{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T04:51:02Z","timestamp":1782190262039,"version":"3.54.5"},"reference-count":41,"publisher":"MDPI AG","issue":"20","license":[{"start":{"date-parts":[[2024,10,19]],"date-time":"2024-10-19T00:00:00Z","timestamp":1729296000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Natural Science Foundation of China","award":["32130084"],"award-info":[{"award-number":["32130084"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Foods"],"abstract":"<jats:p>The demand for food-grade \u03b2-mannanases, ideal for high-temperature baking, is increasing. Using the Komagataella phaffii (P. pastoris) expression system for \u03b2-mannanase production, this study aimed to enhance purification methods. We evaluated better conditions for production and purification of \u03b2-mannanase (PpRmMan134A) from recombinant P. pastoris X-33, focusing on a higher purity and reducing the production of endogenous secretory proteins in fermentation. By adjusting carbon and nitrogen sources, culture time, and temperature, we controlled cell growth to reduce the production of endogenous secretory proteins. The better-evaluated conditions involved culturing recombinant P. pastoris in 70 mL buffered glycerol complex medium for 24 h at 30 \u00b0C, then in modified buffered methanol-complex medium with 0.91% (w\/v) methanol, 0.56% (w\/v) sorbitol, and 0.48% (w\/v) mannitol for another 24 h, which improved the PpRmMan134A yield and reduced endogenous secretory proteins, shortening the fermentation time by 72 h. An affordable purification method using ultrafiltration and salt-out precipitation was utilized. PpRmMan134A showed thermostability up to 100 \u00b0C and effectively degraded locust bean gum into smaller fragments, mainly producing mannotriose. In conclusion, with its enhanced purity due to reduced levels of endogenous secretory proteins, purified PpRmMan134A emerges as a promising enzyme for high-temperature baking applications.<\/jats:p>","DOI":"10.3390\/foods13203324","type":"journal-article","created":{"date-parts":[[2024,10,21]],"date-time":"2024-10-21T08:53:11Z","timestamp":1729500791000},"page":"3324","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Purification and Characterization of \u03b2-Mannanase Derived from Rhizopus microsporus var. rhizopodiformis Expressed in Komagataella phaffii"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-0449-5627","authenticated-orcid":false,"given":"Jinghua","family":"Qu","sequence":"first","affiliation":[{"name":"The State Key Laboratory of Food Science and Resources, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China"},{"name":"School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China"},{"name":"Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, Jiangnan University, Wuxi 214122, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jie","family":"Long","sequence":"additional","affiliation":[{"name":"The State Key Laboratory of Food Science and Resources, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China"},{"name":"School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China"},{"name":"Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, Jiangnan University, Wuxi 214122, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xingfei","family":"Li","sequence":"additional","affiliation":[{"name":"The State Key Laboratory of Food Science and Resources, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China"},{"name":"School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China"},{"name":"Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, Jiangnan University, Wuxi 214122, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xing","family":"Zhou","sequence":"additional","affiliation":[{"name":"The State Key Laboratory of Food Science and Resources, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China"},{"name":"School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7218-2283","authenticated-orcid":false,"given":"Long","family":"Chen","sequence":"additional","affiliation":[{"name":"The State Key Laboratory of Food Science and Resources, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China"},{"name":"School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6427-6124","authenticated-orcid":false,"given":"Chao","family":"Qiu","sequence":"additional","affiliation":[{"name":"The