Functional expression of recombinant hybrid enzymes composed of bacterial and insect’s chitinase domains in E. coli

https://doi.org/10.1016/j.enzmictec.2019.109492Get rights and content

Highlights

  • Hybrid chitinase composed of bacterial and lepidopteran insect’s domains.

  • Functional expression of recombinant hybrid chitinase in Escherichia coli.

  • Enhanced functionality of hybrid chitinase compared with its native enzymes.

Abstract

To elucidate the functional alteration of the recombinant hybrid chitinases composed of bacterial and insect’s domains, we cloned the constitutional domains from chitinase-encoding cDNAs of a bacterial species, Bacillus thuringiensis (BtChi) and a lepidopteran insect species, Mamestra brassicae (MbChi), respectively, swapped one’s leading signal peptide (LSP) - catalytic domain (CD) - linker region (LR) (LCL) with the other’s chitin binding domain (ChBD) between the two species, and confirmed and analyzed the functional expression of the recombinant hybrid chitinases and their chitinolytic activities in the transformed E. coli strains. Each of the two recombinant cDNAs, MbChi’s LCL connected with BtChi’s ChBD (MbLCL-BtChBD) and BtChi’s LCL connected with MbChi’s ChBD (BtLCL-MbChBD), was successfully introduced and expressed in E. coli BL21 strain. Although both of the two hybrid enzymes were found to be expressed by SDS-PAGE and Western blotting, the effects of the introduced genes on the chitin metabolism appear to be dramatically different between the two transformed E. coli strains. BtLCL-MbChBD remarkably increased not only the cell proliferation rate, extracellular and cellular chitinolytic activity, but also cellular glucosamine and N-acetylglucosamine levels, while MbLCL-BtChBD showed about the same profiles in the three tested subjects as those of the strains transformed with each of the two native chitinases, indicating that a combination of the bacterial CD of TIM barrel structure with characteristic six cysteine residues and insect ChBD2 including a conserved six cysteine-rich region (6C) enhances the attachment of the enzyme molecule to chitin compound by MbChBD, and so increases the catalytic efficiency of bacterial CD.

Introduction

Chitin is an abundant polysaccharide compound found in exoskeleton of arthropods including insects and cell walls of various microorganisms such as bacteria, algae and fungi. It is of insoluble and non-digestible linear polymeric structure formed by β-1,4 glycosidic bonds of amino sugar units, glucosamine (GlcN) and N-acetylglucosamine (GlcNAc), showing a very high strength and physicochemical stability [[1], [2], [3], [4], [5], [6], [7]].

Chitinase (EC 3.2.1.14) is an endo-splitting enzyme carrying out the hydrolysis of β-1,4 linkages of glycosidic bonds at the first step of chitinolytic reaction. Lepidopteran insects’ chitinases belonged to glycosyl hydrolase family 18 (GH18) with a conserved glyco-18 catalytic domain are distinguished into Group I (Cht I), Group II (Cht II) and Chitinase-h (Chi-h) [[8], [9], [10], [11]]. The chitinase I (Cht I) is expressed for the degradation of chitin components in cuticle exoskeleton, which occurs during the ecdysis or metamorphic process [[12], [13], [14], [15]]. Cht I is of about 63 kDa with a typical multi-domain structure from N- to C-terminus as follows; the leading signal peptides (LSP) involved in membrane passage, the catalytic domain (CD) for substrate degradation with a glyco-18 catalytic site containing a TIM barrel and a groove-like chitin binding cleft, the linker region (LR) connecting the N-terminal domains to the C-terminal, and finally the chitin binding domain type 2 (ChBD2) with a conventional cysteine-rich region consisting of six cysteine (6C) residues in order to stably attach the enzyme molecule to a substrate compound such as chitin, chitin-oligomer or N-acetylated β-1,4 linked substances [[16], [17], [18]]. Since bacterial chitinases of Bacillus genus are also belonged to glycosyl hydrolase family 18, they have the conserved glyco-18 domain with GH18 chitinase signature sequence F-D-G, and show a serial LSP-CD-LR-CBD multi-domain structure. Similarly to lepidopteran chitinase domain structure, Bacillus bacterial chitinases have a complicated eight-stranded (α/β)8 TIM barrel structure with a chitin insertion domain (CID) organized a blocked tunnel-like substrate binding cleft, and the regular disulfide bonds formed by six cysteine (6C) residues are located in the catalytic region [11,[19], [20], [21], [22]]. As a little different from insects, the ChBD domain of Bacillus thuringiensis has a carbohydrate-binding module 2 (CBM2), a conserved region of about 84 amino acid residues in C-terminal end, showing a broad action as a chitin or cellulose binding domain (CBD) [20,[23], [24], [25]]. Also, it is well known that the bacterial chitinases can act strongly in chitin or cellulose degradation for their physiological and protective functions through extensive chitinolytic reaction [4,6,22,26,27].

Recently, the chitin degrading activity of foreign genes in E. coli was confirmed by transformation and functional overexpression of some insects’ or bacterial native chitinase-encoding cDNAs [[28], [29], [30]], and furthermore, a possible use of chimeric chitinases as a biopesticide, which are of improved insecticidal toxicity due to the recombined insect pathogenic bacterial and viral chitinase constitutive domains has been steadily proposed [[31], [32], [33], [34]]. In the present study, in order to elucidate the functionality of the hybrid chitinases composed of insect and bacterial domains, we cloned the structural domains from chitinases of a typical insect pathogenic bacteria Bacillus thuringiensis and a crop pest lepidopteran insect, the cabbage armyworm, Mamestra brassicae, respectively, and swapped one’s LSP-CD-LR (LCL) region- with the other’s ChBD domain-encoding cDNAs between the two species, and confirmed and analyzed the functional expression of the recombinant hybrid-chitinases and their chitinolytic activities in the transformed E. coli strains.

Section snippets

Organisms and cDNA preparation

Moth, M. brassicae (Mb) larvae were reared at 25 ± 1 °C, 60 % RH under 16L:8D on cabbage. The last instar larvae were collected and chopped by dissecting scissors, and then immediately immersed in TRIzol reagent (GIBCO/BRL). Dissected tissue (≅100 mg) was pulverized in 1 mL of TRIzol by using a micro-pestle, and total RNA was extracted according to the manufacturer's instructions. Double stranded cDNA library was synthesized from the total RNA using Promega Universal Riboclone cDNA Synthesis

Structure of chitinase-encoding cDNAs

The amplified BtChi ORF cDNA (2025 bp) encoding 674 amino acids and MbChi ORF cDNA (1689 bp) encoding 562 amino acids showed typical serial multi-domain structures of LSP-CD-LR-ChBD in both species, which are demarcated according to the previous reports (Table 2).

The hydrophobic leading signal peptide (LSP) domain was composed of 32 in BtChi and 20 amino acid (a.a.) residues in MbChi, respectively [25,29], including some characteristic hydrophobic large apolar amino acid residues such as

Discussion

In previous studies, random recombinations of microbial chitinases have been conducted for the purpose of industrial decomposition and utilization of abandoned chitin compounds, although they were not actually useful because of no superior activity to native chitinase [[40], [41], [42], [43]]. Also, many studies have been performed on the improvement of chitinolytic enzymes in order to develop insect resistant plants or biological prevent of various pests and pathogens through the functional

Declaration of Competing Interest

None.

Acknowledgements

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2012R1A1A2003790), and by 2013 Hannam University Research Fund.

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