Directed Evolution and High-level Expression of <i>α</i>-Amylase from <i>Thermomyces dupontii</i>

JIN Yan; LI Yanxiao; MA Junwen; WANG Yuchuan; YAN Qiaojuan; JIANG Zhengqiang

doi:10.13386/j.issn1002-0306.2021100086

Volume 43 Issue 13

Jun. 2022

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Science and Technology of Food Industry > 2022 > 43(13): 139-147. > DOI: 10.13386/j.issn1002-0306.2021100086

JIN Yan, LI Yanxiao, MA Junwen, et al. Directed Evolution and High-level Expression of α-Amylase from Thermomyces dupontii[J]. Science and Technology of Food Industry, 2022, 43(13): 139−147. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2021100086.

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Directed Evolution and High-level Expression of α-Amylase from Thermomyces dupontii

1.
Key Laboratory of Food Bioengineering (China National Light Industry), College of Engineering, China Agricultural University, Beijing 100083, China
2.
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China
3.
College of Food Science and Engineering, Nanjing University of Finance and Economics/Collaborative Innovation Center for Modern Grain Circulation and Safety, Nanjing 210023, China

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Received Date: October 12, 2021
Available Online: April 30, 2022

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Abstract

Abstract

Objective: The α-amylase from Thermomyces dupontii was modified by direct evolution to improve its thermostability and expression level. Methods: A mutation library of α-amylase (Td-amy) from Thermomyces dupontii was constructed by error-prone PCR. The mutants with higher thermostability and specific activity were selected through high-throughput screening, analysis of the mutants by site-directed mutagenesis and homologous structure simulation, and expression in Pichia pastoris. Results: A positive mutant (mTd-amy) was selected. The optimal temperature of mTd-amy was 60 ℃, which was higher than that of the wild type enzyme Td-amy (55 ℃) by 5 ℃. The specific activity of mTd-amy (466.3 U/mg) was 2.0 times higher than that of Td-amy (227.9 U/mg). Sequence and mutation analysis revealed that four sites (Ala4Val, Ala122Val, Lys194Arg and Ala468Asp) in mTd-amy were mutated, and Ala122 Val and Ala468Asp played a key role in the specific activity and optimal temperature of the mutant. mTd-amy was further expressed in Pichia pastoris, and its expression level was up to 64696 U/mL through high cell density fermentation. Conclusion: Through directed evolution, a positive mutant of with high optimal temperature and specific activity was obtained. It provides a theoretical basis for the molecular modification and industrial application of α-amylase.
- α-amylase,
- directed evolution,
- specific activity,
- site-directed mutation,
- high cell density fermentation,
- Thermomycesdupontii

