Research on Improving the Catalytic Activity of Thermoacidophilic Type III Pullulan Hydrolase TK-PUL by Error-prone PCR

ZENG Jing; GUO Jianjun; YUAN Lin

doi:10.13386/j.issn1002-0306.2021120091

Volume 43 Issue 18

Sep. 2022

Turn off MathJax

Article Contents

Abstract

References

Science and Technology of Food Industry > 2022 > 43(18): 130-136. > DOI: 10.13386/j.issn1002-0306.2021120091

ZENG Jing, GUO Jianjun, YUAN Lin. Research on Improving the Catalytic Activity of Thermoacidophilic Type III Pullulan Hydrolase TK-PUL by Error-prone PCR[J]. Science and Technology of Food Industry, 2022, 43(18): 130−136. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2021120091.

Citation:

PDF (2403 KB)

Research on Improving the Catalytic Activity of Thermoacidophilic Type III Pullulan Hydrolase TK-PUL by Error-prone PCR

Institute of Microbiology, Jiangxi Academy of Sciences, Nanchang 330096, China

More Information

Received Date: December 07, 2021
Available Online: July 10, 2022

Graphical Abstract

Abstract

Abstract

Thermoacidophilic type III pullulan hydrolase TK-PUL can completely hydrolyze starch to starch sugar under liquefaction condition. It has great application potential in the ''one step liquefaction-saccharification'' starch syrup production process. In this study, error-prone PCR technology was used to improve the catalytic activity of TK-PUL. After two sequential error-prone PCR and high-throughput screening, the mutant L538D with improved catalytic activity was obtained. Compared with TK-PUL, the specific activity of L538D with soluble starch as substrate increased by 50%, the specific activity of L538D toward pullulan increased by 21%. And the k_cat/K_m value of L538D for soluble starch, pullulan increased by 44% and 27%, respectively. In other words, the hydrolytic activity of L538D toward α-1,4-glycosidic bond and α-1,6-glycosidic bond were significantly improved, and the hydrolytic activity of L538D toward α-1,4-glycosidic bond increased to a greater degree. Homology modeling showed that replacing Leu538 with Asp in TK-PUL may improve the flexibility of the loop structure including the catalytic site Glu538 by shortening the length of the side chain of amino acid residues and reducing the hydrophobicity of amino acid residues, thus improving the catalytic activity of the enzyme. The results demonstrated that Leu538 in TK-PUL is vital for its catalytic activity.

FullText(HTML)

References (30)

