[1]许锦,徐佳慧,张小濛,等.源自嗜热菌的α-L-鼠李糖苷酶交联聚集体的制备及应用[J].林业工程学报,2020,5(02):122-129.[doi:10.13360/ j.issn.2096-1359.201905036]
 XU Jin,XU Jiahui,ZHANG Xiaomeng,et al.Preparation and application of α-L-rhamnosidase cross-linked aggregates from Thermotoga petrophila DSM 13995[J].Journal of Forestry Engineering,2020,5(02):122-129.[doi:10.13360/ j.issn.2096-1359.201905036]
点击复制

源自嗜热菌的α-L-鼠李糖苷酶交联聚集体的制备及应用()
分享到:

《林业工程学报》[ISSN:1001-8081/CN:32-1160/S]

卷:
5
期数:
2020年02期
页码:
122-129
栏目:
林产化学加工
出版日期:
2020-03-11

文章信息/Info

Title:
Preparation and application of α-L-rhamnosidase cross-linked aggregates from Thermotoga petrophila DSM 13995
文章编号:
2096-1359(2020)02-0122-08
作者:
许锦12徐佳慧1张小濛1卢昌宁1赵林果12*
1. 南京林业大学江苏省林业资源高效加工利用协同创新中心, 南京 210037; 2. 南京林业大学化学工程学院,南京 210037
Author(s):
XU Jin12XU Jiahui1ZHANG Xiaomeng1 LU Changning1 ZHAO Linguo12*
1. Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, Nanjing Forestry University, Nanjing 210037, China; 2. College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China
关键词:
α-L-鼠李糖苷酶 嗜热菌Thermotoga petrophila DSM 13995 Pluronic F127 固定化 循环次数
Keywords:
α-L-rhamnosidases Thermotoga petrophila DSM 13995 Pluronic F127 immobilization reusability
分类号:
Q819
DOI:
10.13360/ j.issn.2096-1359.201905036
文献标志码:
A
摘要:
研究了一种制备嗜热菌Thermotoga petrophila DSM 13995来源的α-L-鼠李糖苷酶交联聚集体的方法。试验结果表明:在酶蛋白与Pluronic F127质量比为1:8、沉淀剂硫酸铵质量浓度为75 mg/mL、戊二醛浓度为40 mmol/L时固定α-L-鼠李糖苷酶,酶活回收率可达49.45%。以硫酸铵-戊二醛(CLEA)的常规顺序制备固定化酶(PAG-R)时,酶活回收率仅为30.87%,而在Pluronic F127-硫酸铵-戊二醛(PL-AS-GL)顺序下制备PAG-R,酶活回收率达到60.90%,固定化剩余酶活力为34 U/mL。此外,研究了固定化酶PAG-R的酶学性质,结果表明PAG-R的最适温度为90 ℃,最适pH为5.0,与游离酶TpeRha的酶学性质相近,但在不同温度和pH条件下PAG-R的比酶活更高。PAG-R在90 ℃保温3 h后剩余酶活力为77.11%,而游离酶TpeRha则完全丧失活力,说明固定化酶的温度稳定性得到了较大的提升。以芦丁为底物,比较了PAG-R与TpeRha催化芦丁生成异槲皮素的产率差异。在温度80 ℃、pH 6.5、加酶量4 U/mL、反应时间90 min时,PAG-R能完全转化3 g/L底物,摩尔转化率为100%,产物的生成量是游离酶的2.31倍。连续反应10次后,芦丁相对转化率仍能保持在60%以上,由此可知固定化后的鼠李糖苷酶其催化效率得到了提高,且重复使用性能良好,更有利于工业化应用。
Abstract:
Immobilization of enzymes is an effective method for improving the stability and catalytic efficiency of enzymes. The ratio of their materials has a decisive effect on the recovery of enzyme activity. α-L-rhamnosidase from Thermotoga petrophila DSM 13995(TpeRha)has high catalytic activity, but its thermal stability is poor. Therefore, it is desired to overcome the drawbacks of TpeRha by immobilization. The cross-linked enzyme aggregate(CLEA)is a kind of technology for protein precipitation and cross-linking to form insoluble and stable immobilized enzyme. Based on the immobilized enzyme(PAG-R)prepared by the immobilization of the conventional ammonium sulfate-glutaraldehyde of CLEA, this study improved the sequence of Pluronic F127-ammonium sulfate-glutaraldehyde(PL-AS-GL). Pluronic F127 is the polyoxyethylene polyoxypropylene ether block copolymer, which can improve the stability and activity of the enzyme through the interaction between the hydrophobic segment of the polymer and the hydrophobic region on the enzyme surface. It is demonstrated that the optimal condition of the immobilization of α-L-rhamnosidase was as follows. When the mass ratios of enzyme to Pluronic F127 were 1:8, the mass concentration of ammonium sulfate was 75 mg/mL and the concentration of glutaraldehyde was 40 mmol/L. At this time, the enzyme activity, protein fixation rate and enzyme activity recovery of the immobilized enzyme were 34 U/mL, 52.53% and 60.90%, respectively. After optimizing the ratio of materials of immobilized enzyme, the enzyme properties were improved significantly, i.e., the residual activity was 77.11% at 90 ℃ for 3 h, while the TpeRha of free enzyme lost its activity completely in 30 min. The residual activity was 72.41% at pH 8 and 70 ℃ for 3 h, while under the same condition, the activity of free enzyme was only 23.35%. Simultaneously, the recovery of enzyme activity(34 U/mL)was 23.2% higher than that of the conventional method. Using rutin(3 g/L)as the substrate, the optimal converting conditions were obtained with the highest enzyme expression, which were the enzyme concentration of 4 U/mL, the induction temperature of 80 ℃, the pH of 6.5, and the induction time of 90 min, and the yield of products was 2.31 times higher than that of free enzymes. After 10 times of repeated reaction, the conversion of rutin remained above 60%, which showed a well improvement of the catalytic efficiency and repeated utilization of the immobilized α-L-rhamnosidase.