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References

[1]	KHLESTKIN V K, PELTEK S E, KOLCHANOV N A. Review of direct chemical and biochemical transformations of starch[J]. Carbohydrate Polymers,2018,181:460−476. doi: 10.1016/j.carbpol.2017.10.035
[2]	何亚洁. α-淀粉酶发酵生产影响因素的研究进展[J]. 现代食品,2020(8):68−69,75. [HE Y J. Research progress on the factors affecting the fermentation production of α-amylase[J]. Modern Food,2020(8):68−69,75. HE Y J. Research progress on the factors affecting the fermentation production of α-amylase[J]. Modern Food, 2020(8): 68-69, 75.
[3]	韩承勇, 刘峰, 韩先卓, 等. 一种双酶法糖化制备超高麦芽糖的方法: 江苏省, CN111041054A[P]. 2020-04-21. HAN C Y, LIU F, HAN X Z, et al. A method for preparing ultra-high maltose by double-enzyme saccharification: Jiangsu Province: CN111041054A[P]. 2020-04-21.
[4]	俞峰, 刘玉春, 柳加润, 等. 耐高温α-淀粉酶在淀粉质酒精生产中的应用研究[J]. 酿酒,2020,47(5):84−88. [YU F, LIU Y C, LIU J R, et al. Research on application of high temperature resistant α-amylase in starch alcohol production[J]. Liquor Making,2020,47(5):84−88. YU F, LIU Y C, LIU J R, et al. Research on application of high temperature resistant α-amylase in starch alcohol production[J]. Liquor Making, 2020, 47(5): 84-88.
[5]	AMOLD F H, VOLKOV A A. Directed evolution of biocatalysts[J]. Current Opinion in Chemical Biology,1999,3(1):54−59. doi: 10.1016/S1367-5931(99)80010-6
[6]	TURNER N J. Directed evolution drives the next generation of biocatalysts[J]. Nature Chemical Biology,2009,5(8):567−573. doi: 10.1038/nchembio.203
[7]	HUANG L, SHAN M Y, MA J Y, et al. Directed evolution of α-amylase from Bacillus licheniformis to enhance its acid-stable performance[J]. Biologia,2019,74:1363−1372. doi: 10.2478/s11756-019-00262-7
[8]	WU H Y, TIAN X J, DONG Z X, et al. Engineering of Bacillus amyloliquefaciens α-amylase with improved calcium independence and catalytic efficiency by error-prone PCR[J]. Starch-starke,2018(70):1700175.
[9]	刘雪莲, 申培立, 牛丹丹, 等. 地衣芽胞杆菌α-淀粉酶耐热耐酸突变体的酶学性质[J]. 食品与发酵工业,2020,46(2):7−10. [LIU X L, SHEN P L, NIU D D, et al. Enzymatic properties of heat and acid tolerant mutants of Bacillus licheniformis α-amylase[J]. Food and Fermentation Industries,2020,46(2):7−10. LIU X L, SHEN P L, NIU D D, et al. Enzymatic properties of heat and acid tolerant mutants of Bacillus licheniformis α-amylase[J]. Food and Fermentation Industries, 2020, 46(2): 7-10.
[10]	赵宁, 王玉川, 易萍, 等. 樟绒枝霉α-淀粉酶在毕赤酵母中的高效表达及在麦芽糖浆制备中的作用[J]. 食品与发酵工业,2019,45(2):1−6. [ZHAO N, WANG Y C, YI P, et al. High-efficiency expression of Cladosporium cinnamomea α-amylase in Pichia pastoris and its role in the preparation of maltose syrup[J]. Food and Fermentation Industries,2019,45(2):1−6. ZHAO N, WANG Y C, YI P, et al. High-efficiency expression of Cladosporium cinnamomea α-amylase in Pichia pastoris and its role in the preparation of maltose syrup[J]. Food and Fermentation Industries, 2019, 45(2) : 1-6.
[11]	WANG Y C, HU H F, MA J W, et al. A novel high maltose-forming a-amylase from Rhizomucor miehei and its application in the food industry[J]. Food Chemistry,2020,305(Feb.1):125447.1−125447.9.
[12]	WANG Y C, ZHAO N, MA J W, et al. High-level expression of a novel α-amylase from Thermomyces dupontiiin Pichia pastoris and its application in maltose syrup production[J]. International Journal of Biological Macromolecules,2019,127:683−692. doi: 10.1016/j.ijbiomac.2019.01.162
