Fluorescent enzyme-coupled activity assay for phenylalanine ammonia-lyases

Abstract Phenylalanine ammonia-lyases (PALs) catalyse the non-oxidative deamination of l-phenylalanine to trans-cinnamic acid, while in the presence of high ammonia concentration the reverse reaction occurs. PALs have been intensively studied, however, their industrial applications for amino acids synthesis remained limited, mainly due to their decreased operational stability or limited substrate specificity. The application of extensive directed evolution procedures to improve their stability, activity or selectivity, is hindered by the lack of reliable activity assays allowing facile screening of PAL-activity within large-sized mutant libraries. Herein, we describe the development of an enzyme-coupled fluorescent assay applicable for PAL-activity screens at whole cell level, involving decarboxylation of trans-cinnamic acid (the product of the PAL reaction) by ferulic acid decarboxylase (FDC1) and a photochemical reaction of the produced styrene with a diaryltetrazole, that generates a detectable, fluorescent pyrazoline product. The general applicability of the fluorescent assay for PALs of different origin, as well as its versatility for the detection of tyrosine ammonia-lyase (TAL) activity have been also demonstrated. Accordingly, the developed procedure provides a facile tool for the efficient activity screens of large mutant libraries of PALs in presence of non-natural substrates of interest, being essential for the substrate-specificity modifications/tailoring of PALs through directed evolution-based protein engineering.

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Chemoenzymatic cascade for stilbene production from cinnamic acid catalyzed by ferulic acid decarboxylase and an artificial metathease

Catalysis Science & Technology ◽

10.1039/c9cy01412h ◽

2019 ◽

Vol 9 (20) ◽

pp. 5572-5576 ◽

Cited By ~ 5

Author(s):

M. A. Stephanie Mertens ◽

Daniel F. Sauer ◽

Ulrich Markel ◽

Johannes Schiffels ◽

Jun Okuda ◽

...

Keyword(s):

Ferulic Acid ◽

Cinnamic Acid ◽

Olefin Metathesis ◽

Metal Contamination ◽

Cascade Reaction ◽

Acid Decarboxylase ◽

Efficient Removal ◽

Ferulic Acid Decarboxylase ◽

Acid Catalyzed ◽

Removal Of Metal

We report a chemoenzymatic cascade reaction for stilbene production combining decarboxylation and olefin metathesis with efficient removal of metal contamination.

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Production of Trans-Cinnamic Acid by Immobilization of the Bambusa oldhamii BoPAL1 and BoPAL2 Phenylalanine Ammonia-Lyases on Electrospun Nanofibers

International Journal of Molecular Sciences ◽

10.3390/ijms222011184 ◽

2021 ◽

Vol 22 (20) ◽

pp. 11184

Author(s):

Pei-Yu Hong ◽

Yi-Hao Huang ◽

GiGi Chin Wen Lim ◽

Yen-Po Chen ◽

Che-Jen Hsiao ◽

...

Keyword(s):

Response Surface ◽

Cinnamic Acid ◽

Catalytic Efficiency ◽

Electrospun Nanofibers ◽

Industrial Applications ◽

Cross Linking ◽

Pal Activity ◽

Surface Models ◽

Dextran Polyaldehyde ◽

Bambusa Oldhamii

Phenylalanine ammonia-lyase (PAL) catalyzes the nonoxidative deamination of phenylalanine to yield trans-cinnamic acid and ammonia. Recombinant Bambusa oldhamii BoPAL1/2 proteins were immobilized onto electrospun nanofibers by dextran polyaldehyde as a cross-linking agent. A central composite design (CCD)-response surface methodology (RSM) was utilized to optimize the electrospinning parameters. Escherichia coli expressed eBoPAL2 exhibited the highest catalytic efficiency among four enzymes. The optimum conditions for fabricating nanofibers were determined as follows: flow rate of 0.10 mL/h, voltage of 13.8 kV, and distance of 13 cm. The response surface models were used to obtain the smaller the fiber diameters as well as the highest PAL activity in the enzyme immobilization. Compared with free BoPALs, immobilized BoPALs can be reused for at least 6 consecutive cycles. The remained activity of the immobilized BoPAL proteins after storage at 4 °C for 30 days were between 75 and 83%. In addition, the tolerance against denaturants of the immobilized BoPAL proteins were significantly enhanced. As a result, the dextran polyaldehyde natural cross-linking agent can effectively replace traditional chemical cross-linking agents for the immobilization of the BoPAL enzymes. The PAL/nylon 6/polyvinyl alcohol (PVA)/chitosan (CS) nanofibers made are extremely stable and are practical for industrial applications in the future.