References

[1]	BELLO-PEREZ L A, FLORES-SILVA P C, AGAMA-ACEVEDO E, et al. Starch digestibility: Past, present, and future[J]. Journal of the Science of Food and Agriculture,2020,100(14):5009−5016. doi: 10.1002/jsfa.8955
[2]	SCHMIELE M, SAMPAIO U M, CLERICI M T P S. Basic principles: Composition and properties of starch[M]. New York: Academic Press, 2019: 1-22.
[3]	OKAFOR D C, OFOEDU C E, NWAKAUDU A, et al. Enzymes as additives in starch processing: A short overview[M]. New York: Academic Press, 2019: 149-168.
[4]	MIAO M, JIANG B, JIN Z, et al. Microbial starch-converting enzymes: Recent insights and perspectives[J]. Comprehensive Reviews in Food Science and Food Safety,2018,17(5):1238−1260. doi: 10.1111/1541-4337.12381
[5]	ZHANG Q G, HAN Y, XIAO H Z. Microbial α-amylase: A biomolecular overview[J]. Process Biochemistry,2017,53:88−101. doi: 10.1016/j.procbio.2016.11.012
[6]	NISHA M, SATYANARAYANA T. Characteristics, protein engineering and applications of microbial thermostable pullulanases and pullulan hydrolases[J]. Applied Microbiology and Biotechnology,2016,100(13):5661−5679. doi: 10.1007/s00253-016-7572-y
[7]	WANG X, NIE Y, XU Y. Industrially produced pullulanases with thermostability: Discovery, engineering, and heterologous expression[J]. Bioresource Technology,2019:360−371.
[8]	AKASSOU M, GROLEAU D. Advances and challenges in the production of extracellular thermoduric pullulanases by wild-type and recombinant microorganisms: A review[J]. Critical Reviews in Biotechnology,2019,39(3):337−350. doi: 10.1080/07388551.2019.1566202
[9]	XIA W, ZHANG K, SU L, et al. Microbial starch debranching enzymes: Developments and applications[J]. Biotechnology Advances,2021:107786.
[10]	HAN T, ZENG F, LI Z, et al. Biochemical characterization of a recombinant pullulanase from Thermococcus kodakarensis KOD1[J]. Letters in Applied Microbiology,2013,57(4):336−343. doi: 10.1111/lam.12118
[11]	AHMAD N, RASHID N, HAIDER M S, et al. Novel maltotriose-hydrolyzing thermoacidophilic type III pullulan hydrolase from Thermococcus kodakarensis[J]. Applied and Environmental Microbiology,2014,80(3):1108−1114. doi: 10.1128/AEM.03139-13
[12]	TOOR K J, AHMAD N, MUHAMMAD M A, et al. TK-PUL, a pullulan hydrolase type III from Thermococcus kodakarensis, a potential candidate for simultaneous liquefaction and saccharification of starch[J]. Amylase,2020,4(1):45−55. doi: 10.1515/amylase-2020-0004
[13]	曾静, 郭建军, 袁林. 嗜热酸性普鲁兰水解酶Ⅲ的高效分泌表达及其酶学性质[J]. 食品工业科技,2020,41(3):98−103, 109. [ZENG J, GUO J J, YUAN L. Efficient secretory expression of thermoacidiphilic type III pullulan hydrolase and its enzymatic properties[J]. Science and Technology of Food industry,2020,41(3):98−103, 109. ZENG J, GUO J J, YUAN L. Efficient secretory expression of thermoacidiphilic type III pullulan hydrolase and its enzymatic properties[J]. Science and Technology of Food industry, 2020, 41(3): 98-103, 109.
[14]	KATARIA A, SHARMA R, SHARMA S, et al. Recent applications of bio-engineering principles to modulate the functionality of proteins in food systems[J]. Trends in Food Science & Technology,2021,113:54−65.
[15]	XU Y, WU Y, LV X, et al. Design and construction of novel biocatalyst for bioprocessing: Recent advances and future outlook[J]. Bioresource Technology,2021:125071.
[16]	ZENG W, GUO L, XU S, et al. High-throughput screening technology in industrial biotechnology[J]. Trends in Biotechnology,2020,38(8):888−906. doi: 10.1016/j.tibtech.2020.01.001
[17]	WU H, TIAN X, DONG Z, et al. Engineering of Bacillus amyloliquefaciens α-amylase with improved calcium independence and catalytic efficiency by error-prone PCR[J]. Starch-Stärke,2018,70(3−4):1700175.
[18]	GAOL M D F L, SATYA A A, PUSPITASARI E, et al. Increasing hydrolytic activity of lipase on palm oil by PCR-based random mutagenesis[J]. International Journal of Oil Palm,2020,3(3):78−87. doi: 10.35876/ijop.v3i3.53
[19]	DAI S, YAO Q, YU G, et al. Biochemical characterization of a novel bacterial laccase and improvement of its efficiency by directed evolution on dye degradation[J]. Frontiers in Microbiology,2021:12.
[20]	SUN Z B, XU J L, LU X, et al. Directed mutation of β-glucanases from probiotics to enhance enzymatic activity, thermal and pH stability[J]. Archives of Microbiology,2020,202(7):1749−1756. doi: 10.1007/s00203-020-01886-z
[21]	SU L, YAO K, WU J. Improved activity of Sulfolobus acidocaldarius maltooligosyltrehalose synthase through directed evolution[J]. Journal of Agricultural and Food Chemistry,2020,68(15):4456−4463. doi: 10.1021/acs.jafc.0c00948
[22]	BASIT A, TAJWAR R, SADAF S, et al. Improvement in activity of cellulase Cel12A of Thermotoga neapolitana by error prone PCR[J]. Journal of Biotechnology,2019,306:118−124. doi: 10.1016/j.jbiotec.2019.09.011
[23]	ANAGNOSTOPOULOS C, SPIZIZEN J. Requirements for transformation in Bacillus subtilis[J]. Journal of Bacteriol,1961,81(5):741−746. doi: 10.1128/jb.81.5.741-746.1961
[24]	GREEN M R, SAMBROOK J. Molecular cloning: A laboratory manual[M]. New York: Cold Spring Harbor Laboratory Press, 2012: 101-200.
[25]	MILLER G L. Use of dinitrosalicylic acid reagent for determination of reducing sugar[J]. Analytical Chemistry,1959,31(3):426−428. doi: 10.1021/ac60147a030
[26]	曾静, 何础阔, 郭建军, 等. N末端结构模块缺失对嗜热酸性III型普鲁兰多糖水解酶TK-PUL酶学性质的影响[J]. 食品科学,2021,42(10):201−208. [ZENG J, HE C K, GUO J J, et al. Effect of truncation of N-terminal structural modules on enzymatic properties of thermoacidiphilic type III pullulan hydrolase TK-PUL[J]. Food Science,2021,42(10):201−208. doi: 10.7506/spkx1002-6630-20200408-099 ZENG J, HE C K, GUO J J, et al. Effect of truncation of N-terminal structural modules on enzymatic properties of thermoacidiphilic type III pullulan hydrolase TK-PUL[J]. Food Science, 2021, 42(10): 201-208. doi: 10.7506/spkx1002-6630-20200408-099
[27]	WATERHOUSE A, BERTONI M, BIENERT S, et al. SWISS-MODEL: Homology modelling of protein structures and complexes[J]. Nucleic Acids Research,2018,46(W1):W296−W303. doi: 10.1093/nar/gky427
[28]	LIU B, PENG Q, SHENG M, et al. Directed evolution of sulfonylurea esterase and characterization of a variant with improved activity[J]. Journal of Agricultural and Food Chemistry,2018,67(3):836−843.
[29]	GUO J, COKER A, WOOD S, et al. Structure and function of the type III pullulan hydrolase from Thermococcus kodakarensis[J]. Acta Crystallographica Section D,2018,74(4):305−314. doi: 10.1107/S2059798318001754
[30]	MØLLER M S, HENRIKSEN A, SVENSSON B. Structure and function of α-glucan debranching enzymes[J]. Cellular and Molecular Life Sciences,2016,73(14):2619−2641. doi: 10.1007/s00018-016-2241-y

