Rational Design of Lipase ROL to Increase Its Thermostability for Production of Structured Tags
1,3-regiospecific lipases are important enzymes that are heavily utilized in the food industries to produce structured triacylglycerols (TAGs). The <i>Rhizopus oryzae</i> lipase (ROL) has recently gained interest because this enzyme possesses high selectivity and catalytic efficiency. Ho...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-08-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/17/9515 |
| _version_ | 1797495426087124992 |
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| author | Jeng Yeong Chow Giang Kien Truc Nguyen |
| author_facet | Jeng Yeong Chow Giang Kien Truc Nguyen |
| author_sort | Jeng Yeong Chow |
| collection | DOAJ |
| description | 1,3-regiospecific lipases are important enzymes that are heavily utilized in the food industries to produce structured triacylglycerols (TAGs). The <i>Rhizopus oryzae</i> lipase (ROL) has recently gained interest because this enzyme possesses high selectivity and catalytic efficiency. However, its low thermostability limits its use towards reactions that work at lower temperature. Most importantly, the enzyme cannot be used for the production of 1,3-dioleoyl-2-palmitoylglycerol (OPO) and 1,3-stearoyl-2-oleoyl-glycerol (SOS) due to the high melting points of the substrates used for the reaction. Despite various engineering efforts used to improve the thermostability of ROL, the enzyme is unable to function at temperatures above 60 °C. Here, we describe the rational design of ROL to identify variants that can retain their activity at temperatures higher than 60 °C. After two rounds of mutagenesis and screening, we were able to identify a mutant ROL_10x that can retain most of its activity at 70 °C. We further demonstrated that this mutant is useful for the synthesis of SOS while minimal product formation was observed with ROL_WT. Our engineered enzyme provides a promising solution for the industrial synthesis of structured lipids at high temperature. |
| first_indexed | 2024-03-10T01:48:30Z |
| format | Article |
| id | doaj.art-c1afe7b9dfeb44a5984226e5f5b7556a |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T01:48:30Z |
| publishDate | 2022-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-c1afe7b9dfeb44a5984226e5f5b7556a2023-11-23T13:12:00ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-08-012317951510.3390/ijms23179515Rational Design of Lipase ROL to Increase Its Thermostability for Production of Structured TagsJeng Yeong Chow0Giang Kien Truc Nguyen1Wilmar Innovation Centre, Wilmar International Limited, 28 Biopolis Road, Singapore 138568, SingaporeWilmar Innovation Centre, Wilmar International Limited, 28 Biopolis Road, Singapore 138568, Singapore1,3-regiospecific lipases are important enzymes that are heavily utilized in the food industries to produce structured triacylglycerols (TAGs). The <i>Rhizopus oryzae</i> lipase (ROL) has recently gained interest because this enzyme possesses high selectivity and catalytic efficiency. However, its low thermostability limits its use towards reactions that work at lower temperature. Most importantly, the enzyme cannot be used for the production of 1,3-dioleoyl-2-palmitoylglycerol (OPO) and 1,3-stearoyl-2-oleoyl-glycerol (SOS) due to the high melting points of the substrates used for the reaction. Despite various engineering efforts used to improve the thermostability of ROL, the enzyme is unable to function at temperatures above 60 °C. Here, we describe the rational design of ROL to identify variants that can retain their activity at temperatures higher than 60 °C. After two rounds of mutagenesis and screening, we were able to identify a mutant ROL_10x that can retain most of its activity at 70 °C. We further demonstrated that this mutant is useful for the synthesis of SOS while minimal product formation was observed with ROL_WT. Our engineered enzyme provides a promising solution for the industrial synthesis of structured lipids at high temperature.https://www.mdpi.com/1422-0067/23/17/9515<i>Rhizopus oryzae</i> lipasethermostabilityenzymatic interesterificationrational designprotein engineeringstructured lipids |
| spellingShingle | Jeng Yeong Chow Giang Kien Truc Nguyen Rational Design of Lipase ROL to Increase Its Thermostability for Production of Structured Tags International Journal of Molecular Sciences <i>Rhizopus oryzae</i> lipase thermostability enzymatic interesterification rational design protein engineering structured lipids |
| title | Rational Design of Lipase ROL to Increase Its Thermostability for Production of Structured Tags |
| title_full | Rational Design of Lipase ROL to Increase Its Thermostability for Production of Structured Tags |
| title_fullStr | Rational Design of Lipase ROL to Increase Its Thermostability for Production of Structured Tags |
| title_full_unstemmed | Rational Design of Lipase ROL to Increase Its Thermostability for Production of Structured Tags |
| title_short | Rational Design of Lipase ROL to Increase Its Thermostability for Production of Structured Tags |
| title_sort | rational design of lipase rol to increase its thermostability for production of structured tags |
| topic | <i>Rhizopus oryzae</i> lipase thermostability enzymatic interesterification rational design protein engineering structured lipids |
| url | https://www.mdpi.com/1422-0067/23/17/9515 |
| work_keys_str_mv | AT jengyeongchow rationaldesignoflipaseroltoincreaseitsthermostabilityforproductionofstructuredtags AT giangkientrucnguyen rationaldesignoflipaseroltoincreaseitsthermostabilityforproductionofstructuredtags |