Mechanism-based site-directed mutagenesis to shift the optimum pH of the phenylalanine ammonia-lyase from Rhodotorula glutinis JN-1

Zhu, Liping; Zhou, Li; Cui, Wenjing; Liu, Zhongmei; Zhou, Zhemin

doi:10.1016/j.btre.2014.06.001

Cited by 12 publications

(5 citation statements)

References 38 publications

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“…[11] Production of both enzymes as either wet (EfPheDC) or dry (AvPAL) whole cell biocatalysts allowed simple investigation of their pH optima. The enzymes were shown to operate under very different conditions in this respect, as consistent with previous studies of PAL enzymes [18,19] and the potential involvement of AADCs with bacterial acid stress responses. [20] As such, initial experiments focussed on the development of a sequential reaction whereby cells, water and ammonium carbamate (required for PAL-mediated amination) were removed via centrifugation and evaporation, leaving a crude isolate [21] for conversion by EfPheDC under separate conditions.…”

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confidence: 88%

Bi‐enzymatic Conversion of Cinnamic Acids to 2‐Arylethylamines

et al. 2020

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The conversion of carboxylic acids, such as acrylic acids, to amines is a transformation that remains challenging in synthetic organic chemistry. Despite the ubiquity of similar moieties in natural metabolic pathways, biocatalytic routes seem to have been overlooked for this purpose. Herein we present the conception and optimisation of a two‐enzyme system, allowing the synthesis of β‐phenylethylamine derivatives from readily‐available ring‐substituted cinnamic acids. After characterisation of both parts of the reaction in a two‐step approach, a set of conditions allowing the one‐pot biotransformation was optimised. This combination of a reversible deaminating and irreversible decarboxylating enzyme, both specific for the amino acid intermediate in tandem, represents a general method by which new strategies for the conversion of carboxylic acids to amines could be designed.

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confidence: 88%

Bi‐enzymatic Conversion of Cinnamic Acids to 2‐Arylethylamines

et al. 2020

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“…However, researches have focused primarily on the possibility to obtain phenylalanine ammonia lyase (E.C.4.3.1.5). As a result of the effect of this enzyme, it is possible to obtain l -phenylalanine, which constitutes the substrate for aspartame production (D’Cunha et al 1996a , 1996b ; Zhu et al 2014 ).…”

Section: Biosynthesis Of Phenylalanine Ammonia Lyase By R mentioning

confidence: 99%

Rhodotorula glutinis—potential source of lipids, carotenoids, and enzymes for use in industries

Kot

Błażejak

Kurcz

et al. 2016

Appl Microbiol Biotechnol

189

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Rhodotorula glutinis is capable of synthesizing numerous valuable compounds with a wide industrial usage. Biomass of this yeast constitutes sources of microbiological oils, and the whole pool of fatty acids is dominated by oleic, linoleic, and palmitic acid. Due to its composition, the lipids may be useful as a source for the production of the so-called third-generation biodiesel. These yeasts are also capable of synthesizing carotenoids such as β-carotene, torulene, and torularhodin. Due to their health-promoting characteristics, carotenoids are commonly used in the cosmetic, pharmaceutical, and food industries. They are also used as additives in fodders for livestock, fish, and crustaceans. A significant characteristic of R. glutinis is its capability to produce numerous enzymes, in particular, phenylalanine ammonia lyase (PAL). This enzyme is used in the food industry in the production of l-phenylalanine that constitutes the substrate for the synthesis of aspartame—a sweetener commonly used in the food industry.

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“…In 2012, bioinformatics was used to identify the regions that are responsible for the stability of Ntn‐hydrolases under alkaline conditions (Suplatov et al, ). Phenylalanine ammonia‐lyase from Rhodotorula glutinis JN‐1 had its optimum pH shifted based on structural analysis and catalytic mechanism (Zhu, Zhou, Cui, Liu, & Zhou, ). Given the previous studies on the enzymatic mechanisms under acidic conditions, the effects of pH on proteins involve not only the global structure but also the local environments.…”

Section: Introductionmentioning

confidence: 99%

Improvement of the acid resistance, catalytic efficiency, and thermostability of nattokinase by multisite‐directed mutagenesis

Liu

Zhao

Han

et al. 2019

Biotech & Bioengineering

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Nattokinase (NK) is a serine protease of the subtilisin family; as a potent fibrinolytic enzyme, it is potentially useful for thrombosis therapy. For NK to be applied as an oral medicine for the treatment of cardiovascular diseases, it must overcome the extremely acidic environments of the gastrointestinal tract despite its limited acidic stability. In this study, three strategies were adopted to improve the acid resistance of NK: (a) Surface charge engineering, (b) sequence alignment, and (c) mutation based on the literature. Eleven variants were constructed and four single-point mutations were screened out for their distinctive catalytic properties: Q59E increased the specific activity; S78T improved the acid stability; Y217K enhanced the acid and thermal stabilities; and N218D improved the thermostability. Based on these observations, multipoint variants were constructed and characterized, and one variant with better acid stability, catalytic efficiency, and thermostability was obtained. Molecular dynamics simulation was carried out to clarify the molecular mechanism of the increased stability of S78T and Y217K mutants under acidic conditions. This study explored effective strategies to engineer acid resistance of NK; moreover, the NK variants with better catalytic properties found in this study have potential applications for the medical industry.

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Mechanism-based site-directed mutagenesis to shift the optimum pH of the phenylalanine ammonia-lyase from Rhodotorula glutinis JN-1

Abstract: HighlightsA residue in active site which affects the optimum pH for catalysis was found.A mutant with an extended optimum pH 7–9 was constructed.The catalytic mechanism explains the extended optimum pH of the mutant.

Cited by 12 publications

References 38 publications

Bi‐enzymatic Conversion of Cinnamic Acids to 2‐Arylethylamines

Bi‐enzymatic Conversion of Cinnamic Acids to 2‐Arylethylamines

Rhodotorula glutinis—potential source of lipids, carotenoids, and enzymes for use in industries

Improvement of the acid resistance, catalytic efficiency, and thermostability of nattokinase by multisite‐directed mutagenesis

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