Screening Of Alginate Lyase Producing Strain And Cloning And Expression Of Its Gene

With the rapid development of society,the demand for natural resources for the production of fuel and food is increasing.Researchers at home and abroad have focused on efficient and sustainable supply of marine algae.In recent years,several studies have demonstrated the feasibility of using brown algae for bioconversion to produce ethanol,but the full potential of brown algae as a raw material for commercial-scale ethanol production is still difficult to achieve,mainly because industrial microbial strains are difficult to effectively degrade alginate to generate its available monosaccharides.Therefore,it is particularly important to look for enzymes that efficiently degrade alginate.In this thesis,strain resources producing alginate lyase and enzyme with high activity and extensive substrate specificity are provided.The results of the study are summarized as follows:?1?Strains of alginate degrading bacteria were isolated from rotted seaweed.BH17,the strain with the highest enzyme activity,was screened through the transparent circle and enzyme activity assay.It was identified as Vibrio alginolyticus by 16S rRNA.Then through the study of its growth characteristics and enzyme production conditions,it was found that the most suitable enzyme production medium for this strain was:0.6%sodium alginate,0.6%?NH₄?₂HPO₄,0.2%K₂HPO₄,4%NaCl,0.1%MgSO₄·7H₂O.The optimum enzyme production pH is 6.5-7.0,the temperature is 20-25 ^oC,and the fermentation time is 48 h.Under the above optimized conditions,the enzyme activity per unit OD₆₂₀ was determined to be 83 U/mL,which is superior to most enzyme activities reported so far.?2?In order to further study enzymatic properties,improve performance and expand the scope of application,we uses a mature E.coli to clone and express the alginate lyase gene.According to the literature,five potential alginate lyase genes on the V.alginolyticus BH17genome were cloned and expressed in E.coli BL21.Five recombinant strains were successfully constructed.Enzyme activity assay showed that rAlgV2 and rAlgV3 had higher enzyme activity,which have industrial application potential.The optimization of inducing conditions for rAlgV3,which had the highest enzyme activity,showed that it was induced at 25 ^oC for 6 h when the OD₆₂₀ of the cultured bacterial cells reached 0.4.The induction of IPTG had little effect on the enzyme activity.After optimization of the inducing conditions,the enzyme activity reached168 U/mL,which was 7.3 times higher than before.?3?In this paper,the recombinant enzyme rAlgV3 with the highest enzyme activity was isolated and purified,and its enzyme properties were characterized.The optimal reaction temperature was 40 ^oC,and the enzyme was relatively stable at 4-20 ^oC.The enzyme had a higher activity at pH 6.5-9.0,in which the optimum pH was 8.0.It showed a wide range of pH that the alginate lyase can exist stably in pH 4.5-9.5.Appropriate concentrations of NaCl and Fe²⁺,Fe³⁺ions have the role of promoting enzyme activity,SDS and Cu²⁺ions can significantly inhibit the enzyme activity.The study also found that the enzyme can degrade Poly-M fragments and Poly-G fragments,with a wide range of substrate properties.The degraded product of sodium alginate of rAlgV3 analyzed by ESI-MS mainly was oligosaccharides with a polymerization degree of 2-3,which means the rAlgV3 was an endo-type alginate lyase.The enzyme plays an important role in the development of third-generation bioethanol and the production of alginate oligosaccharides.