Characterisation of new chitinases produced by halophilic microorganisms. Evaluation of their potential applications

Resumen

A summary, which collects the most relevant aspects exposed in this doctoral thesis and that are discussed in detail in chapters I, II and III, is presented below. Chitin is a linear ß-1,4 linked homopolymer of N-acetyl-D-glucosamine (GlcNAc) units. It is the second most abundant polysaccharide in nature, after cellulose. It is a very abundant renewable source because it is believed that gigatonnes of chitin are annually produced on The Earth, mainly due to the wastes caused by fishing and food industries. It is difficult to degrade, giving rise to an excess of this polysaccharide that can cause an important environmental problem. In spite of being widely distributed in nature, its abundance is higher in the marine environment. However, accumulation of chitin in oceanic sediments is hardly observed, probably due to the existence of marine microorganisms, known as chitinolytic, which are able to hydrolyse chitin in an efficient way. Chitinases are glycoside hydrolase enzymes that degrade chitin catalysing the hydrolysis of the ß-1,4 glucosidic bonds which are present in this polymer. Depending on the amino acids sequence similarity in the catalytic domains, these enzymes are classified into different families, being families 18 and 19 of the glycoside hydrolases (GH18 and GH19) the ones that comprise the highest number of the chitinases known. Moreover, the members of these two families show different three-dimensional structures and mechanisms of action. These enzymes present a wide range of applications and a huge potential. Noteworthy among these applications, the capacity of protection against pathogenic fungi, nematodes and insect plagues, their participation in the obtaining of chitooligosaccharides, glucosamine and GlcNAc, the production of single cell proteins or the formation of protoplasts. Each chitinase presents different biochemical, regulatory and molecular characteristics that can make it interesting for a specific function. The microorganisms that are able to live in extreme conditions can be an important source of new enzymes; this is the case, specifically, of the halophilic microorganisms. It has been described that enzymes from halophiles, above all from extreme halophiles, present a wide spectrum of activity in terms of temperature, pH and NaCl concentration. In industrial processes, and particularly in chitinase applications, the enzymes are used in variable physical and chemical conditions, and so the characterisation of this type of proteins is of high industrial interest and it provides new possibilities in the biocatalytic processes. This information, along with the knowledge of marine microorganisms being efficient degrading chitin, are the reasons that led to deciding the study of the chitinases from two halophilic microorganisms: one extreme, the marine archaeon Halobacterium salinarum, CECT 395 strain, and another one moderate, the marine bacterium Pseudoalteromonas tunicata, CCUG 44952T strain. This selection was carried out after checking that both strains presented extracellular chitinolytic activity through their culture in an optimum medium supplemented with 1% and 2% chitin, respectively. The genomes of H. salinarum CECT 395 and P. tunicata CCUG 44952T are not sequenced yet. Therefore, the analysis of the genome of other strains belonging to the same species that are available in the database, two from H. salinarum, R1 and NRC-1, and one from P. tunicata, D2, were carried out. After the analysis of the genomes of the two strains from H. salinarum, R1 and NRC-1, several open reading frames were found, and, among them, one that could encode for a chitinase. This one was selected in both strains (accession numbers YP_001688891 and NP_279794, respectively) for its study. The analysis of the genome of P. tunicata D2 allowed to find an open reading frame, among others, that would encode a possible chitinase (accession number EAR30107). According to data, the amplification of these fragments was tried in the strains of study. For this, chromosomal DNAs from the microorganisms of study were firstly extracted. Besides, primers were designed, which flanked the genes that encode those possible chitinases, both the full-length sequence and the sequence without the signal peptide that had been previously deduced by the protein analysis with Pred-Signal (for the possible chitinase from H. salinarum CECT 395) and SignalP (for the possible chitinase from P. tunicata CCUG 44952T) programs. It was managed to amplify the gene that would encode for a chitinase in both strains, which let deduce that probably it is present in more strains from the same species. The isolated genes from H. salinarum CECT 395 and P. tunicata CCUG 44952T were called HschiA1 and Ptchi19, respectively. Next step undertaken was the cloning of the genes HschiA1 and Ptchi19, both complete and without the signal peptide, into the expression vectors pET100/D-TOPO® and pETiteTM C-His Kan, respectively. Both vectors allow a directional cloning and the