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Haoran Yu
杭州 | 浙江大学 | 百人计划研究员
  Email   yuhaoran@zju.edu.cn 
TA的实验室:   于浩然课题组
论文

An efficient biocatalytic oxidative dehydroaromatization approach for the construction of quinolines enabled by monoamine oxidase with molecular oxygen

An efficient biocatalytic approach for the oxidative dehydroaromatization of 1,2,3,4-tetrahydroquinolines by employing a monoamine oxidase PpMAO with molecular oxygen under mild conditions.

期刊: Green Chemistry  2023
作者: Huanhuan Jin,Shuyun Ju,Haoran Yu,Lirong Yang,Wenlong Zheng,Jianping Wu
DOI:10.1039/d3gc00858d

Sequence and Structure-Guided Engineering of Urethanase from <i>Agrobacterium tumefaciens</i> d3 for Improved Catalytic Activity

期刊: Journal of Agricultural and Food Chemistry  2022
作者: Mianbin Wu,Haoran Yu,Lirong Yang,Jianping Lin,Tao Zhang,Zhongji Pu,Tingting Kang,Xiumiao Yao
DOI:10.1021/acs.jafc.2c01406

Hot spots-making directed evolution easier

期刊: Biotechnology Advances  2022
作者: Paul A. Dalby,Yiwen Li,Shuang Ma,Haoran Yu
DOI:10.1016/j.biotechadv.2022.107926

Enhancing thermostability of lipase from <i>Pseudomonas alcaligenes</i> for producing <scp>l</scp>-menthol by the CREATE strategy

Rapid enhancement of the thermostability of enzymes by the CREATE strategy.

期刊: Catalysis Science & Technology  2022
作者: Gang Xu,Lirong Yang,Jianping Wu,Zhongji Pu,Kaitong Chen,Tingting Kang,Ziyuan Wang,Zhe Wang,Jinling Xu,Haoran Yu,Zhonglang Yu
DOI:10.1039/d2cy00082b

Enhancing the thermostability of D-allulose 3-epimerase from Clostridium cellulolyticum H10 via a dual-enzyme screening system

期刊: Enzyme and Microbial Technology  2022
作者: Jianping Lin,Haoran Yu,Lirong Yang,Mianbin Wu,Li Zhu,Zhongji Pu,Yun Feng
DOI:10.1016/j.enzmictec.2022.110054

Substrate access path-guided engineering of L-threonine aldolase for improving diastereoselectivity

The L-threonine aldolase from Leishmania major was engineered to improve diastereoselectivity by a CAST/ISM strategy, providing insights into the relationship between physico -chemical properties of substrate access path and diastereoselectivity....

期刊: Chemical Communications  2022
作者: Jianping Wu,Haoran Yu,Li-Rong Yang,Gang Xu,Lanxin Xiao,Zhongji Pu,Wenlong Zheng
DOI:10.1039/d2cc02644a

Computational design of highly stable and soluble alcohol dehydrogenase for NADPH regeneration

AbstractNicotinamide adenine dinucleotide phosphate (NADPH), as a well-known cofactor, is widely used in the most of enzymatic redox reactions, playing an important role in industrial catalysis. However, the absence of a comparable method for efficient NADP+ to NADPH cofactor regeneration radically impairs efficient green chemical synthesis. Alcohol dehydrogenase (ADH) enzymes, allowing the in situ regeneration of the redox cofactor NADPH with high specific activity and easy by-product separation process, are provided with great industrial application potential and research attention. Accordingly, herein a NADP+-specific ADH from Clostridium beijerinckii was selected to be engineered for cofactor recycle, using an automated algorithm named Protein Repair One-stop Shop (PROSS). The mutant CbADH-6M (S24P/G182A/G196A/H222D/S250E/S254R) exhibited a favorable soluble and highly active expression with an activity of 46.3 U/mL, which was 16 times higher than the wild type (2.9 U/mL), and a more stable protein conformation with an enhanced thermal stability: Δ $${T}_{1/2}^{60\mathrm{min}}$$ T 1 / 2 60 min =  + 3.6 °C (temperature of 50% inactivation after incubation for 60 min). Furthermore, the activity of CbADH-6M was up-graded to 2401.8 U/mL by high cell density fermentation strategy using recombinant Escherichia coli, demonstrating its industrial potential. Finally, the superb efficiency for NADPH regeneration of the mutant enzyme was testified in the synthesis of some fine chiral aromatic alcohols coupling with another ADH from Lactobacillus kefir (LkADH).

