Engineering of TM1459 from Thermotoga maritima for Increased Oxidative Alkene Cleavage Activity

Fink, Matthias; Trunk, Sarah; Hall, Mélanie; Schwab, Helmut; Steiner, Kerstin

doi:10.3389/fmicb.2016.01511

Cited by 11 publications

(26 citation statements)

References 38 publications

(37 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The results for the alkene cleavage underline the potential of the PsaPOX as biocatalyst for the generation of aromatic aldehydes with olfactory properties, such as p-anisaldehyde, veratraldehyde, or acetophenone, which are used in the fragrance and flavor industry [1]. Improvement of the conversions and product yields may be accomplished by protein engineering, as has been shown for the alkene cleaving manganese-dependent Cupin TM1459 from Thermotoga maritima [53]. Another application beyond aroma production could be carotene bleaching of whey or wheat dough.…”

Section: Discussionmentioning

confidence: 99%

A DyP-Type Peroxidase of Pleurotus sapidus with Alkene Cleaving Activity

2020

View full text Add to dashboard Cite

Alkene cleavage is a possibility to generate aldehydes with olfactory properties for the fragrance and flavor industry. A dye-decolorizing peroxidase (DyP) of the basidiomycete Pleurotus sapidus (PsaPOX) cleaved the aryl alkene trans-anethole. The PsaPOX was semi-purified from the mycelium via FPLC, and the corresponding gene was identified. The amino acid sequence as well as the predicted tertiary structure showed typical characteristics of DyPs as well as a non-canonical Mn2+-oxidation site on its surface. The gene was expressed in Komagataella pfaffii GS115 yielding activities up to 142 U/L using 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) as substrate. PsaPOX exhibited optima at pH 3.5 and 40 °C and showed highest peroxidase activity in the presence of 100 µM H2O2 and 25 mM Mn2+. PsaPOX lacked the typical activity of DyPs towards anthraquinone dyes, but oxidized Mn2+ to Mn3+. In addition, bleaching of β-carotene and annatto was observed. Biotransformation experiments verified the alkene cleavage activity towards the aryl alkenes (E)-methyl isoeugenol, α-methylstyrene, and trans-anethole, which was increased almost twofold in the presence of Mn2+. The resultant aldehydes are olfactants used in the fragrance and flavor industry. PsaPOX is the first described DyP with alkene cleavage activity towards aryl alkenes and showed potential as biocatalyst for flavor production.

show abstract

Section: Discussionmentioning

confidence: 99%

A DyP-Type Peroxidase of Pleurotus sapidus with Alkene Cleaving Activity

2020

View full text Add to dashboard Cite

show abstract

“…The mutations were introduced by overlap‐extension PCR using standard PCR conditions and a Phusion HF Polymerase. The plasmid pET26b‐TM1459‐C106Q 6 was used as template and the purified PCR products were re‐ligated into the vector pET26b(+) using Nde I and Hind III restriction sites. For subsequent double, triple, quadruple, and quintuple variants, site‐directed mutagenesis was performed using plasmids as templates, which carried already one or more of the desired mutations.…”

Section: Methodsmentioning

confidence: 99%

“…The cell-free lysate was then directly used for high-throughput screening assay. The high-throughput screening assay for oxidative alkene cleavage activity was performed as described before [6]. 130 mL of protein solution (either cell-free lysate or 2 mg mL -1 purified protein) were mixed with 50 mL of substrate mix (46 % ethanol, 36 mM a-methylstyrene (final concentration in reaction 10 mM), 90 mM tert-butyl hydroperoxide (final concentration in reaction 25 mM), and 0.2 % Triton X-100 in 50 mM NaPi, pH 8 buffer) in 96-well microtiter plates.…”

Section: Library Screeningmentioning

confidence: 99%

See 1 more Smart Citation

Engineering TM1459 for Stabilisation against Inactivation by Amino Acid Oxidation

Grill

Pavkov‐Keller

Grininger

et al. 2023

Chemie Ingenieur Technik

Self Cite

View full text Add to dashboard Cite

Oxidative alkene cleavage is a highly interesting reaction to obtain aldehydes and ketones. The Mn‐dependent protein TM1459 from Thermotoga maritima can catalyse alkene cleavage of styrene derivatives in the presence of tert‐butyl hydroperoxide. Despite the high thermal stability of the enzyme, it gets inactivated during the reaction. The data reported here indicate that auto‐oxidation is responsible for the low stability of TM1459 in the oxidative environment required for the alkene cleavage reaction. By targeting the exchange of residues prone to oxidation, this phenomenon was successfully prevented. Importantly, the stability to oxidation conveyed by the amino acid exchanges led to increased enzyme activity. However, the exchanges resulted in slightly modified positions of two of the four metal‐binding amino acids, thereby strongly impacting metal binding.

show abstract

“…Thermotoga neapolitana is a hyperthermophilic Gram-negative bacterium of the order of Thermotogales [94][95][96], which are excellent models for genetic engineering and biotechnological applications [97][98][99][100]. The CTN1690 ORF shows a clear homology of the O 6 -alkylguanine-DNAalkyl-transferase.…”

Section: Pyrococcus Furiosus and Thermotoga Neapolitana Ogtmentioning

confidence: 99%

O6-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability

Mattossovich

Merlo

Miggiano

et al. 2020

IJMS

View full text Add to dashboard Cite

The genome of living cells is continuously exposed to endogenous and exogenous attacks, and this is particularly amplified at high temperatures. Alkylating agents cause DNA damage, leading to mutations and cell death; for this reason, they also play a central role in chemotherapy treatments. A class of enzymes known as AGTs (alkylguanine-DNA-alkyltransferases) protects the DNA from mutations caused by alkylating agents, in particular in the recognition and repair of alkylated guanines in O6-position. The peculiar irreversible self-alkylation reaction of these enzymes triggered numerous studies, especially on the human homologue, in order to identify effective inhibitors in the fight against cancer. In modern biotechnology, engineered variants of AGTs are developed to be used as protein tags for the attachment of chemical ligands. In the last decade, research on AGTs from (hyper)thermophilic sources proved useful as a model system to clarify numerous phenomena, also common for mesophilic enzymes. This review traces recent progress in this class of thermozymes, emphasizing their usefulness in basic research and their consequent advantages for in vivo and in vitro biotechnological applications.

show abstract

Engineering of TM1459 from Thermotoga maritima for Increased Oxidative Alkene Cleavage Activity

Cited by 11 publications

References 38 publications

A DyP-Type Peroxidase of Pleurotus sapidus with Alkene Cleaving Activity

A DyP-Type Peroxidase of Pleurotus sapidus with Alkene Cleaving Activity

Engineering TM1459 for Stabilisation against Inactivation by Amino Acid Oxidation

O6-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability

Contact Info

Product

Resources

About