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Native to designed: microbial α-amylases for industrial applications

PeerJ ◽

10.7717/peerj.11315 ◽

2021 ◽

Vol 9 ◽

pp. e11315

Author(s):

Si Jie Lim ◽

Siti Nurbaya Oslan

Keyword(s):

Protein Engineering ◽

Substrate Specificity ◽

Oxidative Stability ◽

Directed Evolution ◽

Half Life ◽

Rational Design ◽

Survey Methodology ◽

Industrial Applications ◽

Surface Binding ◽

The Future

Background α-amylases catalyze the endo-hydrolysis of α-1,4-D-glycosidic bonds in starch into smaller moieties. While industrial processes are usually performed at harsh conditions, α-amylases from mainly the bacteria, fungi and yeasts are preferred for their stabilities (thermal, pH and oxidative) and specificities (substrate and product). Microbial α-amylases can be purified and characterized for industrial applications. While exploring novel enzymes with these properties in the nature is time-costly, the advancements in protein engineering techniques including rational design, directed evolution and others have privileged their modifications to exhibit industrially ideal traits. However, the commentary on the strategies and preferably mutated residues are lacking, hindering the design of new mutants especially for enhanced substrate specificity and oxidative stability. Thus, our review ensures wider accessibility of the previously reported experimental findings to facilitate the future engineering work. Survey methodology and objectives A traditional review approach was taken to focus on the engineering of microbial α-amylases to enhance industrially favoured characteristics. The action mechanisms of α- and β-amylases were compared to avoid any bias in the research background. This review aimed to discuss the advances in modifying microbial α-amylases via protein engineering to achieve longer half-life in high temperature, improved resistance (acidic, alkaline and oxidative) and enhanced specificities (substrate and product). Captivating results were discussed in depth, including the extended half-life at 100 °C, pH 3.5 and 10, 1.8 M hydrogen peroxide as well as enhanced substrate (65.3%) and product (42.4%) specificities. These shed light to the future microbial α-amylase engineering in achieving paramount biochemical traits ameliorations to apt in the industries. Conclusions Microbial α-amylases can be tailored for specific industrial applications through protein engineering (rational design and directed evolution). While the critical mutation points are dependent on respective enzymes, formation of disulfide bridge between cysteine residues after mutations is crucial for elevated thermostability. Amino acids conversion to basic residues was reported for enhanced acidic resistance while hydrophobic interaction resulted from mutated hydrophobic residues in carbohydrate-binding module or surface-binding sites is pivotal for improved substrate specificity. Substitution of oxidation-prone methionine residues with non-polar residues increases the enzyme oxidative stability. Hence, this review provides conceptual advances for the future microbial α-amylases designs to exhibit industrially significant characteristics. However, more attention is needed to enhance substrate specificity and oxidative stability since they are least reported.

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Directed evolution of prenylated FMN-dependent Fdc supports efficient in vivo isobutene production

Nature Communications ◽

10.1038/s41467-021-25598-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Annica Saaret ◽

Benoît Villiers ◽

François Stricher ◽

Macha Anissimova ◽

Mélodie Cadillon ◽

...

Keyword(s):

Directed Evolution ◽

Mevalonate Pathway ◽

Fold Increase ◽

Binding Pocket ◽

Acid Decarboxylase ◽

Fuel Additive ◽

Decarboxylase Activity ◽

Ferulic Acid Decarboxylase ◽

Acrylic Acids

AbstractIsobutene is a high value gaseous alkene used as fuel additive and a chemical building block. As an alternative to fossil fuel derived isobutene, we here develop a modified mevalonate pathway for the production of isobutene from glucose in vivo. The final step in the pathway consists of the decarboxylation of 3-methylcrotonic acid, catalysed by an evolved ferulic acid decarboxylase (Fdc) enzyme. Fdc belongs to the prFMN-dependent UbiD enzyme family that catalyses reversible decarboxylation of (hetero)aromatic acids or acrylic acids with extended conjugation. Following a screen of an Fdc library for inherent 3-methylcrotonic acid decarboxylase activity, directed evolution yields variants with up to an 80-fold increase in activity. Crystal structures of the evolved variants reveal that changes in the substrate binding pocket are responsible for increased selectivity. Solution and computational studies suggest that isobutene cycloelimination is rate limiting and strictly dependent on presence of the 3-methyl group.

