Self-Emulsifying Biocatalysis Based on an Amphipathic Protein BslA-Modified Esterase Functioning as a Fine-Tuned System for Oil Hydrolysis

Abstract

Traditional heterogeneous enzymatic reactions involving hydrophobic substrates rely on emulsifiers, which pose environmental risks and can destabilize enzymes. To address this problem, a self-emulsifying system based on biofilm surface layer protein A (BslA) was designed and established in this study. Taking the enzymatic hydrolysis of astaxanthin esters as an example, the emulsification capacity and hydrolysis efficiency of BslA-Est3-14 fusion protein, comprising the amphiphilic protein BslA and esterase Est3-14, was investigated. Compared with the group containing no emulsifiers, the addition of BslA-Est3-14 significantly reduced the droplet size from 3.04 μm to 1.46 μm. Additionally, the addition of BslA demonstrated competitive efficacy in maintaining enzyme activity than traditional emulsifiers. Furthermore, the BslA-Est3-14 group increased the yield of free astaxanthin by 52.3%, 78.0%, and 76.9% compared to the Tween-80, Span-20, and ethanol groups, respectively. The highest yield of astaxanthin in emulsion reaction system was finally determined to be 184 μg·mL⁻¹. This method not only mitigates the low efficiency of heterogeneous enzymatic reactions but also eliminates the requirement for surfactants, thereby minimizing environmental footprints and holding significant implications for sustainable bioprocessing in food, pharmaceutical, and green chemistry sectors.

Keywords

astaxanthin / biofilm surface layer protein A / emulsion / enzymatic hydrolysis / self-emulsifying

References

[1] Abdi, G., R. Tendulkar, and C. Thatte, et al. 2024. “Scaling Up Nature's Chemistry: A Guide to Industrial Production of Valuable Metabolites.” In Advances in Metabolomics, edited by V. Singh, 331–375. Springer Nature Singapore Pte Ltd. https://doi.org/10.1007/978-981-97-7459-3_15.

[2] Arnaouteli, S., N. C. Bamford, G. B. Brandani, et al. 2023. “Lateral Interactions Govern Self-Assembly of the Bacterial Biofilm Matrix Protein BslA.” Proceedings of the National Academy of Sciences 120, no. 45: e2312022120. https://doi.org/10.1073/pnas.2312022120.

[3] Arpigny, J. L., and K. E. Jaeger. 1999. “Bacterial Lipolytic Enzymes: Classification and Properties.” Biochemical Journal 343, no. 1: 177–183. https://doi.org/10.1042/bj3430177.

[4] Badmus, S. O., H. K. Amusa, T. A. Oyehan, and T. A. Saleh. 2021. “Environmental Risks and Toxicity of Surfactants: Overview of Analysis, Assessment, and Remediation Techniques.” Environmental Science and Pollution Research 28: 62085–62104. https://doi.org/10.1007/s11356-021-16483-w.

[5] Brandani, G. B., M. Schor, R. Morris, et al. 2015. “The Bacterial Hydrophobin BslA Is a Switchable Ellipsoidal Janus Nanocolloid.” Langmuir 31, no. 42: 11558–11563. https://doi.org/10.1021/acs.langmuir.5b02347.

[6] Cao, Y., L. Yang, X. Qiao, C. Xue, and J. Xu. 2023. “Dietary Astaxanthin: An Excellent Carotenoid With Multiple Health Benefits.” Critical Reviews in Food Science and Nutrition 63, no. 18: 3019–3045. https://doi.org/10.1080/10408398.2021.1983766.

[7] Chen, H., J. Ullah, and J. Jia. 2017. “Progress in Bacillus subtilis Spore Surface Display Technology Towards Environment, Vaccine Development, and Biocatalysis.” Journal of Molecular Microbiology and Biotechnology 27, no. 3: 159–167. https://doi.org/10.1159/000475177.

[8] Debnath, T., T. K. Bandyopadhyay, K. Vanitha, et al. 2023. “Astaxanthin From Microalgae: A Review on Structure, Biosynthesis, Production Strategies and Application.” Food Research International (Ottawa, Ont.) 176: 113841. https://doi.org/10.1016/j.foodres.2023.113841.

[9] Dimitrova, L. M., M. P. Boneva, K. D. Danov, et al. 2016. “Limited Coalescence and Ostwald Ripening in Emulsions Stabilized by Hydrophobin HFBII and Milk Proteins.” Colloids and Surfaces A: Physicochemical and Engineering Aspects 509: 521–538. https://doi.org/10.1016/j.colsurfa.2016.09.066.

