Structural And Functional Study Of Dextranase From Marine Bacteria And Dnazyme

Protease is a kind of biomacromolecule which is produced by cells,and has high specificity and high catalytic ability for the substrate.It is widely used in the clinics,drugs,and production and living.For a long time,it has been considered that protein is the only source of enzyme until the discovery of ribozyme in 1978 and DNAzyme in 1994,which are RNA and DNA with catalytic function,respectively.DNAzyme has stable properties,which is widely used in analytical chemistry,biomedicine,nanoscience,and other fields.The molecular modification of enzyme will be carried out by the relationship between the structure and function of enzyme based on understanding the structure type,folding mode,and active center composition of enzyme(protease or DNAzyme).In turn,the related molecular mechanism can be obtained by comparing the structure of mutant enzyme with that of wild enzyme,which has an important significance to further promote the in-depth scientific research in the related field.In this study,the structure and function of dextranase(protease)from marine bacteria.At the same time,due to the great potential application of GR5 in the Pb2+biosensor in the marine system,the structure,function,and biochemical properties of GR5 DNAzyme were studied.Due to the special marine ecological environment,marine microorganisms have been recognized as potential sources of novel enzymes with better biocatalytic properties than that of terrestrial counterparts.These properties,such as salt tolerance,hyperthermostability,barophilicity,alkali resistance,and low optimum temperature,are necessary for efficient bioprocesses exploitation.Our team successfully screened a strain of dextranase-producing strain from the ocean—Arthrobacter oxidans KQ11.In the second chapter,the whole genome of A.oxidans KQ11 was measured,and the data was analyzed by bioinformatics.The total length of A.oxidans KQ11 genome was 4,811,453 bp.A total of 4,563 genes were encoded with a total length of 4,183,365 bp.A.oxidans KQ11 included 1 chromosome and 3 plasmids.Two GH49 dextranase(EC3.2.1.11)genes(ID:KQ11_GM001677 and KQ11_GM001683)were identified.The KQ11_GM001683(KQ-N for short)gene was used to construct dextranase-producing engineering bacteria.Due to the low ability of natural strains to produce dextranase,and the great challenge to obtain high-purity dextranase,it is particularly important to construct a recombinant strain(high dextranase-producing strain),the dextranase can be easily purified.In the third chapter,the gene KQ-N encoding the dextranase was recombinantly expressed with the plasmid pCold?,and a strain Escherichia coli BL21(DE3)1-5-pColdIII was obtained,which could efficiently product the dextranase.The specific activity of dextranase was 294.43 U/mg after purification,and the purification multiple was 34-fold as such.High-purity protein was obtained,0.1717 mg/mL,which provided an important raw material for the preparation of dextranase crystal.Enzyme derived from the natural environment has been challenged for their weak stability,easily-lost activity,and poor heat tolerance under the heat treatment condition.These greatly limit the development of the native enzyme industry.Therefore,the research and development of dextranase with high activity and thermal stability have a great significance.If we know the structure/function and the thermal stability molecular mechanism of dextranase,we can improve the thermal stability of dextranase by molecular modification based on the protein(enzyme)evolution engineering to meet the needs of industrial applications,such as sugar production.In the fourth chapter,the crystal structure of dextranase from E.coli BL21(DE3)1-5-pCold?(Aodex)was determined at a resolution of 1.4 A(PDB:6NZS).The crystal structure of the conserved Aodex fragment consisted of an N-terminal domain N and a C-terminal domain C.The N-terminal domain N was identified as a ?-sandwich,connected to a righthanded parallel ?-helix at the C-terminus.Aodex was an inverting enzyme,and the catalytic acid and base were Asp439 and Asp420,respectively.The thermostability of the S3 5 7F mutant using semirational design based on B-factors was clearly better than that of wild-type Aodex.This process may promote the aromatic-aromatic interactions that increase the thermostability of mutant S357F.Layered double hydroxides(LDHs)have received widespread attention for their potential applications in catalysis,polymer nanocomposites,pharmaceuticals,and sensors.In the fifth chapter,the mechanism underlying the physiological effects of Mg-Fe layered double hydroxide nanoparticles(Mg-Fe-LDH NPs)on the marine bacterial species A.oxidans KQ11 was investigated.Increased yields of marine dextranase(Aodex)were obtained by exposing A.oxidans KQ11 to Mg-Fe-LDH NPs.Furthermore,the potential effects of Mg-Fe-LDH NPs on bacterial growth and Aodex production were preliminarily investigated.A.oxidans KQ11 growth was not affected by exposure to the Mg-Fe-LDH NPs.In contrast,a U-shaped trend of Aodex production was observed after exposure to NPs at a concentration of 10?g/L-100 mg/L,which was due to competition between Mg-Fe-LDH NP adsorption on Aodex and the promotion of Aodex expression by the NPs.The mechanism underling the effects of Mg-Fe-LDH NPs on A.oxidans KQ11 was investigated.Exposure to 100 mg/L of Mg-Fe-LDH NPs led to NPs adsorption onto Aodex,increased expression of Aodex,and generation of a new Shine-Dalgarno sequence(GGGAG)and sRNAs that both influenced the expression of Aodex.Moreover,the expressions of transcripts related to ferric iron metabolic functions were significantly influenced by treatment.These results provide some valuable information for further investigation of the A.oxidans KQ11 response to Mg-Fe-LDH NPs and will aid in achieving improved marine dextranase production,and even improve such activities in other marine microorganisms.In addition,because of the great potential application of GR5 DNAzyme in Pb2+detection of marine system.Based on the previous research,the structure,function,and related biochemical characteristics of classical GR5 DNAzyme were systematically studied in sixth and seventh chapter.In the sixth chapter,we performed systematic rational evolution experiments to gradually mutate GR5 towards 17E.By using the activity ratio in the presence of Pb2+and Mg2+for defining these two DNAzymes,the critical nucleotide is identified to be T12 in 17E for metal specificity.This study has offered insightful results to link these two classic and widely used DNAzymes,and it will help the field to understand metal binding and catalysis of DNAzymes for biosensor development and design of stimuli-responsive materials.The activity of DNAzymes can be significantly affected by solution conditions.For robust sensing applications,these effects need to be carefully understood and controlled.The effect of pH has been extensively studied due to its mechanistic importance.On the other hand,the effect of salt and buffer species was much less explored.We reason that both cations and anions might affect DNAzyme activity.For this purpose,the GR5 DNAzyme is a good candidate.GR5 is the first ever reported DNAzyme with RNA cleavage activity.In the seventh chapter,GR5 is an optimal sequence.We herein measured the cleavage activity of GR5 in various concentrations of Na+and Mg2+.Both metals inhibited the DNAzyme with Pb2+,and the inhibition constants were 1.8 mM Mg2+and 33.4 mM Na+.For anions,F-inhibited GR5 more strongly than Br-,while Cl-was the least inhibiting anion,which was consistent with the solubility of their lead salts.The reaction can work similarly in many Good's buffers,while phosphate buffer should be avoided.In summary,we have successfully obtained the crystal structure of Aodex(PDB:6NZS),and explained the corresponding functions of catalysis and stability.Based on the semirational design,we have successfully obtained the mutant S357F with the improved thermal stability.The mechanism of the effect of Mg-Fe-LDH NPs on the production of Aodex by A.oxidans KQ11 was investigated.On the other hand,the structure,function,and the related characteristics of GR5 DNAzyme were systematically studied.This whole study not only deepens our understanding of the structure and function of protease and DNAzyme,but also provides an important method to study these two different types of enzyme,which lays an important foundation for their respective application in their respective fields.