Loader

Abstracts - Hugo C. R. de Jesus

EXPRESSION OF A RECOMBINANT CGTASE FROM Paenibacillus pabuli

Hugo C. R. de Jesus1*, Renê P. da Silva1, Dulce H.F. de Souza1, João Batista Fernandes1

1 Department of Chemistry, Federal University of São Carlos

* Correspondence: hugocesar.hb@gmail.com

Introduction

The efficient control of agricultural pests, such as ants, is crucial to meeting the rapidly increasing food and biofuel requirements to support the growing human population with ecological sustainability. However, most of the insect-specific insecticides released into the environment are lost before reaching its target due to physical, chemical or biological factors.1 In this way, methods are needed to improve the slow release of the active compound and to increase their stability and water solubility. The molecular inclusion of insecticides by cyclodextrins (CDs) offers several advantages over conventional agrochemical formulations.2 CD structure is formed by a hydrophilic exterior ‘‘shell’’ and a hydrophobic inner cavity capable of hosting a wide range of organic guest molecules. CDs are produced from starch, glucogen, malto-oligosaccharides, and other dextrins through enzymatic catalysis by cyclodextrin glucosyl transferase (CGTase). In this sense, the present work aims to produce and purify a recombinant CGTase for use in molecular encapsulation process of insecticides.

Methods

CGTase protein-coding gene was isolated from Paenibacillus pabuli strain DSM 3036. The Escherichia coli BL21 Star™ (DE3) and ArcticExpress™ (DE3) strains were used as a host for the pET-22b and pET-SUMO plasmids. Sequence analyses were conducted using Geneious (version 11.0.3; Biomatters Ltd., Auckland, New Zealand) and NCBI database.

Preliminary Data

An alignment between seven known CGTases sequences was performed, which allowed to identify six conserved sequence patterns or motifs:

CGTase from

Consensus region

I

II

III

IV

V

VI

GGDWQGXXXK

GXTAXWISQP

TFGEW

IDNHDMDRF

PXIYYG

ELGNW

T. thermosulfurigenes

GGDWQGIINK

GVTAIWISQP

TFGEW

IDNHDMDRF

PAIYYG

ELGNW

G. stearothermophilus

GGDWQGIINK

GVTAIWISQP

TFGEW

IDNHDMDRF

PNIYYG

ELGNW

Bacillus circulans

GGDWQGLINK

GVTALWISQP

TFGEW

IDNHDMDRF

PAIYYG

ELGNW

Paenibacillus macerans

GGDWQGIIDK

GVTALWISQP

TFGEW

IDNHDMDRF

PAIYYG

ELGSW

Bacillus licheniformis

GGDWQGLVNK

GVTALWISQP

TFGEW

IDNHDMDRF

PAIYYG

ELGNW

Bacillus ohbensis

GGDWQGIIDK

GITAIWISQP

TFGEW

IDNHDMSRF

PTIYYG

ELGNW

Brevibacillus brevis

GGDWQGIINK

GITALWISQP

TFGEW

IDNHDMSRF

PTIYYG

ELGNW

 

The set of consensus regions (I-VI) was used to search for CGTase gene in available microorganisms, leading to identification of the target gene responsible for encoding a 78.2kDa CGTase in Paenibacillus pabuli. Experiments aiming to increase the solubility of the protein and its expression level are in progress.

1. Flaherty, R.J. et al., Chemosphere, 91, p. 912–920, 2013.

2. Tiwari, G. et al., J Pharm Bioallied Sci. 2, p. 2–79, 2010.

Sao Paulo Research Foundation - FAPESP - Proc. 2012/25299-6