GGACTAGTttattttttgtctttgacttcttcaaattcagc |
GroEL F1 | GGAATTCCATatggcagctaaagacgtaaaattcg |
GroEL R1 | GGACTAGTttacatcatgccgcccatgc |
GroES F1 | GGAATTCCATatgaatattcgtccattgcatgatcg |
GroES R1 | GGACTAGTttacgcttcaacaattgccagaatgt |
grpE F1 | GGAATTCCATatgagtagtaaagaacagaaaacgcctg |
grpE R1 | GGACTAGTttaagcttttgctttcgctacagtaacc |
Cht F1 | cttgaggggttttttTctagAatcgagatcgatctcgatcccg |
Cht R1 | actaccggaagcagtgtcgactc |
To F1 | gatgcgtccggcgtagc |
表2 引物列表
Table 2 List of primers
名称 Name | 引物序列Primer sequence(5'-3') |
---|---|
pET22bRihAF | cctcgctgcccagccggcgatggccatgGCACTGCCAATTCTGTTAGATTG |
pET22bRihAR | tgtcgacggagctcgaattcggatccttaAGCGTAAAATTTCAGACGATCAGCC |
FhuD-RihA F1 | gatgaataccgcccacgcggcggctattgatcccaatatgGCACTGCCAATTCTGTTAGA |
FhuD F1 | gactgttaacggcgatggcgctttctccgttgttatggcagatgaataccgcccacgcg |
FhuD F2 | taagaaggagatatacatatgagcggcttacctcttatttcgcgccgtcgactgttaacggcgatggcg |
OmpA-RihA F1 | gcactggctggtttcgctaccgtagcgcaggccatgGCACTGCCAATTCTGTTAG |
OmpA F1 | taagaaggagatatacatatgaaaaagacagctatcgcgattgcagtggcactggctggtttcgc |
OmpC F1 | taagaaggagatatacatatgaaagttaaagtactgtccctcctggtcccagctctgctggtagcag |
OmpC-RihA F1 | ccagctctgctggtagcaggcgcagcaaacgctatgGCACTGCCAATTCTGTTAGA |
OmpF F1 | ttaagaaggagatatacatatgatgaagcgcaatattctggcagtgatcgtccctgctctgttagtagcaggt |
OmpF-RihA F1 | ccctgctctgttagtagcaggtactgcaaacgctatgGCACTGCCAATTCTGTTAG |
OmpT F1 | tttaagaaggagatatacatatgcgggcgaaacttctgggaatagtcctgacaacccctattgcga |
OmpT-RihA F1 | cctgacaacccctattgcgatcagctcttttgctatgGCACTGCCAATTCTGTTAG |
PhoA F1 | tttaagaaggagatatacatatgaaacaaagcactattgcactggcactcttaccgttactgtttacccctgt |
PhoA-RihA F1 | cttaccgttactgtttacccctgtgacaaaagccatgGCACTGCCAATTCTGTT |
PhoE F1 | tttaagaaggagatatacatatgaaaaagagcactctggcattagtggtgatgggcattgtggcatct |
PhoE-RihA F1 | gatgggcattgtggcatctgcatctgtacaggctatgGCACTGCCAATTCTGTTAG |
dnaJ F1 | GGAATTCCATatggctaagcaagattattacgaga |
dnaJ R1 | GGACTAGTttagcgggtcaggtcgtca |
dnaK F1 | GGAATTCCATatgggtaaaataattggtatcgacctgg |
dnaK R1 | GGACTAGTttattttttgtctttgacttcttcaaattcagc |
GroEL F1 | GGAATTCCATatggcagctaaagacgtaaaattcg |
GroEL R1 | GGACTAGTttacatcatgccgcccatgc |
GroES F1 | GGAATTCCATatgaatattcgtccattgcatgatcg |
GroES R1 | GGACTAGTttacgcttcaacaattgccagaatgt |
grpE F1 | GGAATTCCATatgagtagtaaagaacagaaaacgcctg |
grpE R1 | GGACTAGTttaagcttttgctttcgctacagtaacc |
Cht F1 | cttgaggggttttttTctagAatcgagatcgatctcgatcccg |
Cht R1 | actaccggaagcagtgtcgactc |
To F1 | gatgcgtccggcgtagc |
信号肽 Signal peptide | 来源 Source | 氨基酸序列 Amino acid sequence |
---|---|---|
Fhud | Iron(III)hydroxamate ABC transporter periplasmic binding protein | MSGLPLISRRRLLTAMALSPLLWQMNTAHAAAIDPN |
OmpA | Outer-membrane protein A | MKKTAIAIAVALAGFATVAQA |
OmpC | Outer-membrane protein C | MKVKVLSLLVPALLVAGAANA |
OmpF | Outer-membrane protein F | MMKRNILAVIVPALLVAGTANA |
OmpT | Protease 7 | MRAKLLGIVLTTPIAISSFA |
PhoA | Alkaline phosphatase | VKQSTIALALLPLLFTPVTKA |
PhoE | Outer-membrane pore protein F | MKKSTLALVVMGIVASASVQA |
PelB | Pectin lyase | MKYLLPTAAAGLLLLAAQPAMA |
表3 信号肽的序列及来源
Table 3 Sequences and sources of signal peptides
信号肽 Signal peptide | 来源 Source | 氨基酸序列 Amino acid sequence |
---|---|---|
Fhud | Iron(III)hydroxamate ABC transporter periplasmic binding protein | MSGLPLISRRRLLTAMALSPLLWQMNTAHAAAIDPN |
OmpA | Outer-membrane protein A | MKKTAIAIAVALAGFATVAQA |
OmpC | Outer-membrane protein C | MKVKVLSLLVPALLVAGAANA |
OmpF | Outer-membrane protein F | MMKRNILAVIVPALLVAGTANA |