Supplements (0)

Cited By

Cited by

Periodical cited type(10)

1.	毕泗伟. 食品安全监督抽检在食品监管中的作用. 食品安全导刊. 2025(05): 36-38 .
2.	刘绮琪，陈坤才，许静琳，黄德演，余威，张维蔚. 基于多源数据的肉制品食品安全风险评价研究. 职业与健康. 2024(09): 1158-1166 .
3.	杨瑞，赵豪豪，马海军. 食品安全抽样技术发展与提升. 食品安全质量检测学报. 2024(14): 293-298 .
4.	杨子恩. 食品安全抽检数据质量提升策略研究. 食品安全导刊. 2023(04): 72-76 .
5.	曹东丽，翟雨佳，杨栩，刘园，徐慧静. 冷冻饮品网络销售发展现状及对策研究. 质量安全与检验检测. 2023(01): 53-57 .
6.	覃思森. 食品安全监督抽检的效能提升与发展建议. 食品安全导刊. 2023(30): 50-53 .
7.	高超. 食品抽检重复性问题探析. 现代食品. 2023(17): 165-168 .
8.	蓝小飞，谢琳，张丽娟，陈婷，施文婷. 食品安全指数法评价嘉兴市售水产品污染物残留风险. 湖北农业科学. 2023(S1): 200-204 .
9.	郝相帅. 食品抽样环节存在的问题及其对策. 食品安全导刊. 2023(29): 179-181+185 .
10.	范晓燕，张晓佳，易博春，靳亚卫，王艳英. 县域餐饮环节食品安全监管问题及提升策略. 食品安全导刊. 2023(35): 20-23 .