Optimization of methanol‐induced expression and His‐tag purification of Saccharomycopsis fibuligera R64 mutant α‐amylase in Pichia pastoris

Clara Claudia; Elsa Destiana; Rista Awalia; Mia Tria Novianti; Taufik Ramdani Tohari; Dewi Astriany; Shinta Kusumawardani; Muhammad Yusuf; Umi Baroroh

doi:10.22146/ijbiotech.100845

Optimization of methanol‐induced expression and His‐tag purification of Saccharomycopsis fibuligera R64 mutant α‐amylase in Pichia pastoris

https://doi.org/10.22146/ijbiotech.100845

Clara Claudia⁽¹⁾, Elsa Destiana⁽²⁾, Rista Awalia⁽³⁾, Mia Tria Novianti⁽⁴⁾, Taufik Ramdani Tohari⁽⁵⁾, Dewi Astriany⁽⁶⁾, Shinta Kusumawardani⁽⁷⁾, Muhammad Yusuf⁽⁸⁾, Umi Baroroh^(9*)

(1) Department of Biotechnology Pharmacy, Indonesian School of Pharmacy, Bandung, 40266, West Java, Indonesia
(2) Department of Biotechnology Pharmacy, Indonesian School of Pharmacy, Bandung, 40266, West Java, Indonesia
(3) Department of Biotechnology Pharmacy, Indonesian School of Pharmacy, Bandung, 40266, West Java, Indonesia
(4) Research Center for Molecular Biotechnology and Bioinformatics, Jl. Singaperbangsa No. 2, Bandung 40133, West Java, Indonesia
(5) Research Center for Molecular Biotechnology and Bioinformatics, Jl. Singaperbangsa No. 2, Bandung 40133, West Java, Indonesia
(6) Department of Pharmacochemistry, Indonesian School of Pharmacy, Bandung, 40266, West Java, Indonesia
(7) Research Center for Molecular Biotechnology and Bioinformatics, Jl. Singaperbangsa No. 2, Bandung 40133, West Java, Indonesia
(8) Research Center for Molecular Biotechnology and Bioinformatics, Jl. Singaperbangsa No. 2, Bandung 40133, West Java, Indonesia; Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Sumedang, 45363, West Java, Indonesia
(9) Department of Biotechnology Pharmacy, Indonesian School of Pharmacy, Bandung, 40266, West Java, Indonesia
(*) Corresponding Author

Abstract

The Sfamy R64 α‐amylase mutant from Saccharomycopsis fibuligera was expressed in Pichia pastoris to explore its industrial potential. The gene encoding the mutant enzyme was cloned into the pPICZαA vector and transformed into P. pastoris SMD1168. Optimal expression was achieved at 1.5% methanol concentration, with the highest enzyme activity observed at 48 h, reaching 24.06 U/mL. The recombinant protein was purified using Ni‐Sepharose affinity chromatography in native and denaturing conditions. The native conditions retained higher protein integrity and activity, while the denaturing process resulted in partial degradation. Molecular dynamics (MD) simulations conducted to assess the structural stability of the His‐tagged Sfamy R64 α‐amylase mutant and its interaction with the maltose substrate. The simulation confirmed the stable binding of maltose in the active site and the solvent accessibility of the His‐tag, supporting its effectiveness in affinity chromatography. The RMSD, RMSF, and time‐evolution snapshots demonstrated that the protein remained structurally stable over 100 ns at an optimum temperature of 50 °C. The findings suggest that the Sfamy R64 mutant α‐amylase is a promising candidate for industrial applications, combining high expression yields, efficient purification, and stable enzyme‐substrate interactions. The results offer a strong basis for further optimization and large‐scale enzyme production.

Keywords

Affinity chromatography; MD simulation; Methanol induction; Pichia pastoris; Sfamy R64 mutant

Full Text:

SUPPLEMENTARY MATERIALS PDF

References

Abdel-Mageed HM, Radwan RA, AbuelEzz NZ, Nasser HA, El Shamy AA, Abdelnaby RM, EL Gohary NA. 2019. Bioconjugation as a smart immobilization approach for αamylase enzyme using stimuliresponsive EudragitL100 polymer: a robust biocatalyst for applications in pharmaceutical industry. Artif. Cells, Nanomedicine Biotechnol. 47(1):2361–2368. doi:10.1080/21691401.2019.1626414.

Abed FS. 2024. Optimization of Expression and Purification of Recombinant One of Central Nervous System Enzyme (Acetylcholinesterase). Int. Acad. J. Sci. Eng. 11(1):40–47. doi:10.9756/iajse/v11i1/iajse1106.

Ahmad M, Hirz M, Pichler H, Schwab H. 2014. Protein expression in Pichia pastoris: Recent achievements and perspectives for heterologous protein production. Appl. Microbiol. Biotechnol. 98(12):5301– 5317. doi:10.1007/s0025301457325.