[10] Dong, H., X. Li, C. Xue, and X. Mao. 2016. “Astaxanthin Preparation by Fermentation of Esters From Haematococcus pluvialis Algal Extracts With Stenotrophomonas Species.” Biotechnology Progress 32, no. 3: 649–656. https://doi.org/10.1002/btpr.2258.

[11] Gao, K., X. Wang, H. Jiang, J. Sun, and X. Mao. 2021. “Identification of a GDSL Lipase From Streptomyces bacillaris and Its Application in the Preparation of Free Astaxanthin.” Journal of Biotechnology 325: 280–287. https://doi.org/10.1016/j.jbiotec.2020.10.009.

[12] Gao, X., J. Sun, Z. Liu, et al. 2019. “Highly Efficient Preparation of Free All-trans-Astaxanthin From Haematococcus pluvialis Extract by a Rapid Biocatalytic Method Based on Crude Extracellular Enzyme Extract.” International Journal of Food Science & Technology 54, no. 2: 376–386. https://doi.org/10.1111/ijfs.13947.

[13] Grossmann, L., and D. J. McClements. 2023. “Current Insights Into Protein Solubility: A Review of Its Importance for Alternative Proteins.” Food Hydrocolloids 137: 108416. https://doi.org/10.1016/j.foodhyd.2022.108416.

[14] Haeffner, F., and T. Norin. 1999. “Molecular Modelling of Lipase Catalysed Reactions. Prediction of Enantioselectivities.” Chemical and Pharmaceutical Bulletin 47, no. 5: 591–600. https://doi.org/10.1248/cpb.47.591.

[15] Jamdar, S. N., V. Rajalakshmi, M. D. Pednekar, F. Juan, V. Yardi, and A. Sharma. 2010. “Influence of Degree of Hydrolysis on Functional Properties, Antioxidant Activity and ACE Inhibitory Activity of Peanut Protein Hydrolysate.” Food Chemistry 121, no. 1: 178–184. https://doi.org/10.1016/j.foodchem.2009.12.027.

[16] Kim, D., W. Kim, and J. Kim. 2021. “New Bacterial Surface Display System Development and Application Based on Bacillus subtilis YuaB Biofilm Component as an Anchoring Motif.” Biotechnology and Bioprocess Engineering 26, no. 1: 39–46. https://doi.org/10.1007/s12257-020-0397-7.

[17] Liang, L., F. Chen, X. Wang, Q. Jin, E. A. Decker, and D. J. McClements. 2017. “Physical and Oxidative Stability of Flaxseed Oil-in-Water Emulsions Fabricated From Sunflower Lecithins: Impact of Blending Lecithins With Different Phospholipid Profiles.” Journal of Agricultural and Food Chemistry 65, no. 23: 4755–4765. https://doi.org/10.1021/acs.jafc.7b01469.

[18] Lu, P., X. Gao, H. Dong, et al. 2018. “Identification of a Novel Esterase From Marine Environmental Genomic DNA Libraries and Its Application in Production of Free All-trans-Astaxanthin.” Journal of Agricultural and Food Chemistry 66, no. 11: 2812–2821. https://doi.org/10.1021/acs.jafc.7b06062.

[19] Lundberg, B., R. Klemets, and T. Lövgren. 1979. “Effect of Substrate Properties on the Activity of Lysosomal Cholesteryl Ester Hydrolase.” Biochimica et Biophysica Acta (BBA)–Lipids and Lipid Metabolism 572, no. 3: 492–501. https://doi.org/10.1016/0005-2760(79)90156-5.

[20] Ma, Q., H. Zhou, X. Shao, et al. 2024. “Pickering Emulsions Stabilized by Linear Amphiphilic Polymers for Enhancing the Selective Oxidation of Cinnamyl Alcohol.” ACS Applied Polymer Materials 6, no. 16: 10083–10089. https://doi.org/10.1021/acsapm.4c02214.

[21] Ma, W., J. Wang, X. Xu, L. Qin, C. Wu, and M. Du. 2019. “Ultrasound Treatment Improved the Physicochemical Characteristics of Cod Protein and Enhanced the Stability of Oil-in-Water Emulsion.” Food Research International 121: 247–256. https://doi.org/10.1016/j.foodres.2019.03.024.

[22] Ma, X., F. Hou, H. Zhao, et al. 2020. “Conjugation of Soy Protein Isolate (SPI) With Pectin by Ultrasound Treatment.” Food Hydrocolloids 108: 106056. https://doi.org/10.1016/j.foodhyd.2020.106056.

[23] Margesin, R., G. Feller, M. Hämmerle, U. Stegner, and F. Schinner. 2002. “A Colorimetric Method for the Determination of Lipase Activity in Soil.” Biotechnology Letters 24, no. 1: 27–33. https://doi.org/10.1023/A:1013801131553.

