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食品与发酵工业  2021, Vol. 47 Issue (3): 25-30    DOI: 10.13995/j.cnki.11-1802/ts.025498
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
α-L-鼠李糖苷酶AnRhaE在毕赤酵母中的表达及应用
叶德晓1, 黄佳俊1, 卢宇靖2, 林育成2, 李慧灵1, 谭景航1, 周金林1*
1(佛山市汇腾生物技术有限公司,广东 佛山,528225)
2(广东金骏康生物技术有限公司,广东 佛山,528225)
Expression of α-L-rhamnosidase AnRhaE in Pichia pastoris and its application
YE Dexiao1, HUANG Jiajun1, LU Yujing2, LIN Yucheng2, LI Huiling1, TAN Jinghang1, ZHOU Jinlin1*
1(HuiTeng (Foshan) Biotechnology Co.Ltd., Foshan 528225, China)
2(Golden Health (Guangdong) Biotechnology Co.Ltd., Foshan 528225, China)
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摘要 α-L-鼠李糖苷酶对天然产物之间的转化具有重要的作用和应用价值。该研究采用毕赤酵母表达α-L-鼠李糖苷酶AnRhaE,构建pPIC9K-AnRhaE表达载体,电转化至毕赤酵母GS115和KM71H中,经诱导表达,SDS-PAGE电泳显示GS115更有利于生产AnRhaE酶。AnRhaE酶在45~55 ℃时酶活力高,最适温度为55 ℃,对pH的耐受范围较广,pH 4.5~pH 8时酶活性稳定,最适pH为5。AnRhaE能水解α-1,2、α-1,6和2个鼠李糖苷连接的底物,包括柚苷二氢查耳酮、柚苷、芦丁、新橙皮苷、橙皮苷和朝藿定C等天然产物,但是AnRhaE对α-1,2糖苷键连接的底物具有较高的活性,而对α-1,6糖苷键连接的底物活性较差,对朝藿定C的活性较好,能将朝藿定C完全水解生成淫羊藿苷,因此AnRhaE酶在天然产物之间的转化具有较好的应用价值和前景。
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卢宇靖
林育成
叶德晓
黄佳俊
李慧灵
卢宇靖
谭景航
林育成
周金林
李慧灵
谭景航
周金林
关键词:  毕赤酵母  异源表达  α-L-鼠李糖苷酶  生物催化  AnRhaE  天然产物  毕赤酵母  异源表达  生物催化  天然产物    
Abstract: The α-L-rhamnosidase plays an important role in the conversion of natural products, and Pichia pastoris was used to express α-L-rhamnosidase AnRhaE. The pPIC9K-AnRhaE expression vector was constructed and transformed into Pichia pastoris GS115 and KM71H. After induced expression, SDS-PAGE electrophoresis showed that GS115 was more conducive to the production of AnRhaE. High AnRhaE activity was obtained at 45-55 ℃, and the optimum temperature was 55 ℃. AnRhaE was stable at pH 4.5-8, and the optimum pH was 5. AnRhaE could hydrolyze α-1,2, α-1,6 and rhamnosyl-rhamnosyl glycosidic bond, including naringin dihydrogen chalcone to trilobatin, naringin to prunin, rutin to isoquercetin, neohesperidin to hesperetin-7-O-glucoside, hesperidin to hesperetin-7-O-glucoside and epimedin C to icariin. AnRhaE had high activity to α-1,2 glycosidic bond than the α-1,6 glycosidic bond, and could complete hydrolysis epimedin C to icariin. Therefore, AnRhaE is potentially applicable in enzymatic conversion of natural products.
Key words:  α-L-rhamnosidase    AnRhaE    Pichia pastoris    heterologous expression    α-L-rhamnosidase    AnRhaE    biocatalysis    Pichia pastoris    natural products    heterologous expression    biocatalysis    natural products
收稿日期:  2020-08-27      修回日期:  2020-09-16           出版日期:  2021-02-15      发布日期:  2021-03-08      期的出版日期:  2021-02-15
基金资助: 佛山市南海区“蓝海人才计划”项目
作者简介:  硕士(周金林博士为通讯作者,E-mail:david_zhou@goldenhealth.com.cn)
引用本文:    
卢宇靖,林育成,叶德晓,等. α-L-鼠李糖苷酶AnRhaE在毕赤酵母中的表达及应用[J]. 食品与发酵工业, 2021, 47(3): 25-30.
LU Yujing,LIN Yucheng,YE Dexiao,et al. Expression of α-L-rhamnosidase AnRhaE in Pichia pastoris and its application[J]. Food and Fermentation Industries, 2021, 47(3): 25-30.
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http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.025498  或          http://sf1970.cnif.cn/CN/Y2021/V47/I3/25
[1] HUANG G L,LV M J,HU J C,et al.Glycosylation and activities of natural products[J].Mini Reviews in Medicinal Chemistry,2016,16(12):1 013-1 016.