[13]	MILLER G L. Use of dinitrosalicylic acid reagent for determination of reducing sugar[J]. Analytical Biochemistry,1959,31(3):426−428.
[14]	LOWRRY O H, ROSEBROUGH N J, FARR A L, et al. Protein measurement with the folin phenol reagent[J]. Journal of Biological Chemistry,1951(193):265−275.
[15]	赵海燕, 宋晨斌, 刘正亚, 等. 来源于Laceyella sp. 的α-淀粉酶基因克隆、重组表达及酶学性质研究[J]. 生物技术通报,2020,36(8):23−33. [ZHAO H Y, SONG C B, LIU Z Y, et al. The cloning, recombinant expression and enzymatic properties of α-amylase gene from Laceyella sp J]. Biotechnology Bulletin,2020,36(8):23−33.
[16]	LIU S, AHMED S, FANG Y. Cloning, expression and characterization of a novel α-amylase from Salinispora arenicola CNP193[J]. The Protein Journal,2019,38(6):716−722. doi: 10.1007/s10930-019-09870-3
[17]	XIAO Z, WU M, GROSSE S, et al. Genome mining for new α-amylase and glucoamylase encoding sequences and high level expression of a glucoamylase from Talaromyces stipitatus for potential raw starch hydrolysis[J]. Applied Biochemistry and Biotechnology,2014,172:73−86. doi: 10.1007/s12010-013-0460-3
[18]	YANG H, LIU L, SHIN H, et al. Comparative analysis of heterologous expression, biochemical characterization optimal production of an alkaline α-amylase from alkaliphilic Alkalimonas amylolytica in Escherichia coli and Pichia pastoris[J]. Biotechnology Progress,2013(29):39−47.
[19]	丁梦瑶, 谢会芳, 杨江科. 约氏黄杆菌HSL13中新型α-淀粉酶的表达及酶学性质初探[J]. 生物技术,2020,30(6):525−530, 524. [DING M Y, XIE H F, YANG J K. Expression of a novel α-amylase in Flavobacterium johnsoniae HSL13 and preliminary study on its enzymatic properties[J]. Biotechnology,2020,30(6):525−530, 524. DING MY, XIE H F, YANG J K. Expression of a novel α-amylase in Flavobacterium johnsoniae HSL13 and preliminary study on its enzymatic properties[J]. Biotechnology, 2020, 30(6): 525-530, 524.
[20]	LI Y X, YI P, YAN Q J, et al. Directed evolution of a β-mannanase from Rhizomucor miehei to improve catalytic activity in acidic and thermophilic conditions[J]. Biotechnology for Biofuels, 2017, 10: 143.
[21]	MAKHATADZE G I, LOLADZE V V, ERMOLENKO D N, et al. Contribution of surface salt bridges to protein stability: Guidelines for protein engineering[J]. Journal of Molecular Biology,2003,327:1135−1148. doi: 10.1016/S0022-2836(03)00233-X
[22]	PIELA L, NEMETHY G, SCHERAGA H A. Proline-induced constraints in alpha-helices[J]. Biopolymers,1987,26(9):1587−1600. doi: 10.1002/bip.360260910
[23]	陈国仁. 扩展青霉脂肪酶的定点突变及随机突变[D]. 福州: 福建师范大学, 2005. CHEN G R. Site-directed mutation and random mutation of Penicillium extended lipase[D]. Fujian: Normal University, 2005.
[24]	HE X J, CHEN S Y, WU J P, et al. Highly efficient enzymatic synthesis of tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate with a mutant alcohol dehydrogenase of Lactobacillus kefir[J]. Applied Microbiology and Biotechnology,2015,99(21):8963−8975. doi: 10.1007/s00253-015-6675-1
[25]	姚婷, 李华钟, 房耀维, 等. 定点突变提高Thermococcus siculi HJ21高温酸性α-淀粉酶催化活性[J]. 食品科学,2011,32(15):5. [YAO T, LI H Z, FANG Y W, et al. Site-directed mutagenesis improves the catalytic activity of Thermococcus siculi HJ21 high temperature acid alpha-amylase[J]. Food Science,2011,32(15):5. YAO T, LI H Z, FANG Y W, et al. Site-directed mutagenesis improves the catalytic activity of Thermococcus siculi HJ21 high temperature acid alpha-amylase[J]. Food Science, 2011, 32(15): 5.
