Optimization Of Enzyme Production Conditions Of Alginate Lysing Bacteria And Analysis Of Enzymatic Hydrolysates

Fucoidan oligosaccharides have good application prospects in food,health care products,pharmaceutical products,agricultural development and other fields due to their small molecular weight,easy absorption and various pharmacological activities.Alginate lyase is an important tool enzyme for the production of alginate oligosaccharides.In this paper,a strain PLT1 capable of secreting alginate lyase was screened from the soil of Yantai coastal zone,and its enzyme production conditions were optimized based on single factor and multi-factor analysis.On this basis,the alginate lyase secreted by it was purified and its enzymatic properties and main enzymatic hydrolysates were discussed.It laid a foundation for the screening and application of new alginate lyase.The results are as follows:（1）A strain secreting alginate lyase was screened.The strain was identified as Paenibacillus sp.by physiological and biochemical indexes and 16S r DNA analysis,named PLT1.The strain preservation number was CGMCC No.25691.（2）The enzyme production conditions of PLT1 were optimized by single factor and multi-factor analysis.The optimum conditions for enzyme production were as follows:sodium alginate 9.5 g/L,peptone 6.7 g/L,Na Cl 0.5%,p H 7.0.When the inoculation amount was 2%,the culture speed was 170 r/min,and the culture temperature was 31℃,the enzyme production capacity of PLT1 was increased by 1.21 times,and the highest yield was 214.641 U/m L.（3）The alginate lyase produced by PLT1 was purified by ammonium sulfate precipitation.SDS-PAGE analysis showed that the molecular weight of the enzyme was about 68 k Da.The optimum p H and temperature for sodium alginate degradation were 7.0and 50℃,respectively.The enzyme was stable at p H 6.5-8.0,and the enzyme activity only reached 30%of the highest enzyme activity when the temperature exceeded 55℃.A certain concentration of Na⁺,Ca²⁺can promote the enzyme activity,Cu²⁺has obvious inhibitory effect;EDTA could significantly reduce the enzyme activity,and the relative enzyme activity was only 31.63%.It is speculated that the enzyme is a metal ion-dependent alginate lyase.Substrate analysis showed that the enzyme had higher degradation ability for sodium alginate and Poly M,and weaker degradation activity for Poly G.It was speculated that the enzyme was a bifunctional enzyme that preferred to degrade Poly M.（4）Thin layer chromatography（TLC）and liquid chromatography-mass spectrometry（LC-MS）were used to analyze the enzymatic hydrolysates.The results showed that the enzyme could degrade sodium alginate to produce alginate oligosaccharides with a degree of polymerization of 3-5,mainly with a degree of polymerization of 3-4,and the relative ion abundance values accounted for 100%and 90%,respectively.It is speculated that the enzyme is an endonuclease.Infrared absorption spectroscopy（FTIR）analysis showed that the characteristic absorption of Poly M at 889cm^-1and 817 cm^-1was weakened,and the Poly M fragment was reduced,which indicated that the alginate lyase produced by strain PLT1 preferred to degrade Poly M.（5）The promoting effect of the degradation products of sodium alginate on plant growth was preliminarily analyzed.The results showed that 0.05 mg/m L degradation products could significantly promote seed germination and root growth,and root length increased by 38.2%.