参考文献/References:

[1] LIM T, JUNG H, HWANG K T. Bioconversion of cyanidin-3-rutinoside to cyanidin-3-glucoside in black raspberry by crude α-L-rhamnosidase from Aspergillus species[J]. Journal of Microbiology and Biotechnology, 2015, 25(11): 1842-1848. DOI:10.4014/jmb.1503.03098.
[2] DE WINTER K, IMCˇÍKOVÁ D, SCHALCK B, et al. Chemoenzymatic synthesis of α-L-rhamnosidase using recombinant α-L-rhamnosidase from Aspergillus terreus[J]. Bioresource Technology, 2013, 147: 640-644. DOI:10.1016/j.biortech.2013.08.083.
[3] MARKOOVÁ K, WEIGNEROVÁ L, ROSENBERG M, et al. Upscale of recombinant α-L-rhamnosidase production by Pichia pastoris Mut(S)strain[J]. Frontiers in Microbiology, 2015, 6: 1140. DOI:10.3389/fmicb.2015.01140.
[4] YADAV V, YADAV P K, YADAV S, et al. α-L-rhamnosidase: a review[J]. Process Biochemistry, 2010, 45(8): 1226-1235. DOI:10.1016/j.procbio.2010.05.025.
[5] ZVERLOV V V, HERTEL C, BRONNENMEIER K, et al. The thermostable α-L-rhamnosidase RamA of Clostridium stercorarium: biochemical characterization and primary structure of a bacterial α-L-rhamnoside hydrolase, a new type of inverting glycoside hydrolase[J]. Molecular Microbiology, 2000, 35(1): 173-179. DOI:10.1046/j.1365-2958.2000.01691.x.
[6] GE L, CHEN A N, PEI J J, et al. Enhancing the thermostability of α-L-rhamnosidase from Aspergillus terreus and the enzymatic conversion of rutin to isoquercitrin by adding sorbitol[J]. BMC Biotechnology, 2017, 17(1): 21. DOI:10.1186/s12896-017-0342-9.
[7] 柯彩霞, 范艳利, 苏枫, 等. 酶的固定化技术最新研究进展[J]. 生物工程学报, 2018, 34(2): 188-203. DOI:10.13345/j.cjb.170164.
KE C X, FAN Y L, SU F,et al. Recent advances in enzyme immobilization[J]. Chinese Journal of Biotechnology, 2018, 34(2): 188-203. DOI:10.13345/j.cjb.170164.
[8] ROESSL U, NAHELLKA J, NIDETZKY B. Carrier-free immobilized enzymes for biocatalysis[J]. Biotechnology Letters, 2010, 32(3): 341-350. DOI:10.1007/s10529-009-0173-4.
[9] SHEIDON R A. Characteristic features and biotechnological applications of cross-linked enzyme aggregates(CLEAs)[J]. Applied Microbiology & Biotechnology, 2011, 92(3): 467-477. DOI:10.1007/s00253-011-3554-2.
[10] GUPTA M N, RAGHAVA S. Enzyme stabilization via cross-linked enzyme aggregates[J]. Methods in Molecular Biology, 2011, 679: 133-145. DOI:10.1007/978-1-60761-895-9_11.
[11] SUTHIWANGCHAROEN N, NAGARAJIN R. Enhancing enzyme stability by construction of polymer-enzyme conjugate micelles for decontamination of organophosphate agents[J]. Biomacromolecules, 2014, 15(4): 1142-1152. DOI:10.1021/bm401531d.
[12] CHANG S K, SEO J H, DONG G K, et al. Engineered whole-cell biocatalyst-based detoxification and detection of neurotoxic organophosphate compounds[J]. Biotechnology Advances, 2014, 32(3): 652-662. DOI:10.1016/j.biotechadv.2014.04.010.