期刊: Bioresources and Bioprocessing  2021
作者: Lirong Yang,Jianping Wu,Hongyu Zhang,Ziyuan Wang,Tong Deng,Haoran Yu,Haisheng Zhou,Jinling Xu
DOI:10.1186/s40643-021-00362-w

Site‐specifically Incorporate Non‐Canonical Amino Acids into Pseudomonas alcaligenes Lipase to Hydrolyze L‐ menthol propionate among the Eight Isomers

期刊: ChemCatChem  2021
作者: Gang Xu,Lirong Yang,Jianping Wu,Zhe Wang,Haibin Tang,Haoran Yu,Zhonglang Yu
DOI:10.1002/cctc.202100358

Directed Evolution of l-Threonine Aldolase for the Diastereoselective Synthesis of β-Hydroxy-α-amino Acids

期刊: ACS Catalysis  2021
作者: Jianping Wu,Lirong Yang,Gang Xu,Xiuli Cheng,Zhe Wang,Kaitong Chen,Sai Fang,Haoran Yu,Wenlong Zheng
DOI:10.1021/acscatal.0c04949

A beginner's guide to molecular dynamics simulations and the identification of cross-correlation networks for enzyme engineering

期刊: Enzyme Engineering and Evolution: General Methods  2020
作者: Paul A. Dalby,Haoran Yu
DOI:10.1016/bs.mie.2020.04.020

Engineering transketolase to accept both unnatural donor and acceptor substrates and produce α‐hydroxyketones

A narrow substrate range is a major limitation in exploiting enzymes more widely as catalysts in synthetic organic chemistry. For enzymes using two substrates, the simultaneous optimisation of both substrate specificities is also required for the rapid expansion of accepted substrates. Transketolase (TK) catalyses the reversible transfer of a C2‐ketol unit from a donor substrate to an aldehyde acceptor and suffers the limitation of narrow substrate scope for industrial applications. Herein, TK from Escherichia coli was engineered to accept both pyruvate, as a novel donor substrate, and unnatural acceptor aldehydes, including propanal, pentanal, hexanal and 3‐formylbenzoic acid (FBA). Twenty single‐mutant variants were first designed and characterised experimentally. Beneficial mutations were then recombined to construct a small library. Screening of this library identified the best variant with a 9.2‐fold improvement in the yield towards pyruvate and propionaldehyde, relative to wild‐type (WT). Pentanal and hexanal were used as acceptors to determine stereoselectivities of the reactions, which were found to be higher than 98% enantiomeric excess (ee) for the S configuration. Three variants were identified to be active for the reaction between pyruvate and 3‐FBA. The best variant was able to convert 47% of substrate into product within 24 h, whereas no conversion was observed for WT. Docking experiments suggested a cooperation between the mutations responsible for donor and acceptor recognition, which would promote the activity towards both the acceptor and donor. The variants obtained have the potential to be used for developing catalytic pathways to a diverse range of high‐value products.

期刊: The FEBS Journal  2019
作者: Paul A. Dalby,Helen C. Hailes,John M. Ward,Tom D. Sheppard,Armando Cázares‐Körner,Sally Higson,Daniel Méndez‐Sánchez,David Steadman,Roberto Icken Hernández López,Haoran Yu
DOI:10.1111/febs.15108

Enhanced trypsin thermostability in Pichia pastoris through truncating the flexible region

期刊: Microbial Cell Factories  2018
作者: He Huang,Yiru Gan,Zhiyan Wang,Kun Du,Haoran Yu,Lin Liu
DOI:10.1186/s12934-018-1012-x

Exploiting correlated molecular-dynamics networks to counteract enzyme activity–stability trade-off

