Biotechnology and Biological Sciences Doctoral Training Programme
   
   
  

Biotechnology with a pinch of salt - halophilic enzymes in tandem flow reactions

 

Lab rotation project description

The goal of this proposal is to assemble a combined enzymatic system that will   allow two sequential reactions to take place in flow. This system will   generate amines from alcohols via a carbonyl intermediate. Amines are key   functional groups of numerous intermediates for pharma and agrochemical   applications, and enzymatic synthesis will offer a ‘green’ alternative to   traditional methodologies. To overcome the limitations of mesophilic enzymes   such as low stability in organic solvents, a novel system will be based on   two halophilic biocatalysts that offer remarkable stability and excellent   substrate scope. Both enzymes (HvADH2 and HvAAT1) are from the halophilic   archaeon Haloferax volcanii. HvADH2 is exceptionally tolerant to organic   solvents, has an unusually broad substrate scope, and shows enhanced   stability upon covalent immobilization. This makes it an ideal candidate for   flow applications. HvAAT1 has been expressed in H. volcanii and is being   characterised. The gene for HvAAT1 (gabT1) is genetically linked to the adh2   gene, raising the possibility that alcohol substrates converted to aldehydes   and ketones by HvADH2 may be acting as amino acceptor for HvAAT1. Such a   combined system, where ADH and AAT can be easily coupled, would yield the   first completely compatible tandem reaction system using halophilic enzymes.

Fact file

Research theme

IBB

Location

Life Sciences

Rotation

LR1

Contact

2nd supervisor


BBSRC Doctoral Training Partnerships
 

Linked PhD Project Outline

Halophilic   archaea are found in hypersaline lakes such as the Dead Sea, they are able to   grow in molar concentrations of salt. To cope with osmotic stress, halophilic   archaea accumulate salt in their cytoplasm. Consequently, their enzymes are   functional at high salt concentrations, and they have significantly greater   activity in organic solvents than enzymes from non-halophilic organisms.   These facets make halophilic enzymes of great interest to biotechnological   and chemical industries.   

The goal of this proposal is to assemble a combined enzymatic system that   will allow two sequential reactions to take place in flow. We will use this   system to generate amines from alcohols via a carbonyl intermediate. Amines   are key functional groups of numerous intermediates for pharma and   agrochemical applications, and enzymatic synthesis will offer a ‘green’   alternative to traditional methodologies. To overcome the limitations of   mesophilic enzymes such as low stability in organic solvents, we will design   a novel system based on two halophilic biocatalysts that offer remarkable   stability and excellent substrate scope.

The advantages of flow chemistry over batch reactions include efficient   mixing, high throughput, and assembly of multi-step reactions. (Bio)catalysts   can be immobilized on a solid matrix through which the reagents are pumped,   generating a very high ratio of catalyst/reagent with significant economic   benefits, since enzymatic immobilization improves the stability of the   biocatalyst and catalytic performance over time.

Both enzymes (HvADH2 and HvAAT1) are from the halophilic archaeon Haloferax   volcanii. HvADH2 is exceptionally tolerant to organic solvents, has an   unusually broad substrate scope, and shows enhanced stability upon covalent   immobilization. This makes it an ideal candidate for flow applications.   HvAAT1 has been expressed in H. volcanii. The gene for HvAAT1 (gabT1) is   genetically linked to the adh2 gene, raising the possibility that alcohol   substrates converted to aldehydes and ketones by HvADH2 may be acting as   amino acceptor for HvAAT1. Such a combined system, where ADH and AAT can be   easily coupled, would yield the first completely compatible tandem reaction   system using halophilic enzymes.

To achieve the goals of this project, two parallel approaches will be   developed.
1) Sequential immobilization of HvADH2 and HvAAT1 HvADH2 and HvAAT1 will be   covalently immobilized and connected sequentially. While amino transaminases   steadily regenerate their bound cofactor, HvADH2 is NADP(H)-dependent and the   cofactor reacts stoichiometrically with the substrates. NADP(H) is generally   added as a reagent in the reaction mix. In this proposal we will covalently   co-immobilize the cofactor with HvADH2 on epoxy resin for steady availability   and use mildly oxidative conditions in the media to regenerate NADP+ in   situ.

2) H. volcanii whole cell immobilization (co-expression of HvADH2 and   HvAAT1) We will co-express both enzymes in H. volcanii and assemble a whole-cell   bioreactor where the cells are immobilized on nitrocellulose membrane. The   retention of the enzymes within the cell system has been proven to be   efficient and high-yielding. To maintain cell integrity, the reaction will be   studied in various salt concentrations. We will expand the scope to the use   of ionic liquids, which is of high relevance for industrial   applications.

 

Biotechnology and Biological Sciences Doctoral Training Programme

The University of Nottingham
University Park
Nottingham, NG7 2RD

Tel: +44 (0) 115 8466946
Email: bbdtp@nottingham.ac.uk