Hyperthermophilic aldolases as biocatalyst for C–C bond formation: rhamnulose 1-phosphate aldolase from Thermotoga maritima

Abstract

The TM1072 gene from Thermotoga maritima codifies for a putative form of a rhamnulose-1-phosphate aldolase (Rha-1PA Tm). To investigate this enzyme further, its gene was cloned and expressed in Escherichia coli. The purified enzyme was activated by Co(2+) as a divalent metal ion cofactor, instead of Zn(2+) as its E. coli homologue, and exhibited a maximum of activity at 95 °C. Furthermore, the enzyme displayed a high stability against extreme reaction conditions, retaining 90 % of its activity in the presence of 40 % of acetonitrile and showing a half-life greater than 3 h at 115 °C. The kinetic parameters at room temperature (R/T) were also studied; the K M was calculated to be 3.6 ± 0.33 mM, while k cat/K M was found to be 0.7 × 10(3) s(-1) M(-1). Given these characteristics, Rha-1PA Tm is an attractive enzyme for use as a biocatalyst for industrial applications, offering intriguing possibilities for practical biocatalysis.

L‐Rhamnulose‐1‐phosphate Aldolase from Thermotoga maritima in Organic Synthesis: One‐Pot Multistep Reactions for the Preparation of Imino‐ and Nitrocyclitols

“…Thus, reactions were carried out at 25 °C. In spite of its hyperthermophile nature, Rhu1PA Tm retains between 5 and 10% of its maximum activity at 25 °C 10. In these conditions, aldehyde 4 led to nitrocyclitols 5a and 5b and aldehyde (±)‐ 6 led to nitrocyclitols 7a and 7b (Scheme ).…”

“…DMF (30% v/v) was added to the reaction medium to improve the solubility of the aldehydes. Reaction temperature was set up at 45 °C to retain a good activity of the aldolase, while preventing a high degradation rate of the DHAP 10…”

“…Rhu1PA Tm was able to react with these four aldehydes at 45 °C and 80 °C in combination with other mesophilic enzymes, in a one‐pot/two‐steps approach. The reactions were significantly faster at the highest temperature 10…”

“…Thermozymes display outstanding stability against high temperature, as well as against other denaturalisation conditions, such as the presence of organic solvents, detergents or extreme pH values 9. In this sense, we have recently described the heterologous expression of the rhamnulose‐1‐phosphate aldolase from Thermotoga maritima , its functional characterisation and preliminary synthetic properties 10. This enzyme presented an optimum temperature at 95 °C and displayed a great stability to extreme reaction conditions, such as high temperatures (i.e., half‐life of more than 3 h at 115 °C) and in the presence of different organic solvents.…”

Recent Developments in the Preparation of Carbohydrate Derivatives from Achiral Building Blocks by using Aldolases

“…For instance, a new thermostable rhamnulose‐1‐phosphate aldolase from Thermotoga maritima (RhuA1PA Tm ) has been cloned and characterized recently . This catalyst offers outstanding possibilities for industrial applications considering its high stability towards organic solvents even at high temperatures.…”