fusion of the proteins to a hexa-histidine tag that will facilitate the purification of the enzymes. The hexa-histidine tag stayed bound to the N-terminus of HschiA1 and to the C-terminus of Ptchi19. After the cloning, Escherichia coli TOP10 competent cells were transformed with the recombinant plasmids pET-BM-Hs-ChiA1 and pET-BM-Hs-ChiA1-ps, and E. coli 10G competent cells with the recombinant vectors pET-Pt-Chi19 and pET-Pt-Chi19-ps. Once cloned, the genes were sequenced. Thus, it was checked that the gene HschiA1 was comprised of 1641 bp that would encode for a protein of 546 amino acids (HsChiA1p), while the gene Ptchi19 was comprised of 1452 bp that would encode a protein of 483 amino acids (PtChi19p). Once having sequenced, a bioinformatic analysis of the deduced proteins from these gene sequences was carried out. Firstly, an alignment of the cloned proteins with others related to them which are available in the database was completed. Thus, it was observed that HsChiA1p showed homology with the chitinases from the archaea Halobacterium sp. NRC-1 and Halogeometricum borinquense, and identity with the other two archaea. Meanwhile, PtChi19p resulted to have homology with chitinases from the bacteria Pseudoalteromonas tunicata D2, Vibrio vulnificus MO6-24/O, Aeromonas veronii B565 and Pseudoalteromonas flavipulchra, while it showed identity with the ones from the bacterium Streptomyces griseus and from some plants. This result confirmed the possible chitinolytic activity of the proteins of study. After the comparative analysis of the protein sequences, new analysis of the amino acids sequences of HsChiA1p and PtChi19p and of their predicted three-dimensional structures were carried out. Thus, it was checked that the two proteins had a structure formed by several domains. Firstly, it was detected for both proteins the presence of a signal sequence in the N-terminus that agrees with the extracellular chitinase activity that had previously been observed. Another identified domain in the two proteins is a carbohydrate binding module 5 (CBM5), which allows the enzyme binding to the substrate through hydrophobic interactions among some aromatic residues, conserved in HsChiA1p and PtChi19p, and the sugar molecules. However, its position within the sequence varies, being identified after the signal peptide in HsChiA1p, and in the C-terminus in PtChi19p. The consensus sequence AKWWT, which has been described as conserved in bacterial CBM5, was also detected in the archaeon H. salinarum and in the bacterium P. tunicata. Other domains that have been found in several chitinases and bacteria are the ones known as fibronectin type III and of the polycystic kidney disease (PKD) domains, which also seem to participate in the enzyme binding to the substrate. However, only the PKD domain was detected in HsChiA1p and not in PtChi19p. Finally, both chitinases showed a catalytic domain, but the amino acids that comprised them, the three-dimensional structure and their mechanism of action were completely different. While HsChiA1p had a characteristic catalytic domain of GH18 chitinases, the catalytic domain of PtChi19p was the typical one of GH19. It was determined that the catalytic domain of HsChiA1p had a structure known as TIM-barrel and, besides, it could be classified within the subfamily A of prokaryotic chitinases, because of the presence in this domain of an ¿+ß insertion. It has been demonstrated that the chitinase from this archaeon, as well as the ones from bacteria and fungi, has this domain conserved, in particular the DXXDXDXE sequence. This datum, along with directed mutagenesis and crystallographic studies carried out in other chitinases, supports the idea that it plays an important role either in the structure or in the protein activity, and particularly important to mention, the aspartate and glutamate amino acids are essential. Meanwhile, PtChi19p showed an ¿+ß structure, with a high ¿-helical content as it was verified by circular dichroism analysis. In this case, the chitinase from P. tunicata showed several conserved pattern sequences which characterised chitinases within GH19. However, it is in the motif DA[ITV]CK [RK][ES][LAI]A[AT]F[LF]A[NQH][VF][SA][HQ]E[TS]GG[LH]X[YA][VI]VEXN where it has been established that the active centre of these enzymes is located, which also appeared well conserved in PtChi19p. By previous directed mutagenesis and crystallographic studies it was concluded for GH19 chitinases a mechanism of action in which two glutamates would be implicated, one acting as an acid and the other one as a base, that were also located in the PtChi19p sequence. In the predicted structural models of both proteins these possibly implicated amino acids in the mechanism of action of the enzymes were located near the cleft where the substrate is expected to bind. Moreover, the TIM-barrel structure was observed in HsChiA1p while PtChi19p mainly presented a rich ¿-helical structure. After the proteins bioinformatic analysis, E. coli BL21 (DE3) cells were transformed with the previously obtained recombinant vectors with the purpose of intracellularly