Alias NAR, Song AAL, Alitheen NB, Rahim RA, Othman SS, In LLA. 2022. Optimization of Signal Peptide via SiteDirected Mutagenesis for Enhanced Secretion of Heterologous Proteins in Lactococcus lactis. Int. J. Mol. Sci. 23(17):10044. doi:10.3390/ijms231710044.

Anggiani M, Helianti I, Abinawanto A. 2018. Optimization of methanol induction for expression of synthetic gene Thermomyces lanuginosus lipase in Pichia pastoris. In: AIP Conf. Proc., volume 2023. p. 020157–5. doi:10.1063/1.5064154.

Arnau J, Lauritzen C, Petersen GE, Pedersen J. 2006. Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr. Purif. 48(1):1–13. doi:10.1016/j.pep.2005.12.002.

Baroroh U, Kusumawardani S, Novianti MT, Yusuf M, Mardiah I, Azhari RN. 2022. Desain Peta Plasmid Pengkode αAmilase Saccharomycopsis fibuligera R64 Mutan dan Pemodelan Struktur Protein [Design of a Plasmid Map Encoding α-Amylase of Saccharomycopsis fibuligera R64 Mutant and Protein Structure Modeling]. Chim. Nat. Acta 10(3):100– 105. doi:10.24198/cna.v10.n3.42053.

Baroroh U, Yusuf M, Rachman SD, Ishmayana S, Hasan K, Subroto T. 2019. Molecular dynamics study to improve the substrate adsorption of Saccharomycopsis fibuligera R64 alphaamylase by designing a new surface binding site. Adv. Appl. Bioinforma. Chem. 12:1–13. doi:10.2147/AABC.S198110.

Byrne B. 2015. Pichia pastoris as an expression host for membrane protein structural biology. Curr. Opin. Struct. Biol. 32:9–17. doi:10.1016/j.sbi.2015.01.005.

Case DA, Cheatham TE, Darden T, Gohlke H, Luo R, Merz KM, Onufriev A, Simmerling C, Wang B, Woods RJ. 2005. The Amber biomolecular simulation programs. J. Comput. Chem. 26(16):1668–1688. doi:10.1002/jcc.20290.

Fuwa H. 1954. A new method for microdetermination of amylase activity by the use of amylose as the substrate. J. Biochem. 41(5):583–603. doi:10.1093/oxfordjournals.jbchem.a126476.

Gaffar S, Permana D, Natalia D, Subroto T, Soemitro S. 2015. Secretory Expression of Saccharomycopsis fibuligera R64 αAmylase with Native Signal Peptide in Pichia pastoris. Procedia Chem. 17:177–183. doi:10.1016/j.proche.2015.12.113.

Gasser B, Steiger MG, Mattanovich D. 2015. Methanol regulated yeast promoters: Production vehicles and toolbox for synthetic biology. Microb. Cell Fact. 14(1):196. doi:10.1186/s1293401503871.

Gopinath SCB, Anbu P, Arshad MKM, Lakshmipriya T, Voon CH, Hashim U, Chinni SV. 2017. Biotechnological Processes in Microbial Amylase Production. Biomed. Res. Int. 2017:1272193. doi:10.1155/2017/1272193.

Goyal N, Gupta JK, Soni SK. 2005. A novel raw starch digesting thermostable αamylase from Bacillus sp. I3 and its use in the direct hydrolysis of raw potato starch. Enzyme Microb. Technol. 37(7):723–734. doi:10.1016/j.enzmictec.2005.04.017.

Green MR, Sambrook J. 2012. Molecular Cloning: A Laboratory Manual, 4th edition. Cold Spring Harb. Lab. Press 33(1):2,028.

Hasan K, Tirta Ismaya W, Kardi I, Andiyana Y, Kusumawidjaya S, Ishmayana S, Subroto T, Soemitro S. 2008. Proteolysis of αamylase from Saccharomycopsis fibuligera: Characterization of digestion products. Biologia (Bratisl). 63(6):1044–1050. doi:10.2478/s117560080167z.

Hernández-Moreno AV, Perdomo-Abúndez FC, Pérez-Medina Martínez V, Luna-Bárcenas G, Villaseñor-Ortega F, Pérez NO, López-Morales CA, Flores-Ortiz LF, Medina-Rivero E. 2015. Structural and functional characterization of a recombinant leucine aminopeptidase. J. Mol. Catal. B Enzym. 113:39–46. doi:10.1016/j.molcatb.2014.12.013.

Jin LT, Hwang SY, Yoo GS, Choi JK. 2004. Sensitive silver staining of protein in sodium dodecyl sulfatepolyacrylamide gels using an azo dye, calconcarboxylic acid, as a silverion sensitizer. Electrophoresis 25(15):2494–2500. doi:10.1002/elps.200306002.

