The Salt Tolerance Mechanism Of Novel Marine Esterases H8 And E40 And The Catalytic Mechanism Of Carbohydrate Esterase H22

Lipolytic enzymes are involved in catalyzing the hydrolysis and synthesis of esters and are found in plants,animals and microorganisms.Owing to their wide substrate specificity,exquisite chemoselectivity,regioselectivity,no cofactor requirement,and high stability in organic solvents,lipolytic enzymes are currently used in a broad array of industrial applications.Carbohydrate esterases are a large group of carbohydrate-active enzymes that catalyse the removal of ester substituents from the glycan chains of polysaccharides.These polysaccharides are from different organisms,including peptidoglycan from microorganisms,chitin from animals,xylan from plants.Some lipolytic enzymes also have the activity of carbohydrate esterases.Most of the marine environments have special characteristics as low temperatures,high pressure,high salinity,darkness and oligotrophication.During the long-term process of marine microorganisms adapting to the extreme marine environment,the extracellular organic carbon degradation enzymes secreted by marine microorganisms have evolved special characteristics to adapt to the marine environment,such as cold-adaption,salt-tolerance,high pressure resistance and unique substrate selectivity.Marine microorganisms provide a huge resource for obtaining novel enzymes with unique properties and potential for industrial application.Metagenomics,a cultivation-independent method,has been developed to discover new functional genes from both cultured and uncultured microorganisms.Currently,only a few lipolytic enzymes are reported to be halotolerant or halophilic.Meanwhile,due to the lack of structure,the molecular basis for the halotolerance of halotolerant or halophilic lipolytic enzymes is still unclear.Studies on halotolerant and halophilic enzymes found that they may have diverse salt-adapted strategies.In this thesis,the salt tolerance mechanisms of esterase H8 and HSL esterase E40 from marine sediments and the catalytic mechanism of a novel carbohydrate esterase H22 from marine bacterium were investigated.?.Screening of lipolytic enzymes from deep-sea surface sediments from the South China Sea based on functional metagenomic analysisFosmid metagenomic libraries were constructed from the South China Sea deep-sea surface sediment samples at Sites 63(water depth of 3939 m)and Site 100(water depth of 4240 m),respectively.Site 63 library contained 24,000 fosmid clones,and site 100 library contained 7,200 clones.Functional assays were carried out to screen active lipolytic enzymes in these two fosmid libraries,and a total of 66 positive clones were identified.Based on the construction of subcloning libraries,screening and subsequent sequencing,the gene sequences encoding lipolytic enzymes on 11 fosmids were determined(P6-2F,P49-7F,P194-7C,P24-3C,P43-6C,P92-7D,P103-3B,P194-1G,P138-1B,P138-8D,P43-5H).Among them,fosmids P6-2F,P49-7F and P194-7C,fosmids P103-3B and P194-1G carried the same lipolytic enzyme gene,respectively.And finally,8 lipolytic enzyme genes were obtained,which were named H1,H2,H3,H4,H5,H6,H7 and H8,respectively.Based on the further sequence analysis,the lipolytic enzyme families that these eight enzymes belong to were defined.Lipolytic enzymes H1,H4,H5 and H7 belong to lipolytic enzyme Family ?,H2,H3 and H6 belong to Family ?,and H8 belongs to Family ?.?.Identification and characterization of the novel salt-tolerant esterase H8A lipolytic enzyme gene H8 screened from deep-sea sediment Site 100 fosmid library from the South China Sea was expressed in Escherichia coli,purified and analysed.Among the characterized lipolytic enzymes,H8 showed the highest sequence identity(46%)to a family V esterase(Est 16)from a microbial consortium.Phylogenetic analysis also showed that H8 is a new member of family V of bacterial lipolytic enzymes.Based on sequence alignments with other proteins from family V,the catalytic triad of H8 was identified,which is composed of Ser120,Asp247,and His275.H8 could effectively hydrolyze short-chain monoesters(C4-C10),with the highest activity toward p-nitrophenyl hexanoate(69.0 U/mg).H8 showed a limited ability to degrade pNP esters longer than 10 carbon atoms,indicating that H8 is an esterase.The optimal temperature and pH for H8 activity were 35? and pH 10.0,respectively,the catalysis by H8 did not require metal ions.H8 had high salt tolerance,still having full activity in NaCl with a concentration as high as 4.0 M and remaining stable after 1 h incubation in 4.6 M NaCl,which suggests that H8 is well adapted to the marine saline environment and that H8 may have industrial potentials.Unlike reported halophilic/halotolerant enzymes with high acidic/basic residue ratios and low pI values,H8 contains a large number of basic residues,leading to its high basic/acidic residue ratio and high predicted pI(9.09).By searching NCBI nr database using the H8 sequence as a query,more than 10 homologs of H8 are found to have more basic residues