Study On A Hyaluronidase From Arthrobacter Sp

Hyaluronic acid（HA）is a kind of polymeric acidic polysaccharide,which is widely found in the extracellular matrix（ECM）of vertebrate tissues.HA plays an important role in inflammation,wound healing,angiogenesis and so on.HA has a wide range of molecular weights,ranging from 10 kDa to 500 kDa.The physiological function of HA is closely related to its molecular weight size and under what circumstances.High molecular weight HA is anti-angiogenesis,while low molecular weight HA promotes angiogenesis.At present,Commercial HA is mainly obtained by microbial fermentation,and very few are extracted from animal tissues.The HA obtained by these methods are generally high molecular weight and molecular weight above 100 kDa.Low molecular weight HA（LMW-HA）was obtained by physical or chemical methods.The reaction conditions of these methods are severe,often destroy the structure of monosaccharide residues,at the same time,it is difficult to specifically degrade the glycosidic bond,so it is impossible to obtain uniform HA.The preparation of small molecule HA by enzymatic hydrolysis has the advantage of mildly specific degradation of glycosidic bonds,and high quality HA can be obtained.Hyaluronidase is a class of glycosidases that predominantly degrades HA,as a drug penetrant to promote drug absorption,postoperative edema dissipation and local anesthesia effect.In addition to animal testis and venom,more and more microorganisms have been reported to produce hyaluronidase.Because Hyaluronidase from animals are limited,the preparation of hyaluronidase by microbial fermentation is receiving more and more attention.Although it is found that many microorganisms can produce hyaluronidase,but the enzyme activity is generally low,the main reason is the low enzyme production.A strain producing hyaluronidase was screened from contaminated sodium hyaluronate solution.Although the activity is good,it is very difficult to separate and purify.Our lab wants to experiment with the exogenous expression of this enzyme.In this study,the genomic sequences of the strains were sequenced and the amino acid sequences of the coding hyaluronidase were obtained by genome analysis.The sequences were verified by SDS-PAGE,Native-PAGE and mass spectrometry.The recombinant vector was designed and the expression and purification of hyaluronidase were completed.Finally,the degradation products were studied by fluorescence assisted carbohydrate electrophoresis（FACE）.1.Genome sequencing and sequence analysis of strainsA gene encoding hyaluronidase was obtained by Genomic Sequence Analysis.The protein has a molecular weight of 83 kDa and pI 6.55.2.electrophoresis and mass spectrometry to verify the annotation sequenceThe fermentation broth was incubated for 48 h and subjected to ultrafiltration centrifugation.The results of SDS-PAGE showed that there was a protein band near 83kDa.The results of active electrophoresis showed that the fermentation supernatant had the activity of hyaluronidase and only one active band,indicating that the strain only expressed a hyaluronidase.Analysis of extracellular protein mass spectrometry showed that the bacteria expressed the protein encoded by the annotation gene.The gene obtained by genomic analysis is a gene encoding hyaluronidase.3.The structural bioinformatics analysis of active site architectureAt present,the hyaluronidases have been classified into 2 families according to sequence similarity.The analysis of the sequence and structure of the active architecture region is of great significance for the study of the mechanism of hyaluronidase.In this paper,the amino acid of the active architecture region of these two families was analyzed and the active center sequence spectra were prepared.4.Exogenous expression and purification of hyaluronidaseSelecting pET-28a as vector,Escherichia coli as engineering bacteria.we successfully induced the expression of soluble hyaluronidase at 20℃,in 1 mM IPTG.The nickel column was eluted with different concentrations of imidazole eluate and eluted in 100 mM imidazole elution to obtain electrophoretic pure hyaluronidase,which could be used for later studies.5.Enzymatic Properties of Recombinant Hyaluronidase（1）Effects of temperature and pH on recombinant HAase The optimum reaction temperature of recombinant hyaluronidase was 42℃,and the activity was high between 37℃ and 42℃,and the stability was good.The enzyme was incubated at 50℃ for 10 min,and the enzyme activity was only 10%of the maximum enzyme activity.The optimal pH of recombinant hyaluronidase was 6.5,and the stability of the enzyme was higher at pH 5.5-8.0.（2）Effects of Different Concentrations of NaCl and NaH₂PO₄-Na₂HPO₄ Buffer on recombinant HAaseLow concentration of NaH₂PO₄-Na₂HPO₄ buffer slightly promoted enzyme activity,high concentrations of NaH₂PO₄-Na₂HPO₄ buffer inhibited enzyme activity.The addition of NaCl to the substrate solution can promote enzymatic activity and the highest activity was at 5 mM.（3）Effects of Metal Ions,EDTA and Surfactants on recombinant HAase Most of the investigated metal ions,including Ca²⁺,Mg²⁺ and Ba²⁺,had positive effects on the activity of recombinant HAase.Recombinant HAase showed the highest activity in 100 mmol/L BaCl₂ with activity increased to 168%.Cu²⁺（10 mmol/L）and Zn²⁺（10 mmol/L）severely inhibited the activity of recombinant HAase by more than 80%.EDTA（10 mmol/L）did not affect the activity of the enzyme,but when the concentration increased to 100 mmol/L,the activity of recombinant hyaluronidase was significantly reduced,about 80%,but did not completely lose the enzyme activity.Non-ionic surfactants（Triton X-100）moderately inhibit recombinant HAase activity at different concentrations.However,ionic detergent（SDS）completely inhibited the enzyme activity at any concentration.（4）Substrate Specificity of Recombinant Hyaluronidase The recombinant HAase degraded HA Specificity,and can not degraded other glycosaminoglycans.The Michaelis constant（K_m）and maximum velocity(V_max)were measured to 4.985 mg/mL and 0.4071 μm/mL/min respectively.