Juturu V, Wu JC. 2018. Heterologous Protein Expression in Pichia pastoris: Latest Research Progress and Applications. ChemBioChem 19(1):17–21. doi:10.1002/cbic.201700460.

Kielkopf CL, Bauer W, Urbatsch IL. 2021. Expression of cloned genes in Pichia pastoris using the methanolinducible promoter AOX1. Cold Spring Harb. Protoc. 2021(1):pdb.prot102160. doi:10.1101/pdb.prot102160.

Laskowski RA, MacArthur MW, Moss DS, Thornton JM. 1993. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26(2):283–291. doi:10.1107/s0021889892009944.

Love KR, Shah KA, Whittaker CA, Wu J, Bartlett MC, Ma D, Leeson RL, Priest M, Borowsky J, Young SK, Love JC. 2016. Comparative genomics and transcriptomics of Pichia pastoris. BMC Genomics 17(1):550. doi:10.1186/s128640162876y.

Mohanty S, Babbal, Khasa YP. 2023. Heterologous Gene Expression in Pichia pastoris: Success Stories and Commercial Ventures. Singapore: Springer Nature Singapore. p. 513––569.

Schartner J, Güldenhaupt J, Katharina Gaßmeyer S, Rosga K, Kourist R, Gerwert K, Kötting C. 2018. Highly stable protein immobilization via maleimidothiol chemistry to monitor enzymatic activity. Analyst 143(10):513–569. doi:10.1039/c8an00301g.

Sembiring ER, Fuad AM, Suryadi H. 2024. Expression and purification of recombinant human granulocyte colonystimulating factor (rGCSF) from Pichia pastoris. Indones. J. Biotechnol. 29(4):205. doi:10.22146/ijbiotech.93609.

Sinha J, Plantz BA, Inan M, Meagher MM. 2005. Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: Case study with recombinant ovine interferonτ. Biotechnol. Bioeng. 89(1):102–112. doi:10.1002/bit.20318.

Sobolev OV, Afonine PV, Moriarty NW, Hekkelman ML, Joosten RP, Perrakis A, Adams PD. 2020. A Global Ramachandran Score Identifies Protein Structures with Unlikely Stereochemistry. Structure 28(11):1249–1258.e2. doi:10.1016/j.str.2020.08.005.

Tantray JA, Mansoor S, Wani RFC, Nissa NU. 2023. Preparation of competent cells by CaCl2 treatment. Academic Press. p. 117–121. doi:10.1016/b9780 443191749.000271.

Tsuda M, Nonaka K. 2024. Recent progress on heterologous protein production in methylotrophic yeast systems. World J. Microbiol. Biotechnol. 40(7):200. doi:10.1007/s11274024040089.

Unver Y, Dagci I. 2024. Komagataella phaffii (Pichia pastoris) as a Powerful Yeast Expression System for Biologics Production. Front. Biosci. (Elite Ed.) 16(2):19. doi:10.31083/j.fbe1602019.

Wang C, Jiang W, Yu C, Xia J. 2024. Transcriptional Downregulation of Methanol Metabolism Key Genes During Yeast Death in Engineered Pichia pastoris. Biotechnol. J. 19(10):e202400328. doi:10.1002/biot.202400328.

Webb B, Sali A. 2016. Comparative protein structure modeling using MODELLER. Curr. Protoc. Bioinforma. 54(1):5.6.1–5.6.37. doi:10.1002/cpbi.3.

Wingfield PT. 2015. Overview of the Purification of Recombinant Proteins. Curr. Protoc. Protein Sci. 80(1):6.1.1–6.1.35. doi:10.1002/0471140864.ps0601s80.

Yan Z, Zhu Q, Ma L, Li G, Su E, Zeng J, Chen Y, Meng E, Deng S. 2023. Effects of HisTag Length on the Soluble Expression and Selective Immobilization of DAmino Acid Oxidase from Trigonopsis variabilis: A Preliminary Study. Processes 11(6):1588. doi:10.3390/pr11061588.

Yasokawa D, Murata S, Iwahashi Y, Kitagawa E, Nakagawa R, Hashido T, Iwahashi H. 2010. Toxicity of methanol and formaldehyde towards Saccharomyces cerevisiae as assessed by DNA microarray analysis. Appl. Biochem. Biotechnol. 160(6):1685–1698. doi:10.1007/s120100098684y.

Zahrl RJ, Mattanovich D, Gasser B. 2018. The impact of ERAD on recombinant protein secretion in Pichia pastoris (Syn Komagataella spp.). Microbiol. (United Kingdom) 164(4):453–463. doi:10.1099/mic.0.000630.

DOI: https://doi.org/10.22146/ijbiotech.100845