expressing the fusion proteins. The expression of the recombinant plasmids that contained the genes of the chitinases from H. salinarum and P. tunicata occurred under control of the T7 promoter and it was induced by IPTG. To continue with the study, the recombinant strains that showed the highest expression levels were selected: BL21-Hs-ChiA1 (which expressed the H. salinarum¿s gene without the signal peptide) and BL21-Pt-Chi19 (which expressed the complete gene of P. tunicata). E. coli does not accumulate salt at the intracellular level, and so it is not considered an optimal host for the expression of halophilic proteins which need this compound for their correct folding and activity. However, in this study it has been demonstrated that the expression of active halophilic enzymes in this host is possible by lysis and subsequent dialysis of the cellular extracts at the suitable NaCl conditions, this being the first time an extreme halophilic enzyme has been expressed as an active form in E. coli. Proteins purification process was carried out by affinity and size exclusion chromatography. The size of the purified protein from H. salinarum, HsChiA1p, was approximately 66.5 kDa and from P. tunicata, PtChi19p, was approximately 51 kDa, which agrees with the theoretical deduction and they are included within the range of the chitinases characterised so far. The two enzymes showed their chitinase activity by degrading the natural substrates, colloidal chitin and crystalline chitin, being HsChiA1p more effective on them. On the other hand, chitinase activity was also checked against synthetic substrates. Thus, both HsChiA1p and PtChi19p hydrolysed p-NP N-acetyl-ß-D-glucosaminide (pNP-(GlcNAc)), HsChiA1p also degraded p-NP ß-D-N,N¿,N¿¿-triacetylchitotriose (pNP-(GlcNAc)3) and none of the two enzymes acted on p-NP N,N¿-diacetyl-ß-D-chitobioside (pNP-(GlcNAc)2). It was decided to use the chromogenic substrate pNP-(GlcNAc) for the subsequent experiments. After the purifications of HsChiA1p and PtChi19p and the substrate selection, it was checked that their specific activity was eight times higher than the one from the crude extracts, and the effects of different physico-chemical factors on their chitinase activity were also determined. HsChiA1p showed activity between 15-45 °C and between pH values of 6-9. Moreover, it resulted to be highly stable at suboptimal conditions. Concretely, after 40 min at temperatures below (25 °C and 30 °C) the optimum (40 °C), the protein maintained more than a 90% of its activity. Regarding pH, more than a 90% of activity was conserved after 40 min at pH values below and over (6.5, 8, 8.5) the optimum (7.3). Its activity was stimulated by the metals Mg2+, K+ and Ca2+, and strongly inhibited by Mn2+. Meanwhile, PtChi19p resulted to be active in the range of 20-50 °C and pH values 6-9.5. In regard of its stability, after 40 min at lower temperatures (25 °C and 30 °C) than the optimum (43 °C), its activity increased between 20-40%. It was also stable at pH values different than the optimum (7.5), although less than HsChiA1p, preserving more than a 70% of activity after 40 min at pH 6.5 and 8.5. None of the tested metals stimulated its activity but it was inhibited by Mn2+, although less than HsChiA1p, as well as by several heavy metals. As it would be expected, especially for HsChiA1p, both enzymes showed dependence in salt concentration, resulting to be active in a wide range of NaCl concentrations (0-3.5 M for HsChiA1p and 0-2.5 M for PtChi19p). It is important to indicate that most of the enzymes of extreme halophiles are inactivated and denaturalised at NaCl concentrations lower than 1 M. However, HsChiA1p conserved a 15% of its chitinase activity in the absence of this compound, while PtChi19p maintained a 20%. In previous studies, it has been suggested that the hexa-histidine tag, to which HsChiA1p and PtChi19p are fused, could be helping in the folding and stability of the proteins. This wide action range, in terms of pH, temperature and NaCl requirements, as well as their stability at suboptimal conditions, will provide them with a high versatility in numerous applications. Once the proteins were biochemically characterised and because it is known that fungal cell walls are composed by chitin in a high percentage, the antifungal activity spectrum of the recombinant strains of E. coli BL21 (DE3), BL21-Hs-ChiA1 (which expressed the HschiA1 gene without the signal peptide of H. salinarum CECT 395) and BL21-Pt-Chi19 (which expressed the Ptchi19 full-length sequence of P. tunicata CCUG 44952T), was studied. The two strains were able to inhibit the growth of phytopathogenic fungi, such as Fusarium oxysporum or Armillaria mellea, of dermatophytes, such as Trichophyton mentagrophytes or Microsporum gypseum, and of the phytopathogen and human pathogen fungus Aspergillus niger. Besides, the antifungal effect that both strains performed together was studied in order to know whether a synergic action was observed. This synergy was detected against A. mellea, showing higher growth inhibition of this fungus when the two proteins acted together than separately. The analysis of all the results exposed, related to the biochemical characteristics and stability, allows proposing HsChiA1p and PtChi19p as new chitinases of interest for the degradation of chitin residues and the obtaining of oligosaccharides and N-acetylglucosamine. Besides, the proven antifungal activity makes them potential biological fungicides that could be used, either individually or combined with other compounds, as an alternative to chemical antifungal compounds. However, as it occurs with the rest of enzymes with a potential applied interest, the successful use of these chitinases in real applications will depend on whether the optimisation of the expression and culture conditions and the obtaining of overproducer strains allow a high production yield that makes the process profitable. With this purpose, the effects of different parameters on the functional expression of HsChiA1p and PtChi19p in the strains BL21-Hs-ChiA1 and BL21-Pt-Chi19, respectively, were comparatively studied. Significant differences between conditions tested in each assay were found. The first factor to be studied was the effect of the cellular density of the culture before induction. Optical densities at 600 nm of 1 for BL21-Hs-ChiA1 and of 0.7 for BL21-Pt-Chi19 were found to give rise to the highest levels of active protein. On the other hand, the production of active HsChiA1p and PtChi19p was also affected by the amount of inducer and the duration of induction. Thus, IPTG concentrations of 0.5 mM and 0.25 mM and induction times of 5 h and 1 h for the strains BL21-Hs-ChiA1 and BL21-Pt-Chi19, respectively, allowed obtaining the highest amount of active protein. The shortest culture times as well as the reduction in the inducer concentration and induction time are of great interest because they reduce the cost of the process and decrease the possibility of protein instability or proteolysis induction. Induction temperature is another important factor to consider when optimising as it has a notable effect on the folding and stability of the proteins. The highest active protein levels were obtained after induction of BL21-Hs-ChiA1 at 30-37 °C and BL21-Pt-Chi19 at 25 °C. These results let conclude that the effect of each parameter is specific for each enzyme of study. Moreover, in the last few years different host strains have been developed in order to increase the production of recombinant proteins in E. coli. In this study, five E. coli strains compatible with pET expression systems were tested. The best result, a significant increase of 60% in the expression of active HsChiA1p was observed with the strain BL21 Star (DE3) that is characterised by having a mutation in the gene rne (rne131) which encodes an endonuclease implicated in mRNA degradation. This datum shows that this mutation improves very effectively the stability of the mRNA of the archaeon chitinase. On the other hand, a rare codons bioinformatic analysis for the genes HschiA1 and Ptchi19 indicated that none of the two DNA sequences were the optimal in order to obtain a maximal expression in E. coli. On the basis of this analysis, the effect on the production of active HsChiA1p and PtChi19p of the E. coli strains BL21 RP (DE3) and Rosetta 2 (DE3), which possess extra copies of some tRNA genes for rare codons, was checked. A significant increase of 40% in the active protein expression was the best result for PtChi19p and it was obtained with the strain BL21 RP (DE3). On the other hand, a significant increase of 40% in the active HsChiA1p production was reached using the strain Rosetta 2 (DE3). In spite of the important improvement in the production of active HsChiA1p and PtChi19p, part of the proteins still remained insoluble. Therefore, as the last step, the establishment of the best conditions, in each case, for the recovery of biologically active proteins from inclusion bodies was proposed. By different refolding dilutions and times it was possible to refold part of those proteins. In particular, the highest recovered activities were obtained, for HsChiA1p after a 1:2 dilution of the solubilised protein and 8 h of folding, and for PtChi19p after a 1:4 dilution of the solubilised protein and 20 h of refolding. The study of the chitinases HsChiA1p, from the extremely halophilic archaeon H. salinarum CECT 395, and PtChi19p, from the moderately halophilic bacterium P. tunicata CCUG 44952T, has demonstrated that they are robust and versatile enzymes with interest for different applications. In spite of the importance of halophilic enzymes, their use is still unusual mainly due to the difficulties to produce them from wild microorganisms. Among these problems, the synthesis regulation in these strains and, above all, the necessity of culturing the halophilic microorganisms at high NaCl concentrations are highlighted. In this work, the conditions for the heterologous expression of the two halophilic enzymes of study were optimised, using E. coli as host. This allowed the increase of their production in more simple media, without the mentioned NaCl concentration requirements, and, above all, in a faster way.