OmpT | Protease 7 | MRAKLLGIVLTTPIAISSFA |
PhoA | Alkaline phosphatase | VKQSTIALALLPLLFTPVTKA |
PhoE | Outer-membrane pore protein F | MKKSTLALVVMGIVASASVQA |
PelB | Pectin lyase | MKYLLPTAAAGLLLLAAQPAMA |
图3 pET22b-RihA质粒的构建及验证 A:rihA基因PCR扩增(M:DNA marke,1:rihA);B:重组质粒pET22b-RihA的菌落PCR验证(M:DNA marker,1:rihA);C:重组质粒pET22b-RihA双酶切验证(M:DNA marker,1:pET22b-RihA)
Fig. 3 Construction and validation of pET22b-RihA plasmid A:PCR amplification product of rihA(M:DNA marker,1:rihA). B:Colony PCR validation of recombinant plasmid pET22b-RihA(M:DNA marker,1:rih A). C:Double digestion verification of recombinant plasmid pET22b-RihA(M:DNA marker,1:pET22b-RihA)
图4 菌株A、菌株B和BL21菌株表达产物的SDS-PAGE分析 M:蛋白标准品;F:发酵液上清;U:超声破碎上清;P:超声破碎沉淀
Fig.4 SDS-PAGE analysis of expression products of strain A,B and BL21 M:Molecular weight marker;F:fermentation supernatant;U:ultrasonication supernatant;P:precipitation
图5 9株菌在不同时间胞外和胞内RihA酶活力 A:发酵液上清中RihA酶活力;B:菌体超声破碎上清RihA酶活力
Fig.8 Extracellular and intracellular RihA activities of 9 strains at different time A:Extracellular RihA enzyme activity. B:Intracellular RihA enzyme activity
图6 7种信号肽质粒pET22b-Fhud/OmpA/OmpC/OmpF/OmpT/PhoA/PhoE-RihA 菌落PCR验证
Fig.6 Colony PCR verification of seven kinds of signal pep-tide plasmid pET22b-Fhud/OmpA/OmpC/OmpF/OmpT/PhoA/PhoE-RihA M:DNA marker;1:Fhud-rihA(1 088 bp);2:OmpA-rihA(1 043 bp);3:OmpC-rihA(1 043 bp);4:OmpF-rihA(1 047 bp);5:OmpT-rihA(1 042 bp);6:PhoA-rihA(1 045 bp);7:PhoE-rihA(1 045 bp)
图8 串联分子伴侣质粒的构建及验证 A:不同分子伴侣表达盒的PCR扩增,M:DNA marker,1:T7P-dnaJ-T7T(1 401 bp),2:T7P-dnaK-T7T(2 187 bp),3:T7P-groEL-T7T(1 917 bp),4:T7P-groES-T7T(564 bp),5:T7P-grpE-T7T(864 bp);B:串联分子伴侣质粒的PCR验证,M:DNA marker,1:groES-groEL(2 429 bp),2:dnaK-dnaJ-grpE(4 348 bp),3:dnaK-dnaJ-grpE-groES-groEL(6 725 bp)
Fig.8 Construction and validation of tandem molecular chaperone plasmids A:PCR amplification of different chaperone expression boxes,M:DNA marker,1:T7P-dnaJ-T7T(1 401 bp),2:T7P-dnaK-T7T(2 187 bp),3:T7P-groEL-T7T(1 917 bp),4:T7P-groES-T7T(564 bp),5:T7P-grpE-T7T(864 bp). B:PCR validation of tandem molecular chaperone plasmids,M:DNA marker,1:groES-groEL(2 429 bp),2:dnaK-dnaJ-grpE(4 348 bp),3:dnaK-dnaJ-grpE-groES-groEL(6 725 bp)
图9 不同菌株表达产物的SDS-PAGE分析 M:蛋白标准品;F:发酵液上清;U:超声破碎上清;P:超声破碎沉淀
Fig.9 SDS-PAGE analysis of expression products of differ-ent strains M:Molecular weight marker;F:fermentation supernatant;U:ultrasonication supernatant;P:precipitation
投底物量 Amount of the substrate | 发酵液中初始尿苷浓度 Initial uridine concentration of fermentation broth/(g·L-1) | 尿嘧啶产量 Uracil yield/(g·L-1) | 尿嘧啶产率 Uracil productive rate /% |
---|---|---|---|
1倍体积/20 mL One time the volume/20 mL | 163.02±4.50 | 73.45±1.03 | 98.16±1.38 |
1.5倍体积/30 mL 1.5 times the volume/30 mL | 195.60±6.01 | 88.09±1.33 | 98.12±1.48 |
2倍体积/40 mL Two times the volume/40 mL | 218.78±3.95 | 97.90±2.17 | 97.49±2.16 |
表4 不同底物浓度转化时尿嘧啶产量及产率
Table 4 Uracil yield and uracil productive rate transformed with different substrate concentration
投底物量 Amount of the substrate | 发酵液中初始尿苷浓度 Initial uridine concentration of fermentation broth/(g·L-1) | 尿嘧啶产量 Uracil yield/(g·L-1) | 尿嘧啶产率 Uracil productive rate /% |
---|---|---|---|
1倍体积/20 mL One time the volume/20 mL | 163.02±4.50 | 73.45±1.03 | 98.16±1.38 |
1.5倍体积/30 mL 1.5 times the volume/30 mL | 195.60±6.01 | 88.09±1.33 | 98.12±1.48 |
2倍体积/40 mL Two times the volume/40 mL | 218.78±3.95 | 97.90±2.17 | 97.49±2.16 |
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