基于 D-阿洛糖生物传感器的 D-阿洛糖 3-酰亚胺酶半理性工程，可同时提高催化活性和热稳定性。

IF 3.2 3区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Biotechnology Journal Pub Date : 2024-08-21 DOI:10.1002/biot.202400280

Zijie Li, Yangfan Hu, Cheng Yu, Kangqing Fei, Liqun Shen, Yishi Liu, Hideki Nakanishi

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引用次数: 0

摘要

背景：D- 阿洛糖是最著名的稀有糖类之一，广泛应用于食品、化妆品和制药行业。生产 D-阿洛糖最常用的方法是在 D-阿洛糖 3-酰亚胺酶（DAEase）的催化下从 D-果糖转化而来。针对野生型DAEase普遍存在的催化效率低、热稳定性差的问题，本研究采用D-阿洛糖生物传感器，建立了一种方便、高效的DAEase变体高通量筛选方法：结果：通过半合理的分子改造，Caballeronia insecticola DAEase的催化活性和耐热性同时得到了提高。与野生型相比，DAEaseS37N/F157Y变体的催化活性提高了14.7%，65°C时的半衰期（t1/2）从1.60小时提高到27.56小时，提高了17.23倍。令我们欣喜的是，表达该 DAEase 变体的枯草芽孢杆菌 WB800 全细胞在 1 小时内从 500 g L-1 D-果糖中转化出 33.6% 的 D-阿洛糖。此外，还对细胞固定化的实用性进行了评估，固定化细胞的相对活性从第二周期到第七周期都保持在 80% 以上：所有这些结果表明，DAEaseS37N/F157Y 变体将成为工业化生产 D-纤维素的潜在候选者。

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Semi-rational engineering of D-allulose 3-epimerase for simultaneously improving the catalytic activity and thermostability based on D-allulose biosensor

Background

D-Allulose is one of the most well-known rare sugars widely used in food, cosmetics, and pharmaceutical industries. The most popular method for D-allulose production is the conversion from D-fructose catalyzed by D-allulose 3-epimerase (DAEase). To address the general problem of low catalytic efficiency and poor thermostability of wild-type DAEase, D-allulose biosensor was adopted in this study to develop a convenient and efficient method for high-throughput screening of DAEase variants.

Results

The catalytic activity and thermostability of DAEase from Caballeronia insecticola were simultaneously improved by semi-rational molecular modification. Compared with the wild-type enzyme, DAEase^S37N/F157Y variant exhibited 14.7% improvement in the catalytic activity and the half-time value (t_1/2) at 65°C increased from 1.60 to 27.56 h by 17.23-fold. To our delight, the conversion rate of D-allulose was 33.6% from 500-g L⁻¹ D-fructose in 1 h by Bacillus subtilis WB800 whole cells expressing this DAEase variant. Furthermore, the practicability of cell immobilization was evaluated and more than 80% relative activity of the immobilized cells was maintained from the second to seventh cycle.

Conclusion

All these results indicated that the DAEase^S37N/F157Y variant would be a potential candidate for the industrial production of D-allulose.

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来源期刊

Biotechnology Journal Biochemistry, Genetics and Molecular Biology-Molecular Medicine

CiteScore

8.90

自引率

2.10%

发文量

123

审稿时长

1.5 months

期刊介绍： Biotechnology Journal (2019 Journal Citation Reports: 3.543) is fully comprehensive in its scope and publishes strictly peer-reviewed papers covering novel aspects and methods in all areas of biotechnology. Some issues are devoted to a special topic, providing the latest information on the most crucial areas of research and technological advances. In addition to these special issues, the journal welcomes unsolicited submissions for primary research articles, such as Research Articles, Rapid Communications and Biotech Methods. BTJ also welcomes proposals of Review Articles - please send in a brief outline of the article and the senior author''s CV to the editorial office. BTJ promotes a special emphasis on: Systems Biotechnology Synthetic Biology and Metabolic Engineering Nanobiotechnology and Biomaterials Tissue engineering, Regenerative Medicine and Stem cells Gene Editing, Gene therapy and Immunotherapy Omics technologies Industrial Biotechnology, Biopharmaceuticals and Biocatalysis Bioprocess engineering and Downstream processing Plant Biotechnology Biosafety, Biotech Ethics, Science Communication Methods and Advances.