Sortase A-mediated crosslinked short-chain dehydrogenases/reductases as novel biocatalysts with improved thermostability and catalytic efficiency

Abstract (S)-carbonyl reductase II (SCRII) from Candida parapsilosis is a short-chain alcohol dehydrogenase/reductase. It catalyses the conversion of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol with low efficiency. Sortase was reported as a molecular “stapler” for site-specific protein conj...

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Autores principales:	Kunpeng Li, Rongzhen Zhang, Yan Xu, Zhimeng Wu, Jing Li, Xiaotian Zhou, Jiawei Jiang, Haiyan Liu, Rong Xiao
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/9daad771d7d84bdcae96adb32fc21141
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spelling	oai:doaj.org-article:9daad771d7d84bdcae96adb32fc211412021-12-02T11:52:23ZSortase A-mediated crosslinked short-chain dehydrogenases/reductases as novel biocatalysts with improved thermostability and catalytic efficiency10.1038/s41598-017-03168-z2045-2322https://doaj.org/article/9daad771d7d84bdcae96adb32fc211412017-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-03168-zhttps://doaj.org/toc/2045-2322Abstract (S)-carbonyl reductase II (SCRII) from Candida parapsilosis is a short-chain alcohol dehydrogenase/reductase. It catalyses the conversion of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol with low efficiency. Sortase was reported as a molecular “stapler” for site-specific protein conjugation to strengthen or add protein functionality. Here, we describe Staphylococcus aureus sortase A-mediated crosslinking of SCRII to produce stable catalysts for efficient biotransformation. Via a native N-terminal glycine and an added GGGGSLPETGG peptide at C-terminus of SCRII, SCRII subunits were conjugated by sortase A to form crosslinked SCRII, mainly dimers and trimers. The crosslinked SCRII showed over 6-fold and 4-fold increases, respectively, in activity and k cat/K m values toward 2-hydroxyacetophenone compared with wild-type SCRII. Moreover, crosslinked SCRII was much more thermostable with its denaturation temperature (Tm) increased to 60 °C. Biotransformation result showed that crosslinked SCRII gave a product optical purity of 100% and a yield of >99.9% within 3 h, a 16-fold decrease in transformation duration with respect to Escherichia coli/pET-SCRII. Sortase A-catalysed ligation also obviously improved Tms and product yields of eight other short-chain alcohol dehydrogenases/reductases. This work demonstrates a generic technology to improve enzyme function and thermostability through sortase A-mediated crosslinking of oxidoreductases.Kunpeng LiRongzhen ZhangYan XuZhimeng WuJing LiXiaotian ZhouJiawei JiangHaiyan LiuRong XiaoNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-11 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Kunpeng Li Rongzhen Zhang Yan Xu Zhimeng Wu Jing Li Xiaotian Zhou Jiawei Jiang Haiyan Liu Rong Xiao Sortase A-mediated crosslinked short-chain dehydrogenases/reductases as novel biocatalysts with improved thermostability and catalytic efficiency
description	Abstract (S)-carbonyl reductase II (SCRII) from Candida parapsilosis is a short-chain alcohol dehydrogenase/reductase. It catalyses the conversion of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol with low efficiency. Sortase was reported as a molecular “stapler” for site-specific protein conjugation to strengthen or add protein functionality. Here, we describe Staphylococcus aureus sortase A-mediated crosslinking of SCRII to produce stable catalysts for efficient biotransformation. Via a native N-terminal glycine and an added GGGGSLPETGG peptide at C-terminus of SCRII, SCRII subunits were conjugated by sortase A to form crosslinked SCRII, mainly dimers and trimers. The crosslinked SCRII showed over 6-fold and 4-fold increases, respectively, in activity and k cat/K m values toward 2-hydroxyacetophenone compared with wild-type SCRII. Moreover, crosslinked SCRII was much more thermostable with its denaturation temperature (Tm) increased to 60 °C. Biotransformation result showed that crosslinked SCRII gave a product optical purity of 100% and a yield of >99.9% within 3 h, a 16-fold decrease in transformation duration with respect to Escherichia coli/pET-SCRII. Sortase A-catalysed ligation also obviously improved Tms and product yields of eight other short-chain alcohol dehydrogenases/reductases. This work demonstrates a generic technology to improve enzyme function and thermostability through sortase A-mediated crosslinking of oxidoreductases.
format	article
author	Kunpeng Li Rongzhen Zhang Yan Xu Zhimeng Wu Jing Li Xiaotian Zhou Jiawei Jiang Haiyan Liu Rong Xiao
author_facet	Kunpeng Li Rongzhen Zhang Yan Xu Zhimeng Wu Jing Li Xiaotian Zhou Jiawei Jiang Haiyan Liu Rong Xiao
author_sort	Kunpeng Li
title	Sortase A-mediated crosslinked short-chain dehydrogenases/reductases as novel biocatalysts with improved thermostability and catalytic efficiency
title_short	Sortase A-mediated crosslinked short-chain dehydrogenases/reductases as novel biocatalysts with improved thermostability and catalytic efficiency
title_full	Sortase A-mediated crosslinked short-chain dehydrogenases/reductases as novel biocatalysts with improved thermostability and catalytic efficiency
title_fullStr	Sortase A-mediated crosslinked short-chain dehydrogenases/reductases as novel biocatalysts with improved thermostability and catalytic efficiency
title_full_unstemmed	Sortase A-mediated crosslinked short-chain dehydrogenases/reductases as novel biocatalysts with improved thermostability and catalytic efficiency
title_sort	sortase a-mediated crosslinked short-chain dehydrogenases/reductases as novel biocatalysts with improved thermostability and catalytic efficiency
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/9daad771d7d84bdcae96adb32fc21141
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Sortase A-mediated crosslinked short-chain dehydrogenases/reductases as novel biocatalysts with improved thermostability and catalytic efficiency

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