than acidic residues and high pI values,suggesting that H8 and its-homologs may represent an uncharacterized group of lipolytic enzymes rich in basic residues.According to multiple sequence alignment,we selected five conserved basic residues(Arg195,Arg203,Arg216,Arg236,and Arg263)for site-directed mutation to acidic Glu to investigate their roles in H8 halotolerance.The effect of NaCl on the activities and stabilities of the mutants was measured and compared to WT H8.Mutation of Arg195,Arg203 or Arg236 to acidic Glu significantly decreased the activity and/or stability of H8 under high salts,suggesting that these basic residues play a role in the salt tolerance of H8.?.Structural and mechanistic insights into the improvement of the halotolerance of the marine microbial esterase E40 from the HSL familyLipolytic enzymes were screened from a previously constructed deep-sea surface sediment E505 fosmid library from the South China Sea,an HSL cold-adapted esterase E40 was identified.By introducing hydrophobic residues,we obtained several mutants with improved thermostability(I203F,S202W/I203F and M3+S202W/I203F).Here,we compared the halotolerance of E40 and its mutants and found that most of the mutants had higher halotolerance than wild-type(WT)E40.The halotolerance of these proteins followed this order:M3+S202W/I203F>S202W/I203F>I203F>E40.The circular dichroism(CD)spectra and the fluorescence spectra analyses suggested that the secondary and tertiary structures were affected by the introduced hydrophobic residues in buffers containing NaCI.However,gel filtration analysis indicated that the quaternary structure of E40 was less stable than that of the M3+S202W I203F mutant in 0.5 M NaC1.We solved the crystal structures of the S202W 1203F and M3+S202W I203F mutants to reveal the structural basis for their improved halotolerance.Like other HSLs,E40 monomer contains a CAP domain and a catalytic domain.Structural analysis revealed that the introduction of hydrophobic residues Trp202 and Phe203 in ?7 significantly improved E40 halotolerance by strengthening intradomain hydrophobic interactions between residues Phe203 and Trp202 and other residues in the catalytic domain.By further introducing hydrophobic residues in loop1,the M3+S202W I203F mutant became more rigid and halotolerant due to the formation of additional interdomain hydrophobic interactions in loop 1 and ?7.During the process of adapting to the high salt marine environment,halophilic proteins have evolved to contain a decreased number of large hydrophobic amino acid residues and have reduced hydrophobic interactions on the surface and in the hydrophobic cores.However,our study showed that E40 become more rigid and more stable than the wild type under high-salt conditions due to the increased intradomain and interdomain hydrophobic interactions.These results broaden our understanding of the halotolerance mechanisms of enzymes and may be helpful in protein engineering targeting to improve the stability of enzymes with industrial potential against both high salt concentrations?.Structure and catalytic mechanism insights into the action of an SGNH hydrolase H22The strain Arcticibacterium luteifluviistationis SM1504 was isolated from the surface seawater from the King's Fjord,Svalbard.Previous studies have completed the complete genome sequencing of the strain and the corresponding gene annotation Based on this,an SGNH hydrolase gene,H22,was screened.Among the characterized enzymes,H22 showed the highest sequence identity(24%)to the SGNH acetyl xylo-oligosaccharide esterase Axe2 from Geobacillus stearothermophilus,which indicating that H22 is a novel SGNH hydrolase.Based on the phylogenetic analysis,H22 and its homologs may represent a new family of carbohydrate esterases.The substrate specificities towards different acetylated oligosaccharides of H22 were investigated.H22 showed an ability to degrade many kinds of acetylated oligosaccharides with the maximal activity toward 1,2,3,5-Tetra-O-acetyl-D-xylofuranose,suggesting that H22 is an acetyl xylo-oligosaccharide esterase.TLC analysis showed that H22 had no selectivity for acetylation sites on substrate 1,2,3,5-Tetra-O-acetyl-D-xylofuranose.The optimal temperature for H22 activity was 25?,and it retained more than 80%activity during 0-20?,suggesting that it is a cold-adapted enzyme.The optimal for H22 activity was pH 9.0,the catalysis by H22 may not require metal ions.H22 had high salt tolerance,still having full activity in NaCl with a concentration as high as 3.0 M and remaining stable after 1 h incubation in 4.8 M NaCl.The structures with high resolution of H22(2.5 A)and S18A-acetate complex(1.53A)were solved to elucidate the structural basis of H22 degrading acetylated xylose substrates.Combined with structural analysis,mutation and biochemical validation,the catalytic triad residues(Ser18,Asp 186,and His189)and the oxyanion hole residues(Gly55 and Asn84)of H22 were identified,the potential molecular mechanism of the action of H22 degrading 1,2,3,5-Tetra-O-acetyl-D-xylofuranose was proposed.