Gábor Hornyánszky

Expression and properties of the highly alkalophilic phenylalanine ammonia-lyase of thermophilic Rubrobacter xylanophilus

The sequence of a phenylalanine ammonia-lyase (PAL; EC: 4.3.1.24) of the thermophilic and radiotolerant bacterium Rubrobacter xylanophilus (RxPAL) was identified by screening the genomes of bacteria for members of the phenylalanine ammonia-lyase family. A synthetic gene encoding the RxPAL protein was cloned and overexpressed in Escherichia coli TOP 10 in a soluble form with an N-terminal His6-tag and the recombinant RxPAL protein was purified by Ni-NTA affinity chromatography. The activity assay of RxPAL with l-phenylalanine at various pH values exhibited a local maximum at pH 8.5 and a global maximum at pH 11.5. Circular dichroism (CD) studies showed that RxPAL is associated with an extensive α-helical character (far UV CD) and two distinctive near-UV CD peaks. These structural characteristics were well preserved up to pH 11.0. The extremely high pH optimum of RxPAL can be rationalized by a three-dimensional homology model indicating possible disulfide bridges, extensive salt-bridge formation and an excess of negative electrostatic potential on the surface. Due to these properties, RxPAL may be a candidate as biocatalyst in synthetic biotransformations leading to unnatural l- or d-amino acids or as therapeutic enzyme in treatment of phenylketonuria or leukemia.

Synthesis of cycloalkanoindoles, the carba analogs of physostigmine

The title compounds 9 were prepared by combined aza-Claisen rearrangement/intramolecular ring-closure reaction of N-allylaniline derivatives 3, followed by BBr 3 mediated cleavage of methoxy group and subsequent formation of the phenylcarbamyl derivatives.

A convenient method for the preparation of cyclohepta[b]indole derivatives

Cyclohepta[b]indole derivatives 7 were prepared by subsequent aza-Claisen rearrangement and intramolecular ring-closure of (cycloheptenylmethyl)benzenamine (3). The mechanisms of the reactions are also discussed.

Facile synthesis of cycloalkanoindole derivatives by aza-Claisen rearrangement

Carba analogs of physostigmine were prepared from aniline derivatives with BF3.Et2O catalyzed cyclization. Suzuki coupling reactions of the new compounds are also discussed.Graphical abstract.

Preparation of novel hexythiazox analogues

Novel analogues of the title compound were prepared in several steps: via addition of methylmagnesium iodide to an acetamide derivative to yield diastereomeric amino alcohols, followed by hydrolysis, cyclization to the corresponding oxazole or thiazole derivative, and a coupling reaction with isocyanates. Results from acaricidal tests showed the compounds to be 100 times less active than hexythiazox.

Co-immobilized Whole Cells with ω-Transaminase and Ketoreductase Activities for Continuous-Flow Cascade Reactions

An improved sol–gel process involving the use of hollow silica microspheres as a supporting additive was applied for the co‐immobilization of whole cells of Escherichia coli with Chromobacterium violaceum ω‐transaminase activity and Lodderomyces elongisporus with ketoreductase activity. The co‐immobilized cells with two different biocatalytic activities could perform a cascade of reactions to convert racemic 4‐phenylbutan‐2‐amine or heptan‐2‐amine into a nearly equimolar mixture of the corresponding enantiomerically pure R amine and S alcohol even in continuous‐flow mode. The novel co‐immobilized whole‐cell system proved to be an easy‐to‐store and durable biocatalyst.

CHAPTER 15: SynBiocat: Protein Purification, Immobilization and Continuous-flow Processes

The activity of SynBiocat Ltd, a small spin-off company, is related to various areas of synthetic chemistry, biotechnology and enzymology. This chapter gives an overview of the knowledge and technologies of the company covering the development of suitable biocatalysts and bioreactor solutions for synthetic biotransformations. In co-operation with Bioorganic Chemistry Group (BCG) at Budapest University of Technology and Economics and Fermentia Ltd, SynBiocat can offer a wide selection of enzyme immobilization methods for known or novel enzymes to select the most suitable variant for a desired technology. Our company also offers custom immobilization development as a service to develop the optimal immobilized form of the biocatalyst of customers. For immediate screening, kits of immobilization supports or immobilized enzymes such as lipases, proteases, ketoreductases (as whole-cell biocatalysts), phenylalanine ammonia-lyases, phenylalanine 2,3-aminomutases are accessible. To enable screening of continuous-flow biotransformations, a selection of immobilized biocatalyst-filled minireactors are available off-the-shelf.