State Key Laboratory of Food Science and Resources, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China"},{"name":"School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7802-9722","authenticated-orcid":false,"given":"Zhengyu","family":"Jin","sequence":"additional","affiliation":[{"name":"The State Key Laboratory of Food Science and Resources, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China"},{"name":"School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China"},{"name":"Collaborative Innovation Center of Food Safety and Quality Control in Jiangsu Province, Jiangnan University, Wuxi 214122, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,10,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Davy, P., Kirkman, T., Scarlett, C.J., and Vuong, Q. (2022). Characterisation of a High Fibre Flour Prepared from Soy Milk By-Product and Its Potential Use in White Wheat Bread. Foods, 11.","DOI":"10.3390\/foods11233921"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Tian, B., Zhou, C., Li, D., Pei, J., Guo, A., Liu, S., and Li, H. (2021). Monitoring the Effects of Hemicellulase on the Different Proofing Stages of Wheat Aleurone-Rich Bread Dough and Bread Quality. Foods, 10.","DOI":"10.3390\/foods10102427"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Dawood, A., and Ma, K. (2020). Applications of Microbial \u03b2-Mannanases. Front. Bioeng. Biotechnol., 8.","DOI":"10.3389\/fbioe.2020.598630"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Rueckel, M., Janson, S., Solbak, A., and Fickler, A. (2024). Spatial Activity Mapping of SS-Mannanase on Soybean Seeds. Sci. Rep., 14.","DOI":"10.1038\/s41598-024-51494-w"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"112215","DOI":"10.1016\/j.lwt.2021.112215","article-title":"\u03b2-Mannanase BoMan26B from Bacteroides Ovatus Produces Mannan-Oligosaccharides with Prebiotic Potential from Galactomannan and Softwood \u03b2-Mannans","volume":"151","author":"Bhattacharya","year":"2021","journal-title":"LWT"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"107822","DOI":"10.1016\/j.carres.2019.107822","article-title":"Structural Diversity and Prebiotic Potential of Short Chain \u03b2-Manno-Oligosaccharides Generated from Guar Gum by Endo-\u03b2-Mannanase (ManB-1601)","volume":"486","author":"Mary","year":"2019","journal-title":"Carbohydr. Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e155","DOI":"10.1002\/cpz1.155","article-title":"Practical Methods for Expression of Recombinant Protein in the Pichia Pastoris System","volume":"1","author":"Mohammadzadeh","year":"2021","journal-title":"Curr. Protoc."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1016\/j.biortech.2018.01.138","article-title":"High-Level Expression of an Engineered \u03b2-Mannanase (mRmMan5A) in Pichia Pastoris for Manno-Oligosaccharide Production Using Steam Explosion Pretreated Palm Kernel Cake","volume":"256","author":"Li","year":"2018","journal-title":"Bioresour. Technol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1016\/j.procbio.2020.09.034","article-title":"High-Level Expression of a Glycoside Hydrolase Family 26 \u03b2-Mannanase from Aspergillus Niger in Pichia Pastoris for Production of Partially Hydrolysed Fenugreek Gum","volume":"100","author":"Wang","year":"2021","journal-title":"Process Biochem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2073","DOI":"10.2478\/s11756-020-00525-8","article-title":"Functional Expression and Characterization of an Endo-1,4-\u03b2-Mannosidase from Triticum Aestivum in Pichia Pastoris","volume":"75","author":"Zhang","year":"2020","journal-title":"Biologia"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Ren, M., Zhang, Y., Wang, X., and Qiu, H. (2022). Catalytic Hydrogenation of CO2 to Methanol: A Review. Catalysts, 12.","DOI":"10.3390\/catal12040403"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Zhu, T., Sun, H., Wang, M., and Li, Y. (2019). Pichia Pastoris as a Versatile Cell Factory for the Production of Industrial Enzymes and Chemicals: Current Status and Future Perspectives. Biotechnol. J., 14.","DOI":"10.1002\/biot.201800694"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"5867","DOI":"10.1002\/jcp.29583","article-title":"Pichia Pastoris: A Highly Successful Expression System for Optimal Synthesis of Heterologous