[13] MINKYU K, MANOS G, AARON H, et al. Enhanced activity and stability of organophosphorus hydrolase via interaction with an amphiphilic polymer[J]. Chemical Communications, 2014, 50(40): 5345-5348. DOI:10.1039/C3CC47675H.
[14] CHENG H, ZHAO Y, LUO X, et al. Cross-linked enzyme-polymer conjugates with excellent stability and detergent-enhanced activity for efficient organophosphate degradation[J]. Bioresources and Bioprocessing, 2018, 5(1): 49. DOI:10.1186/s40643-018-0236-2.
[15] XIAO L W, JUN G, JING Y Z, et al. A general method for synthesizing enzyme-polymer conjugates in reverse emulsions using Pluronic as a reactive surfactant[J]. Chemical Communications, 2015, 51(47): 9674-9677. DOI:10.1039/C5CC01776A.
[16] 曾伟秀, 田清青, 赵昕, 等.交联血管紧张素转化酶聚集体的制备及性质[J]. 应用化学, 2013, 30(7): 815-820. DOI:10.3724/SP.J.1095.2013.20412.
ZENG W X, TIAN Q Q, ZHAO X, et al. Preparation and properties of cross-linked angiotensin converting enzyme aggregates[J]. Chinese Journal of Applied Chemistry, 2013, 30(7): 815-820. DOI:10.3724/SP.J.1095.2013.20412.
[17] 黎克纯, 卢建芳, 周菊英, 等. 具有菲环骨架的高分子载体固定淀粉酶交联聚集体[J]. 食品科学, 2017, 38(14): 112-119. DOI:10.7506/spkx1002-6630-201714017.
LI K C, LU J F, ZHOU J Y,et al. Immobilization of cross-linked amylase aggregates on polymer containing phenanthrene skeleton[J]. Food Science, 2017, 38(14): 112-119. DOI:10.7506/spkx1002-6630-201714017.
[18] CUI J D, SUN L M, LI L L, et al. A simple technique of preparing stable CLEAs of phenylalanine ammonia lyase using coaggregation with starch and bovine serum albumin[J]. Applied Biochemistry & Biotechnology, 2013, 170(8): 1827-1837. DOI:10.1007/s12010-013-0317-9.
[19] LI L, LI H X, YAN B, YU S T. Preparation of a reversible soluble-insoluble beta-D-glucosidase with perfect stability and activity[J]. Journal of Biotechnology, 2019, 291: 46-51. DOI:10.1016/j.jbiotec.2018.12.014.
[20] YOSHIDA E, HIDAKA M, FUSHINOBU S, et al. Role of a PA14 domain in determining substrate specificity of a glycoside hydrolase family 3 beta-glucosidase from Kluyveromyces marxianus[J]. Biochemical Journal, 2010, 431(1): 39-49. DOI:10.1042/BJ20100351.
[21] YANG Y, ZHANG X, QIANG Y, et al. A mechanism of glucose tolerance and stimulation of GH1 β-glucosidases[J]. Scientific Reports, 2015, 5: 17296. DOI:10.1038/srep17296.
[22] WU T, PEI J J, GE L, et al. Characterization of a α-L-rhamnosidase from bacteroides thetaiotaomicron with high catalytic efficiency of epimedin C[J]. Bioorganic Chemistry, 2018, 81:461-467.DOI:10.1016/j.bioorg.2018.08.004.

备注/Memo

备注/Memo:
收稿日期:2019-05-22 修回日期:2019-12-05
基金项目:国家自然科学基金(31570565); “十三五”国家重点研发计划(2016YFD0600805)。
作者简介:许锦,女,研究方向为酶工程。通信作者:赵林果,男,教授。E-mail:lgzhao@njfu.edu.cn
更新日期/Last Update: 2020-03-10