The directed evolution of enzymes for improved activity or substrate specificity commonly leads to a trade-off in stability. We have identified an activity–stability trade-off and a loss in unfolding cooperativity for a variant (3M) of Escherichia coli transketolase (TK) engineered to accept aromatic substrates. Molecular dynamics simulations of 3M revealed increased flexibility in several interconnected active-site regions that also form part of the dimer interface. Mutating the newly flexible active-site residues to regain stability risked losing the new activity. We hypothesized that stabilizing mutations could be targeted to residues outside of the active site, whose dynamics were correlated with the newly flexible active-site residues. We previously stabilized WT TK by targeting mutations to highly flexible regions. These regions were much less flexible in 3M and would not have been selected a priori as targets using the same strategy based on flexibility alone. However, their dynamics were highly correlated with the newly flexible active-site regions of 3M. Introducing the previous mutations into 3M reestablished the WT level of stability and unfolding cooperativity, giving a 10.8-fold improved half-life at 55 °C, and increased midpoint and aggregation onset temperatures by 3 °C and 4.3 °C, respectively. Even the activity toward aromatic aldehydes increased up to threefold. Molecular dynamics simulations confirmed that the mutations rigidified the active-site via the correlated network. This work provides insights into the impact of rigidifying mutations within highly correlated dynamic networks that could also be useful for developing improved computational protein engineering strategies.

期刊: Proceedings of the National Academy of Sciences  2018
作者: Paul A. Dalby,Haoran Yu
DOI:10.1073/pnas.1812204115

Coupled molecular dynamics mediate long- and short-range epistasis between mutations that affect stability and aggregation kinetics

Multiple mutations are typically required to significantly improve protein stability or aggregation kinetics. However, when several substitutions are made in a single protein, the mutations can potentially interact in a nonadditive manner, resulting in epistatic effects, which can hamper protein-engineering strategies to improve thermostability or aggregation kinetics. Here, we have examined the role of protein dynamics in mediating epistasis between pairs of mutations. With Escherichia coli transketolase (TK) as a model, we explored the epistatic interactions between two single variants H192P and A282P, and also between the double-mutant H192P/A282P and two single variants, I365L or G506A. Epistasis was determined for several measures of protein stability, including the following: the free-energy barrier to kinetic inactivation, ∆∆G‡; thermal transition midpoint temperatures, Tm; and aggregation onset temperatures, Tagg. Nonadditive epistasis was observed between neighboring mutations as expected, but also for distant mutations located in the surface and core regions of different domains. Surprisingly, the epistatic behaviors for each measure of stability were often different for any given pairwise recombination, highlighting that kinetic and thermodynamic stabilities do not always depend on the same structural features. Molecular-dynamics simulations and a pairwise cross-correlation analysis revealed that mutations influence the dynamics of their local environment, but also in some cases the dynamics of regions distant in the structure. This effect was found to mediate epistatic interactions between distant mutations and could therefore be exploited in future protein-engineering strategies.

期刊: Proceedings of the National Academy of Sciences  2018
作者: Paul A. Dalby,Haoran Yu
DOI:10.1073/pnas.1810324115

Two strategies to engineer flexible loops for improved enzyme thermostability

期刊: Scientific Reports  2017
作者: Paul A. Dalby,Cheng Zhang,Yihan Yan,Haoran Yu
DOI:10.1038/srep41212

A Novel Strategy for Thermostability Improvement of Trypsin Based on N-Glycosylation within the ��-Loop Region

期刊: Journal of Microbiology and Biotechnology  2016
作者: He Huang,Yiru Gan,Kun Du,Haoran Yu,Ye Liu,Chao Guo
DOI:10.4014/jmb.1512.12070

The role of proline substitutions within flexible regions on thermostability of luciferase

期刊: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics  2015
作者: He Huang,Yiru Gan,Chao Guo,Yang Zhao,Haoran Yu
DOI:10.1016/j.bbapap.2014.10.017

Engineering proteins for thermostability through rigidifying flexible sites

期刊: Biotechnology Advances  2014
作者: He Huang,Haoran Yu
DOI:https://doi.org/10.1016/j.biotechadv.2013.10.012

Soluble Expression of Active Recombinant Firefly Luciferase in Escherichia coli

期刊: Journal of Pure and Applied Microbiology  2013
作者: He Huang,Guangbo Kang,Xinya Liang,Liujiehuo,Yanjie Liu,Haoran Yu

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