Proteins","volume":"235","author":"Karbalaei","year":"2020","journal-title":"J. Cell. Physiol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Liu, Z., Ning, C., Yuan, M., Fu, X., Yang, S., Wei, X., Xiao, M., Mou, H., and Zhu, C. (2020). High-Efficiency Expression of a Superior \u03b2-Mannanase Engineered by Cooperative Substitution Method in Pichia Pastoris and Its Application in Preparation of Prebiotic Mannooligosaccharides. Bioresour. Technol., 311.","DOI":"10.1016\/j.biortech.2020.123482"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1016\/j.ymben.2018.04.017","article-title":"Metabolic Engineering of Pichia Pastoris","volume":"50","author":"Gasser","year":"2018","journal-title":"Metab. Eng."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"178","DOI":"10.1016\/j.pep.2013.06.005","article-title":"High-Level Expression of Staphylococcal Protein A in Pichia Pastoris and Purification and Characterization of the Recombinant Protein","volume":"90","author":"Hao","year":"2013","journal-title":"Protein Expr. Purif."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"178","DOI":"10.1016\/j.fbp.2023.04.002","article-title":"Membrane-Based Approach for the Removal of Pigment Impurities Secreted by Pichia Pastoris","volume":"139","author":"Kongsinkaew","year":"2023","journal-title":"Food Bioprod. Process."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Kastilan, R., Boes, A., Spiegel, H., Voepel, N., Chudobov\u00e1, I., Hellwig, S., Buyel, J.F., Reimann, A., and Fischer, R. (2017). Improvement of a Fermentation Process for the Production of Two PfAMA1-DiCo-Based Malaria Vaccine Candidates in Pichia Pastoris. Sci. Rep., 7.","DOI":"10.1038\/s41598-017-11819-4"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1061","DOI":"10.1007\/s00449-016-1584-y","article-title":"Oxygen Transfer as a Tool for Fine-Tuning Recombinant Protein Production by Pichia Pastoris under Glyceraldehyde-3-Phosphate Dehydrogenase Promoter","volume":"39","year":"2016","journal-title":"Bioprocess Biosyst. Eng."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1007\/s00253-017-8612-y","article-title":"Integrating Metabolic Modeling and Population Heterogeneity Analysis into Optimizing Recombinant Protein Production by Komagataella (Pichia) Pastoris","volume":"102","author":"Theron","year":"2018","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"834","DOI":"10.1126\/science.abm7649","article-title":"Metabolic Scaling Is the Product of Life-History Optimization","volume":"377","author":"White","year":"2022","journal-title":"Science"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2175","DOI":"10.1038\/ismej.2017.86","article-title":"Applying Allometric Theory to Fungi","volume":"11","author":"Rillig","year":"2017","journal-title":"ISME J."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1002\/yea.3556","article-title":"Cross-Talk between Autophagy and Sporulation in Saccharomyces Cerevisiae","volume":"38","author":"Barve","year":"2021","journal-title":"Yeast"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1453","DOI":"10.1021\/acssynbio.7b00078","article-title":"Control of the Yeast Mating Pathway by Reconstitution of Functional \u03b1-Factor Using Split Intein-Catalyzed Reactions","volume":"6","author":"Siu","year":"2017","journal-title":"ACS Synth. Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e8558","DOI":"10.1002\/ece3.8558","article-title":"Infection by dsRNA Viruses Is Associated with Enhanced Sporulation Efficiency in Saccharomyces Cerevisiae","volume":"12","author":"Skirgaila","year":"2022","journal-title":"Ecol. Evol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1126\/science.276.5309.122","article-title":"A General Model for the Origin of Allometric Scaling Laws in Biology","volume":"276","author":"West","year":"1997","journal-title":"Science"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2248","DOI":"10.1126\/science.1061967","article-title":"Effects of Size and Temperature on Metabolic Rate","volume":"293","author":"Gillooly","year":"2001","journal-title":"Science"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1376","DOI":"10.1016\/j.bbagen.2018.03.016","article-title":"Structural and Biochemical Insights into the Substrate-Binding Mechanism of a Novel Glycoside Hydrolase Family 134 \u03b2-Mannanase","volume":"1862","author":"You","year":"2018","journal-title":"Biochim. Et Biophys. Acta (BBA)-Gen. Subj."