Improving the Specific Activity and Stability of Alkaline Pectinase PEL3 Through SpyTag/SpyCatcher Cyclization

Overview

Journal Biotechnol Lett

Specialties Biochemistry
Biotechnology

Date 2023 May 12

PMID 37171698

Authors

Chao Du

Siqin Tan

Lin Liu

Yuling Zhou

Pan Wu

Guimin Zhang

Affiliations

Soon will be listed here.

Abstract

Objectives: PEL3, an alkaline pectinase, exhibited the highest activity among documented alkaline pectate lyases reported in our early study. Unfortunately, undesired thermal stability hampering its industrial application. The purpose of this study is to enhance the performance of wild-type PEL3 (W-PEL3) based on SpyTag/SpyCatcher-mediated cyclization.

Results: The cyclized PEL3 (C-PEL3) was observed to fold correctly and generate a spatial conformation in a head-to-tail manner in E. coli. C-PEL3 exhibited comparable optimum pH and temperature to those of W-PEL3. Moreover, the catalytic activity of C-PEL3 increased by 23% compared to W-PEL3, and the k/K of C-PEL3 was 1.5-fold greater than that of the W-PEL3. Importantly, C-PEL3 showed improved stability compared to W-PEL3. Firstly, C-PEL3 displayed a 65% increase in residual activity after treatment at 55 °C for 30 min. Secondly, C-PEL3 was prone to resist heat-induced protein aggregation. Thirdly, C-PEL3 exhibited metal ion stability. Circular dichroism analysis revealed that C-PEL3 was more capable of maintaining its secondary structures than W-PEL3 upon heat treatment.

Conclusions: C-PEL3, the initial example of a circular pectinase through SpyTag/SpyCatcher cyclization, exhibits superior performance and represents a highly encouraging contender for industrial utilization.

Citing Articles

Current status and emerging frontiers in enzyme engineering: An industrial perspective.

Ndochinwa O, Wang Q, Amadi O, Nwagu T, Nnamchi C, Okeke E Heliyon. 2024; 10(11):e32673.

PMID: 38912509 PMC: 11193041. DOI: 10.1016/j.heliyon.2024.e32673.

Construction of a versatile fusion protein for targeted therapy and immunotherapy.

Huang X, Yang L, Yang K, Zhou H, Abudureheman T, Zheng W Protein Sci. 2024; 33(4):e4944.

PMID: 38501479 PMC: 10949329. DOI: 10.1002/pro.4944.

Correction to: Improving the specific activity and stability of alkaline pectinase PEL3 through SpyTag/SpyCatcher cyclization.

Du C, Tan S, Liu L, Zhou Y, Wu P, Zhang G Biotechnol Lett. 2023; 45(9):1245-1247.

PMID: 37436534 DOI: 10.1007/s10529-023-03409-4.

References

Aboye T, Camarero J . Biological synthesis of circular polypeptides. J Biol Chem. 2012; 287(32):27026-32. PMC: 3411040. DOI: 10.1074/jbc.R111.305508. View

Anbar M, Gul O, Lamed R, Sezerman U, Bayer E . Improved thermostability of Clostridium thermocellum endoglucanase Cel8A by using consensus-guided mutagenesis. Appl Environ Microbiol. 2012; 78(9):3458-64. PMC: 3346478. DOI: 10.1128/AEM.07985-11. View

Bhattacharya M, Jain N, Bhasne K, Kumari V, Mukhopadhyay S . pH-Induced conformational isomerization of bovine serum albumin studied by extrinsic and intrinsic protein fluorescence. J Fluoresc. 2010; 21(3):1083-90. DOI: 10.1007/s10895-010-0781-3. View

Dinu D, Nechifor M, Stoian G, Costache M, Dinischiotu A . Enzymes with new biochemical properties in the pectinolytic complex produced by Aspergillus niger MIUG 16. J Biotechnol. 2007; 131(2):128-37. DOI: 10.1016/j.jbiotec.2007.06.005. View

Ewert J, Gluck C, Strasdeit H, Fischer L, Stressler T . Influence of the metal ion on the enzyme activity and kinetics of PepA from Lactobacillus delbrueckii. Enzyme Microb Technol. 2018; 110:69-78. DOI: 10.1016/j.enzmictec.2017.10.002. View