[24] Martínez-Delgado, A. A., S. Khandual, and S. J. Villanueva–Rodríguez. 2017. “Chemical Stability of Astaxanthin Integrated Into a Food Matrix: Effects of Food Processing and Methods for Preservation.” Food Chemistry 225: 23–30. https://doi.org/10.1016/j.foodchem.2016.11.092.

[25] Mei, J., X. Zhao, Y. Yi, Y. Zhang, X. Wang, and G. Ying. 2020. “Preparation of Astaxanthin by Lipase-Catalyzed Hydrolysis From Its Esters in a Slug-Flow Microchannel Reactor.” Process Biochemistry 98: 241–246. https://doi.org/10.1016/j.procbio.2020.06.017.

[26] Mesa, M., J. A. Pereañez, L. M. Preciado, and C. Bernal. 2018. “How the Triton X-100 Modulates the Activity/Stability of the Thermomyces lanuginose Lipase: Insights From Experimental and Molecular Docking Approaches.” International Journal of Biological Macromolecules 120: 2410–2417. https://doi.org/10.1016/j.ijbiomac.2018.09.009.

[27] Oliveira, C. M., F. H. Xavier, A. R. V. Morais, et al. 2019. “Hydrophobin-Stabilized Nanoemulsion Produced by a Low-Energy Emulsification Process: A Promising Carrier for Nutraceuticals.” Food Hydrocolloids 89: 749–757. https://doi.org/10.1016/j.foodhyd.2018.11.057.

[28] Partschefeld, C., U. Schwarzenbolz, S. Richter, and T. Henle. 2007. “Crosslinking of Casein by Microbial Transglutaminase and Its Resulting Influence on the Stability of Micelle Structure.” Biotechnology Journal 2, no. 4: 456–461. https://doi.org/10.1002/biot.200600232.

[29] Sokolik, V. V., V. S. Chursina, A. A. Artemchuk, A. F. Artemchuk, and G. Bozhko. 2006. “Depression of Serum Esterase and Lipoprotein Lipase Activities in Acute and Longitudinal Actions of Ethanol.” Biomeditsinskaia khimiia 52, no. 1: 95–100.

[30] Sui, X., S. Bi, B. Qi, et al. 2017. “Impact of Ultrasonic Treatment on an Emulsion System Stabilized With Soybean Protein Isolate and Lecithin: Its Emulsifying Property and Emulsion Stability.” Food Hydrocolloids 63: 727–734. https://doi.org/10.1016/j.foodhyd.2016.10.024.

[31] Tauer, K., C. Wei, A. Tripathi, and O. Kiryutina. 2018. “Experiments and Thoughts on Mass Transfer During Emulsification.” Polymer Reaction Engineering of Dispersed Systems II, no. 281: 23–43. https://doi.org/10.1007/12_2017_18.

[32] Tian, Q., J. Zhang, J. Yan, S. Zhang, and Z. Yu. 2023. “Enzyme Anchoring Amphiphilic Polymer Nanoparticles for Enhanced Pickering Interfacial Biocatalysis.” ACS Applied Polymer Materials 5, no. 3: 1747–1755. https://doi.org/10.1021/acsapm.2c01868.

[33] Vicaria, J. M., O. Herrera-Márquez, M. Serrano-Haro, A. Vidal, E. Jurado, and J. L. Jiménez-Pérez. 2022. “Optimization of Surfactants Formulations to Stabilise Proteases and Amylases.” Chemical Engineering Science 260: 117858. https://doi.org/10.1016/j.ces.2022.117858.

[34] Yang, L., F. Li, X. Cao, X. Qiao, C. Xue, and J. Xu. 2022. “Stability and Bioavailability of Protein Matrix-Encapsulated Astaxanthin Ester Microcapsules.” Journal of the Science of Food and Agriculture 102, no. 5: 2144–2152. https://doi.org/10.1002/jsfa.11556.

[35] Zhao, T., X. Yan, L. Sun, et al. 2019. “Research Progress on Extraction, Biological Activities and Delivery Systems of Natural Astaxanthin.” Trends in Food Science & Technology 91: 354–361. https://doi.org/10.1016/j.tifs.2019.07.014.

[36] Zhong, J., and J. S. Francisco. 2020. “Catalytic and Autocatalytic Chemical Processes in the Atmosphere.” Annual Reports in Computational Chemistry 16: 157–185. https://doi.org/10.1016/bs.arcc.2020.07.003.

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2025 The Author(s). Food Bioengineering published by John Wiley & Sons Australia, Ltd on behalf of State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology.