Characterization Of Chitin Degradation Of Pseudoalteromonas And Novel Chitinases

The ocean is the largest ecosystem on the earth,and marine microorganisms play an important role in the marine ecosystem and the geobiochemical circulation.In the ocean,chitin is the most abundant organic carbon,and marine chitinolytic bacteria play an essential role in marine chitin degradation and recycling.Although chitin can be degraded by marine microorganisms,the underlying chitin degradation mechanism is still unclear in many aspects.Bacteria of the genus Pseudoalteromonas is ocean-specific and widely distributed in the global marine environments from surface water to deep-sea sediments.Recently,based on bioinformatic analysis,a high percentage of Pseudoalteromonas strains have been found to contain at least one chitin degradation cluster(CDC)in their genomes,suggesting the potential role of the Pseudoalteromonas genus in marine chitin degradation.However,until now,only limited reports on chitinolytic Pseudoalteromonas strains are available.Due to the lack of the systematic study on chitin degradation by Pseudoalteromonas at the genus level,the contribution of Pseudoalteromonas to marine chitin degradation in marine environments is still unknown.Chitinases play an important role in the microbial degradation of marine chitin,which can hydrolyse chitin to chitin-oligosaccharides or monosaccharides.Glycoside hydrolase family 18(GH18)chitinases are the most extensively studied due to their large amount in nature and efficient degradation of crystalline chitin.Among GH18 chitinases,in contrast to the extensive studies on the exochitinases,only a limited number of bacterial endochitinases are characterized.The catalytic mechanism of GH18 endochitinase is still unclear.In this dissertation,the chitin degradation by Pseudoalteromonas at the genus level was systematically analyzed,demonstrating that the genus Pseudoalteromonas plays an important role in marine chitin degradation.To further reveal the mechamism for chitin degradation by Pseudoalteromonas,the key chitinolytic enzyme genes of P.flavipulchra DSM 14401T involved in the crystalline chitin degradation were identified and characterized in detail.In addition,a novel GH18 endochitinase Chi23 was identified from the chitinolytic P.aurantic DSM 6057T,and its crystal structure was solved.Based on biochemical,structural and mutational analyses,the catalytic mechanism of the GH18 endochitinase Chi23 was proposed.This study on Pseudoalteromonas strains and their chitin-degrading enzymes will provide a theoretical basis for clarifying the underlying mechanism for marine chitin degradation by marine microorganisms.1.Analysis of chitin degradation ability of Pseudoalteromonas and its role in marine chitin degradationIn order to study the role of the genus Pseudoalteromonas in marine chitin degradation,we analyzed the chitin degradation ability of 26 type strains of Pseudoalteromonas from different seas around the world and 139 non-type Pseudoalteromonas strains randomly isolated from different marine environments of the Yellow Sea of China.16S rRNA phylogeny suggested that the above-mentioned 165 Pseudoalteromonas strains belong to at least 35 Pseudoalteromonas species.Meanwhile,we also analyzed the chitin degradation ability of two Pseudoalteromonas strains(SM9913 and CF6-2)from deep sea.Our results showed that more than 20%of 167 Pseudoalteromonas strains(covering 74%of Pseudoalteromonas species)could degrade colloidal chitin.Chitinolytic Pseudoalteromonas strains degrade chitin into mainly(GlcNAc)1-3 extracellularly,and then the resulting chitiooligosaccharides can be further catabolized by Pseudoalteromonas.These chitinolytic strains are mainly from surface water and alga samples,demonstrating that Pseudoalteromonas is an important group of chitin-degraders in marine environments,especially in upper seawater.Almost all chitinolytic strains showed obvious hydrolytic activities against both the long-chain colloidal chitin and the chitotriose analog 4-MU-(G1cNAc)2,indicating that endo-and/or exo-chitinases are indeed involved in chitin degradation by chitinolytic Pseudoalteromonas strains.Moreover,all the chitinolytic strains could hydrolyze 4-MU-GlcNAc,a typical substrate of N-acetylglucosaminase,suggesting that N-acetylglucosaminases in addition to chitinases also contribute to the chitin degradation by chitinolytic Pseudoalteromonas strains.The growth and chitinase production of chitinolytic Pseudoalteromonas strains on chitopentose,a larger chitooligosaccharide than chitobiose,were also determined to mimic chitin substrates in nature.All the tested strains could grow on chitopentoses as the sole carbon source The chitinase production curves of these strains were consistent with their growth curves on chitopentoses.The extracellular chitinase activity gradually increased with the increase of cell numbers,reaching the maximum value in a range of 0.4-3.2 U/mL at the stable phase.In addition,the potential key enzyme genes involving in chitin degradation by Pseudoalteromonas were identified through genomic analysis.The chitinolytic Pseudoalteromonas strains assayed in this study contain one or two conserved CDC clusters in their genomes.In addition to the CDC cluster,a considerable number of additional chitinase-encoding genes are also present in most chitinolytic strains.The presence of so many chitinase genes in chitinolytic Pseudoalteromonas strains may be helpful to efficiently degrade the different forms of chitin they encounter through synergistic action.Based on SignalP and Psortb analysis,most of the predicted chitinases of the chitinolytic Pseudoalteromonas strains belong to the GH18 family,and all the LPMOs belong to the auxiliary activity family(AA10).2.Identification and functional analysis of chitinolytic enzyme genes in Pseudoalteromonas flavipulchra DSM 14401TThe type strain Pseudoalteromonas flavipulchra DSM 14401T isolated from the surface seawater of France Nice,could quickly degrade both colloidal chitin and crystalline chitin,displaying efficient chitin-degrading ability.In order to study the mechanism for chitin degradation by Pseudoalteromonas,the chitinolytic enzyme genes in P.flavipulchra DSM 14401T were identified and studied.Genomic analysis showed that P.flavipulchra DSM 14401T contains four potential chitinase genes(pflab0431,pflab0434,pflaa4287 and pflaa2822)belonging to GH18,one potential chitinase gene(pflab0889)belonging to GH19 and two potential LPMO gene(pflab0432 and pflab1147)belonging to AA10 family,among which pflab0431,pflab0432 and pflab0434 constitute a CDC cluster.Comparative transcriptomic analysis showed that,except for the gene pflab1147,all the other six genes were significantly up-regulated when the strain was cultivated with crystalline chitin or N-acetylglucosamine as the sole carbon source.In addition,the proteins encoded by the six genes can be detected with a high abundance in the extracellular proteome of the strain when crystalline chitin was used as the sole carbon source.Therefore,these results indicated that these six genes participate in the crystallin chitin degradation of P.flavipulchra DSM 14401T.Then,to further study the function of these enzymes in chitin degradation,these genes were over-expressed in Escherichia coli BL21(DE3)and their encoding proteins were purified.Except for the LPMO gene,the other five chitinases were successfully expressed.Biochemical characterization results showed that PfChib0889 and PfChia2822 only degraded colloidal chitin,and PfChia4287,PfChib0431 and PfChib0434 could hydrolyze both crystalline and colloidal chitin,suggesting that chitinases PfChia4287,PfChib0431 and PfChib0434 play a key role in crystalline chitin degradation by P.flavipulchra DSM 14401T.The chitinases PfChib0431,PfChib0434 and PfChia4287 are all mesophilic enzymes,and their optimal temperatures are 50-60℃.In addition,they have good thermostability and can exist in the marine environment for a long time.Therefore,the presence of these chitinases can provide nutrients for the growth of their source strain and other marine bacteria through degrading the marine chitin.3.Analysis of the catalytic properties of a novel GH18 endochitinase Chi23 from Pseudoalteromonas aurantia DSM 6057TBased on the genomic analysis of several chitinolytic Pseudoalteromonas strains and functional characterization of key chitinolytic enzyme genes of P.flavipulchra DSM 14401T,it is revealed that,similar to other chitinolytic bacteria,GH18 chitinases play an important role in the chitin degradation by Pseudoalteromonas.Based on sequence similarity,GH18 bacterial chitinases are grouped into three subfamilies,A,B and C.In contrast to the extensive studies on subfamily A chitinases,only a limited number of bacterial chitinases from subfamily B and C have been studied.In this study,we characterized a GH18 endochitinase Chi23 from a chitinolytic type strain Pseudoalteromonas aurantia DSM 6057T.Phylogenetic analysis showed that Chi23 belongs to the subfamily B of the GH18 family.Among the characterized chitinases,Chi23 is most closely related to the GH18 endochitinase PPL2 from Parkia platycephala seeds with a low sequence identity of only 30%,suggesting that Chi23 is a novel GH18 chitinase.Chi23 was predicted to be a single-domain enzyme,containing only a GH18 catalytic domain.Subsequently,Chi23 was over-expressed and purified.Gel filtration analysis showed that Chi23 presented as monomers in solution.Recombinant Chi23 could hydrolyze both crystalline and colloidal chitin,generating(GlcNAc)2 and(GlcNAc)3 as predominant products.To reveal the action mode of Chi23,time course of(GlcNAc)6 hydrolysis by Chi23 was performed.Degradation products from(GlcNAc)6 by Chi23 were(GlcNAc)2,(GlcNAc)3 and(GlcNAc)4 at first,and the resulting(GlcNAc)4 was subsequently hydrolyzed into(GlcNAc)2,which further indicated that Chi23 is an endochitinase.Different from the reported endochitinase which are all modular enzymes,Chi23 is a single-domain enzyme of GH18 with crystalline chitin-degrading activity.The optimal temperature for Chi23 activity was 60℃.Chi23 was stable at 60℃,and retained 40%of its maximal activity after 1 h incubation at 70℃.Chi23 had the highest activity at pH 5.0 and showed good stability in 4.5 M NaCl.Therefore,Chi23 is a thermostable,halotolerant,and acidic chtinase.4.Structural insight into chitin degradation and thermostability of a novel GH18 endochitinase Chi23 from Pseudoalteromonas aurantia DSM 6057TAlthough potential residues involved in substrate binding and catalysis for the GH18 endochitinases have been suggested based on the limited structures,their contributions to chitin degradation,especially for crystalline chitin,are still unclear.To clarify the catalytic mechanism of the GH18 endochitinases,the crystal structure of Chi23 was solved at 1.8-A resolution.Consistent with sequence analysis,structural analysis also showed that Chi23 contains only a catalytic domain.Like the catalytic domains of the other chitinases from the GH18 subfamily B,the overall structure of Chi23 adopts the classical(β/α)8 TIM-barrel fold.However,unlike other bacterial endochitinases of the GH18 family,Chi23 lacks the chitin-binding domain(CBD)and the β-hairpin subdomain,and harbors short loops.We modeled the structure of the wild-type Chi23 in complex with(GlcNAc)5 by molecular docking.Based on the structural,mutational and biochemical analyses,the key residues of Chi23 involved in substrate binding and catalysis in chitin degradation were revealed.Furthermore,the three unique residues Asn9,Asp229 and Gln261 in Chi23 were found to play an important role in chitin degradation,and their cumulative roles in substrate binding and catalysis contribute to the activity of the single-domain Chi23 against both crystalline and soluble chitin,making its efficiency of chitin degradation come up to the multi-domain endochitinases.In addition,we firstly analyzed the contribution of identified substrate-binding residues to the thermostability of GH18 endochitinase.Our results show that the substrate-binding residues(Tyr 189,Asnl 90,Asp229,Trp260 and Gln261)at the-1 and-2 subsites play important roles in maintaining thermostability of Chi23.Among them,residues Asp229 and Gln261 can stabilize the protein structure of Chi23 through forming a hydrogen bond network between its side-chain and surrounding residues.Our structure-function analysis on the novel endochitinase Chi23 broadens our knowledge on the molecular mechanisms of the GH18 endochitinases for chitin degradation,and offers a basis for developing the industrial applications of Chi23 and other GH18 chitinases.