[26]	陈春, 宿玲恰, 夏伟, 等. 定向进化提高来源于Arthrobacter ramosus的MTHase的热稳定性[J]. 生物技术通报,2021,37(3):84−91. [CHEN C, SU L Q, XIA W, et al. Directed evolution improves the thermal stability of MTHase derived from Arthrobacter ramosus[J]. Biotechnology Bulletin,2021,37(3):84−91. CHEN C, SU L Q, XIA W, et al. Directed evolution improves the thermal stability of MTHase derived from Arthrobacter ramosus[J]. Biotechnology Bulletin, 2021, 37(3): 84-91.
[27]	史然, 张登娅, 谷懿寰, 等. 地杆菌α-L-岩藻糖苷酶的分子改造及其在合成2'-岩藻糖基乳糖中的应用[J]. 食品科学,2021,42(18):135−142. [SHI R, ZHANG D Y, GU Y H, et al. Direct evolution of the α-L-fucosidase from Pedobacter sp. and its application in the synthesis of 2’-fucosyllactose[J]. Food Science,2021,42(18):135−142. SHI R, ZHANG D Y, GU Y H, et al. Direct evolution of the α-L-fucosidase from Pedobacter sp. and its application in the synthesis of 2’-fucosyllactose[J]. Food Science, 2021, 42(18): 135-142.
[28]	TRABELSI S, SAHNOUN M, ELGHARBI F, et al. Aspergillus oryzae S2 AmyA amylase expression in Pichia pastoris: Production, purification and novel properties[J]. Molecular Biology Reports,2018(46):921−932.
[29]	HE Z, ZHANG L, MAO Y, et al. Cloning of a novel thermostable glucoamylase from thermophilic fungus Rhizomucor pusillus and high-level co-expression with α-amylase in Pichia pastoris[J]. BMC Biotechnology,2014,14:114. doi: 10.1186/s12896-014-0114-8
[30]	WANG J R, LI Y Y, LIU D N, et al. Codon optimization significantly improves the expression level of α-amylase gene from Bacillus licheniformis in Pichia pastoris[J]. BioMed Research International,2015,2015:248680. doi: 10.1155/2015/248680
[31]	何正贵, 尹燕辰, 毛佑志, 等. 微小根毛霉耐热α-淀粉酶基因的克隆与高效表达[J]. 南方农业学报,2014,45(2):165−172. [HE Z G, YIN Y C, MAO Y Z, et al. Cloning and high-efficiency expression of heat-resistant α-amylase gene from Rhizomucor pusillus[J]. Southern Journal of Agricultural Sciences,2014,45(2):165−172. HE Z G, YIN Y C, MAO Y Z, et al. Cloning and high-efficiency expression of heat-resistant α-amylase gene from Rhizomucor pusillus[J]. Southern Journal of Agricultural Sciences, 2014, 45(2): 165-172.
[32]	LI S, ZUO Z, NIU D, et al. Gene cloning, heterologous expression, and characterization of a high maltose-producing α-amylase of Rhizopus oryzae.[J]. Applied Biochemistry Biotechnology,2011,164(5):581−592. doi: 10.1007/s12010-011-9159-5
[33]	ZHU W, CAO Y, LI W, et al. The error-prone PCR of alpha-amylase from Bacillus amyloliquefaciens toward enhanced acid tolerance and higher specific activity[J]. Journal of Pure and Applied Microbiology,2013,7(3):1489−1496.
[34]	马银凤, 申培立, 王彩喆, 等. 中温α-淀粉酶的分子进化及酶学性质[J]. 食品与发酵工业,2021,47(11):14−18. [MA Y F, SHEN P L, WANG C Z, et al. Molecular evolution and biochemical characterization of Bacillus amyloliquefaciens α-amylase[J]. Food and Fermentation Industries,2021,47(11):14−18. MA Y F, SHEN P L, WANG C Z, et al. Molecular evolution and biochemical characterization of Bacillus amyloliquefaciens α-amylase[J]. Food and Fermentation Industries, 2021, 47(11): 14-18.

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