Fan X, Li X, Zhou Y, Mei M, Liu P, Zhao J . Quantitative Analysis of the Substrate Specificity of Human Rhinovirus 3C Protease and Exploration of Its Substrate Recognition Mechanisms. ACS Chem Biol. 2019; 15(1):63-73. DOI: 10.1021/acschembio.9b00539. View

Gao D, Sun X, Liu M, Liu Y, Zhang H, Shi X . Characterization of Thermostable and Chimeric Enzymes via Isopeptide Bond-Mediated Molecular Cyclization. J Agric Food Chem. 2019; 67(24):6837-6846. DOI: 10.1021/acs.jafc.9b01459. View

Goldenberg D, Creighton T . Circular and circularly permuted forms of bovine pancreatic trypsin inhibitor. J Mol Biol. 1983; 165(2):407-13. DOI: 10.1016/s0022-2836(83)80265-4. View

Gomez B, Gullon B, Remoroza C, Schols H, Parajo J, Alonso J . Purification, characterization, and prebiotic properties of pectic oligosaccharides from orange peel wastes. J Agric Food Chem. 2014; 62(40):9769-82. DOI: 10.1021/jf503475b. View

10.

Yang L, Zhang J, Liu D, Han T, Qin Q, Wang A . Ancestral glycoprotein hormone and its cognate receptor present in primitive chordate ascidian: Molecular identification and functional characterization. Int J Biol Macromol. 2023; 229:401-412. DOI: 10.1016/j.ijbiomac.2022.12.297. View

11.

John J, Kaimal K, Smith M, Rahman P, Chellam P . Advances in upstream and downstream strategies of pectinase bioprocessing: A review. Int J Biol Macromol. 2020; 162:1086-1099. DOI: 10.1016/j.ijbiomac.2020.06.224. View

12.

Klug-Santner B, Schnitzhofer W, Vrsanska M, Weber J, Agrawal P, Nierstrasz V . Purification and characterization of a new bioscouring pectate lyase from Bacillus pumilus BK2. J Biotechnol. 2005; 121(3):390-401. DOI: 10.1016/j.jbiotec.2005.07.019. View

13.

Liang C, Gui X, Zhou C, Xue Y, Ma Y, Tang S . Improving the thermoactivity and thermostability of pectate lyase from Bacillus pumilus for ramie degumming. Appl Microbiol Biotechnol. 2014; 99(6):2673-82. DOI: 10.1007/s00253-014-6091-y. View

14.

Lojkowska E, Masclaux C, Boccara M, Hugouvieux-Cotte-Pattat N . Characterization of the pelL gene encoding a novel pectate lyase of Erwinia chrysanthemi 3937. Mol Microbiol. 1995; 16(6):1183-95. DOI: 10.1111/j.1365-2958.1995.tb02341.x. View

15.

Ni Z, Li N, Xu P, Guo Z, Zong M, Lou W . Enhancement of thermostability and catalytic properties of ammonia lyase through disulfide bond construction and backbone cyclization. Int J Biol Macromol. 2022; 219:804-811. DOI: 10.1016/j.ijbiomac.2022.07.213. View

16.

Peng X, Chen G, Han Z, Yang J . High-level secretive expression of a novel achieved Talaromyces cellulolyticus endo-polygalacturonase in Pichia pastoris by improving gene dosage for hydrolysis of natural pectin. World J Microbiol Biotechnol. 2019; 35(6):84. DOI: 10.1007/s11274-019-2657-2. View

17.

Polizzi K, Bommarius A, Broering J, Chaparro-Riggers J . Stability of biocatalysts. Curr Opin Chem Biol. 2007; 11(2):220-5. DOI: 10.1016/j.cbpa.2007.01.685. View

18.

Schoene C, Bennett S, Howarth M . SpyRing interrogation: analyzing how enzyme resilience can be achieved with phytase and distinct cyclization chemistries. Sci Rep. 2016; 6:21151. PMC: 4748275. DOI: 10.1038/srep21151. View

19.

Shi X, Zhang L, Wu F, Fu Y, Yin S, Si Y . Kinetics for Cu(2+) induced Sepia pharaonis arginine kinase inactivation and aggregation. Int J Biol Macromol. 2016; 91:926-33. DOI: 10.1016/j.ijbiomac.2016.06.049. View

20.

Si M, Xu Q, Jiang L, Huang H . SpyTag/SpyCatcher Cyclization Enhances the Thermostability of Firefly Luciferase. PLoS One. 2016; 11(9):e0162318. PMC: 5033358. DOI: 10.1371/journal.pone.0162318. View