[2] YADAV V,YADAV P K,YADAV S,et al.α-L-Rhamnosidase:A review[J].Process Biochemistry,2010,45(8):1 226-1 235.
[3] 王艳君, 刘同军,曹涛,等.α-L-鼠李糖苷酶的研究进展[J].中国酿造,2010(10):11-15
WANG Y J,LIU T J,CAO T,et al.Research progress of α-L-rhamnosidase[J].China Brewing,2010(10):11-15.
[4] WANG M Y,LI H,XU F X,et al.Diterpenoid lead stevioside and its hydrolysis products steviol and isosteviol:Biological activity and structural modification[J].European Journal of Medicinal Chemistry,2018,156:885-906.
[5] SZEJA W,GRYNKIEWICZ G,RUSIN A.Isoflavones,their glycosides and glycoconjugates.Synthesis and biological activity[J].Current Organic Chemistry,2017,21(3):218-235.
[6] KITTI N,PLUMB G W,BERRIN J G,et al.Deglycosylation by small intestinal epithelial cell β-glucosidases is a critical step in the absorption and metabolism of dietary flavonoid glycosides in humans[J].European Journal of Nutrition,2003,42(1):29-42.
[7] LOMBARD V,GOLACONDA R H,DRULA E,et al.The carbohydrate-active enzymes database (CAZy) in 2013[J].Nucleic Acids Research.2014,42:490-495.
[8] SUDHIR K,GLEN S,KOICHIRO T.MEGA7:Molecular evolutionary genetics analysis Version 7.0 for bigger datasets[J].Molecular Biology and Evolution,2016,33(7):1 870-1 874.
[9] DANIELA G,BARBORA F,PETR H,et al.Recombinant α-L-rhamnosidase from Aspergillus terreus in selective trimming of rutin[J].Process Biochemistry,2012,47(5):828-835.
[10] NIELSEN H.Predicting secretory proteins with Signal P[J].Methods in Molecular Biology,2017,1 611:59-73.
[11] LENKA W,PETR M,DANIELA G,et al.Preparatory production of quercetin-3-β-D-glucopyranoside using alkali-tolerant thermostable α-L-rhamnosidase from Aspergillus terreus[J].Bioresource Technology,2011,115:222-227.
[12] FUJIMOTO Z,JACKSON A,MICHIKAWA M,et al.The structure of a Streptomyces avermitilis α-L-rhamnosidase reveals a novel carbohydrate-binding module CBM67 within the six-domain arrangement[J].Journal of Biological Chemistry,2013,288(17):12 376-12 385.
[13] GUILLOTIN L,KIM H,TRAORE Y,et al.Biochemical characterization of the α-L-Rhamnosidase DtRha from Dictyoglomus thermophilum:Application to the selective derhamnosylation of natural flavonoids[J].Acs Omega,2019,4(1):1 916-1 922.
[14] CUI Z,MARUYAMA Y,MIKAMI B,et al.Crystallization and preliminary crystallographic analysis of the family GH78 α-L-rhamnosidase RhaB from Bacillus sp.GL1[J].Acta Crystallographica,2006,62(7):646-648.
[15] LI B,JI Y,LI Y,et al.Characterization of a glycoside hydrolase family 78 α-L-rhamnosidase from Bacteroides thetaiotaomicron VPI-5482 and identification of functional residues[J].Biotech,2018,8(2):120.
[16] ELLIS C O'N,CLARE E M S,MICHAEL J P,et al.Crystal structure of a novel two domain GH78 family α-rhamnosidase from Klebsiella oxytoca with rhamnose bound[J].Proteins,2015;83(9):1 742-1 749.
[17] NDEH D,ROGOWSKI A,CARTMELL A,et al.Complex pectin metabolism by gut bacteria reveals novel catalytic functions[J].Nature,2017,544(7 648):65-70
[18] TERRY B,DRIZA D V,IZZO V,et al.Crystal structures and directed evolution of the α-L-rhamnosidase RHA-P from Novosphingobium sp.PP1Y[J].The FASEB Journal,2020,34(S1):1.
[19] PACHL P,SKERLOVÁ J,SIMCÍKOVÁ D,et al.Crystal structure of native α-L-rhamnosidase from Aspergillus terreus[J].Acta Crystallographica Section D Structural Biology,2018,74(11):1 078-1 084.
[20] 章如安, 杨晟,邱荣德,等.巴斯德毕赤酵母表达体系研究及进展[J].微生物学通报,2000,27(5):371-373.
ZHANG R A,YANG S,QIU R D,et al.Research and progress on the expression system of Pichia pastoris[J].Microbiology China,2000,27(5):371-373.
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