Heterologous Expression And Characterization Of Alginate Lyase Gene From Marine Vibrio Sp.SS-1

Alginate lyase is a key enzyme for the preparation of multifunctional alginate-derived oligosaccharides?ADO?using alginate as substrate,it is important for seaweed resources utilization and aquaculture industry.In this paper,a highly producing alginate dominant strain lyase was screened from the intestinal tract of haliotis discus hannai.The strains were identified by molecular biotechnology.At the same time,the optimum fermentation conditions,the best culture formula,the heterologous expression of Alg gene and the enzymatic properties of the strain were studied in detail,as follows:1)Screening,identification and optimization of fermentation conditions for producing alginate lyase strain.A strain named SS-1 producing alginate lyase was isolated from the liver and pancreas gut of Haliotis discus hannai by transparent circle method,using sodium alginate as the sole carbon source on agar medium.Strain SS-1 was initially identified as Vibrio sp.by the results of morphological observation and 16 S rRNA gene sequence analysis,and then submitted to NCBI to get Genbank number KX266239.The optimal fermentation conditions of SS-1 were 30?,initial pH 7.2,it produced the highest activity of alginate enzyme of about 38.6 U/m L and the specific activity was 138.9 U/mg at 28 h.2)Determined the fermentation medium formulation of marine Vibrio sp.SS-1.Under the optimum fermentation conditions of strain SS-1,the significant factors?maltose,yeast powder and dipotassium hydrogen phosphate?were determined by Plackett-Burman experiment with single carbon source,nitrogen source and inorganic salt.And then used the steepest climbing experiment approached the maximum response area to found the best fermentation medium formula of marine Vibrio sp.SS-1 by Box-Behnken center combination experiment.Experiments show that the best fermentation medium formula of marine Vibrio sp.SS-1 was maltose 11.86 g/L,yeast powder 16.72 g/L,K2HPO₄ 1.36 g/L,MgSO₄ · 7H₂ O 1.5 g/L,FeSO₄ · 7H₂ O 0.1 g/L,NaCl 6.75 g/L,respectively.Under the optimum culture conditions,the enzyme activity was verified to be 191.8 U/mL and the specific activity was 167.3 U/mg,which was 4.97 times higher than that before optimization.3)The heterologous expression of Alg of alginate lyase gene of marine Vibrio sp.SS-1.The purpose of this study was to clone,express and characterize a new alginate lyase Alg from marine Vibrio sp.SS-1 by gene technology constructed the expression of E.coli BL21?DE5??/p ET28a?+?-Alg.The expression of recombinant protein was induced to achieve the efficient expression by 1 mmol/L IPTG for 6 h.The fermentation broth was ultrasonically broken?15W,4s / 8s,15min?and was purificated by Ni-NTA resin affinity chromatography.The results show that the recombinant alginate lyase protein molecular weight was 38.0 k D,the purification ratio was 51.01 times,and the recovery rate of pure protein was 26%.Its was determinated the recombinant enzyme activity of was 187.5 U/mL and the specific activity was 138.9 U/mg.4)Study on enzymatic properties of recombinant alginate lyase were from marine Vibrio sp.SS-1.The enzymatic properties were studied in terms of reaction temperature and stability,pH and stability,metal ion,substrate specificity,enzyme kinetic equation and so on.The results showed that the optimum pH of the recombinant enzyme was 8.0,and the relative enzyme activity was maintained at 60% under the condition of pH was kept 4.0-9.0 and the temperature was maintained 30?for 2 h.The optimum temperature was 30 ?,and the thermal stability experiment showed that the residual enzyme activity was more than 40% when the temperature was higher than 30? for 2 h.At the concentration of 1 mmol/L,K+,Na+ and Mg2+ had significant stimulation on the enzyme activity,while Cu2+,Fe3+,Ba2+,Fe2+,Al3+,SDS and EDTA had inhibitory effects on the enzyme.The kinetic parameters Km and Vmax of recombinant alginate lyase were 5.93 mg/m L and 826.44 mg/?mL·min?,respectively.ESI-MS analysis showed that the degradation of alginate was mainly DP2,DP3 and DP4,et al.Recombinant enzyme was a bifunctional enzyme with good enzymatic properties and an effective bottom effect of Poly?M?degradation to produce disaccharides,which can be used for the preparation of alginate-derived oligosaccharide.At the same time,the analysis of physico-chemical properties of recombinant protein showed that 344 amino acids were identified by the Alg encoding gene of Vibrio sp.SS-1,which had a molecular weight of about 38.8 kD and an isoelectric point p I of 4.80.The total number of negatively charged amino acids Asp and Glu was 50,and the total number of positively charged amino acids Arg and Lys was 34,which can be deduced as an alkaline protease.This was consistent with the physical and chemical properties of recombinant proteins in enzymatic properties.The spatial conformational analysis of protein molecules showed that the recombinant protease was a low-clustered single-chain protein molecule with a homology of 51.08% by used online software SWISS-MODEL.It was inferred that the stability of the recombinant enzyme was poor,which infered the main reason was that most of the irregular linear curl?about 44%?and a small amount of ?-helix?about 27%?and ?-fold?about 27%?were scattered arrangement.The recombinant alginate lyase had highly conserved sequence of "NTKYARSELRA","GQIH",which determined the key amino acid residues in the catalytic activity of the recombinant enzyme.Based on this point that we can use site directed mutagenesis modification of alginate lyase enzyme activity increased value to provide a good basis.It was similar 54.13% with the amino acid sequence of Vibrio sp.QY101 that concluded the recombinant enzyme may be a novel protease.With the deepening of the research of alginate lyase,more potential value would be explored.The study provides a good technical basis for the analysis of the enzymatic properties of alginate lyase.It provided good preparation for fucoidan of the enzyme resources.