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Directed evolution of CotA laccase for increased substrate specificity using Bacillus subtilis spores

Protein Engineering Design and Selection ◽

10.1093/protein/gzq036 ◽

2010 ◽

Vol 23 (8) ◽

pp. 679-682 ◽

Cited By ~ 46

Author(s):

Nirupama Gupta ◽

Edgardo T. Farinas

Keyword(s):

Bacillus Subtilis ◽

Substrate Specificity ◽

Directed Evolution ◽

Cota Laccase

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Mechanistic insights into the catalytic reaction of ferulic acid decarboxylase from Aspergillus niger: a QM/MM study

Physical Chemistry Chemical Physics ◽

10.1039/c6cp08811b ◽

2017 ◽

Vol 19 (11) ◽

pp. 7733-7742 ◽

Cited By ~ 16

Author(s):

Ge Tian ◽

Yongjun Liu

Keyword(s):

Aspergillus Niger ◽

Ferulic Acid ◽

Catalytic Reaction ◽

Acid Decarboxylase ◽

Rate Limiting Step ◽

Limiting Step ◽

Ferulic Acid Decarboxylase ◽

Rate Limiting

QM/MM calculations reveal the cofactor prFMNiminiumto be the catalytically relevant species compared with prFMNketamine. The protonation of the intermediate is the rate-limiting step, and the prolonged leaving of the generated CO2can facilitate this process.

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Screening and directed evolution of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae

10.1101/166678 ◽

2017 ◽

Author(s):

Chenlu Zhang ◽

Ligia Acosta-Sampson ◽

Vivian Yaci Yu ◽

Jamie H. D. Cate

Keyword(s):

Saccharomyces Cerevisiae ◽

Directed Evolution ◽

Plant Biomass ◽

Carbon Sources ◽

Industrial Applications ◽

Yeast Saccharomyces Cerevisiae ◽

Cellulosic Biofuel ◽

Economic Production ◽

Report Analysis ◽

Selection Medium

AbstractThe economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters.

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Directed Evolution of a Homodimeric Laccase from Cerrena unicolor BBP6 by Random Mutagenesis and In Vivo Assembly

International Journal of Molecular Sciences ◽

10.3390/ijms19102989 ◽

2018 ◽

Vol 19 (10) ◽

pp. 2989 ◽

Cited By ~ 3

Author(s):

Ji Zhang ◽

Fuying Ma ◽

Xiaoyu Zhang ◽

Anli Geng

Keyword(s):

Directed Evolution ◽

Laccase Activity ◽

Catalytic Properties ◽

Random Mutagenesis ◽

Catalytic Performance ◽

Industrial Applications ◽

Subunit Interaction ◽

Cerrena Unicolor ◽

Protein Model

Laccases have great potential for industrial applications due to their green catalytic properties and broad substrate specificities, and various studies have attempted to improve the catalytic performance of these enzymes. Here, to the best of our knowledge, we firstly report the directed evolution of a homodimeric laccase from Cerrena unicolor BBP6 fused with α-factor prepro-leader that was engineered through random mutagenesis followed by in vivo assembly in Saccharomyces cerevisiae. Three evolved fusion variants selected from ~3500 clones presented 31- to 37-fold increases in total laccase activity, with better thermostability and broader pH profiles. The evolved α-factor prepro-leader enhanced laccase expression levels by up to 2.4-fold. Protein model analysis of these variants reveals that the beneficial mutations have influences on protein pKa shift, subunit interaction, substrate entrance, and C-terminal function.

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Reshaping the Active Pocket of Esterase Est816 for Resolution of Economically Important Racemates

Catalysis Science & Technology ◽

10.1039/d1cy01028j ◽

2021 ◽

Author(s):

Xiaolong Liu ◽

Meng Zhao ◽

Xinjiong Fan ◽

Yao Fu

Keyword(s):

Substrate Specificity ◽

Industrial Applications ◽

Pure Compounds ◽

Optically Pure ◽

Wide Substrate Specificity

One of the most important industrial applications of bacterial esterases is the production of optically pure compounds. However, the contradiction between the wide substrate specificity and high enantioselectivity of natural...

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