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1016\/S1046-5928(03)00136-0","article-title":"Production of the Active Antifungal Pisum Sativum Defensin 1 (Psd1) in Pichia Pastoris: Overcoming the Inefficiency of the STE13 Protease","volume":"31","author":"Cabral","year":"2003","journal-title":"Protein Expr. Purif."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"10235","DOI":"10.1021\/acs.jafc.1c04124","article-title":"Efficient Secretory Expression and Purification of Food-Grade Porcine Myoglobin in Komagataella Phaffii","volume":"69","author":"Zhang","year":"2021","journal-title":"J. Agric. Food Chem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1042\/BA20070267","article-title":"Prevention of Human Granulocyte Colony-Stimulating Factor Protein Aggregation in Recombinant Pichia Pastoris Fed-Batch Fermentation Using Additives","volume":"52","author":"Bahrami","year":"2009","journal-title":"Biotechnol. Appl. Biochem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1002\/bit.22543","article-title":"Metabolic Flux Analysis for Recombinant Protein Production by Pichia Pastoris Using Dual Carbon Sources: Effects of Methanol Feeding Rate","volume":"105","author":"Oliver","year":"2010","journal-title":"Biotechnol. Bioeng."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1429","DOI":"10.1007\/s12010-014-1387-z","article-title":"High-Level Extracellular Production of Glucose Oxidase by Recombinant Pichia Pastoris Using a Combined Strategy","volume":"175","author":"Gu","year":"2015","journal-title":"Appl. Biochem. Biotechnol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"125709","DOI":"10.1016\/j.foodchem.2019.125709","article-title":"Preparation, Characterization, and Prebiotic Activity of Manno-Oligosaccharides Produced from Cassia Gum by a Glycoside Hydrolase Family 134 \u03b2-Mannanase","volume":"309","author":"Li","year":"2020","journal-title":"Food Chem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1186\/s13065-017-0251-y","article-title":"Citric Acid: Emerging Applications of Key Biotechnology Industrial Product","volume":"11","author":"Ciriminna","year":"2017","journal-title":"Chem. Cent. J."},{"key":"ref_36","unstructured":"WHO (2004). Safety Evaluation of Certain Food Additives and Contaminants, WHO."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"585","DOI":"10.1016\/S1389-1723(01)80321-2","article-title":"Non-Repressing Carbon Sources for Alcohol Oxidase (AOX1) Promoter of Pichia Pastoris","volume":"92","author":"Inan","year":"2001","journal-title":"J. Bioscience Bioeng."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1002\/bit.26440","article-title":"Development of a General Defined Medium for Pichia Pastoris","volume":"115","author":"Matthews","year":"2018","journal-title":"Biotechnol. Bioeng."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"206","DOI":"10.1016\/j.jconrel.2011.02.010","article-title":"Casein-Based Formulations as Promising Controlled Release Drug Delivery Systems","volume":"153","author":"Elzoghby","year":"2011","journal-title":"J. Control. Release"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"452","DOI":"10.1016\/j.jfoodeng.2010.04.030","article-title":"Computational Fluid Dynamics (CFD) Modeling of an Electrical Heating Oven for Bread-Baking Process","volume":"100","author":"Chhanwal","year":"2010","journal-title":"J. Food Eng."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.biotechadv.2016.11.001","article-title":"Production, Properties, and Applications of Endo-\u03b2-Mannanases","volume":"35","author":"Srivastava","year":"2017","journal-title":"Biotechnol. Adv."}],"container-title":["Foods"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2304-8158\/13\/20\/3324\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T16:16:51Z","timestamp":1760113011000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2304-8158\/13\/20\/3324"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,10,19]]},"references-count":41,"journal-issue":{"issue":"20","published-online":{"date-parts":[[2024,10]]}},"alternative-id":["foods13203324"],"URL":"https:\/\/doi.org\/10.3390\/foods13203324","relation":{},"ISSN":["2304-8158"],"issn-type":[{"value":"2304-8158","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,10,19]]}}}