Heterologous Expression And Application Of Alginate Lyases From Pseudoalteromonas Carrageenovora ASY5

Alginate lyase is a tool enzyme with wide application prospects in many industries.This kind of enzyme mainly degrades alginate to produce alginate oligosaccharides by the elimination reaction.In order to explore novel alginate lyase with excellent properties,three alginate lyase genes,alg1281,alg2491 and alg23 from P.carrageenovora ASY5 strain were heterologously expressed in E.coli and afterwards purified.The enzymatic properties and enzymatic hydrolysis products of these three recombinant alginate lyases were determined.The results were as follows:(1)Based on our previous work on the whole genome sequencing of P.carrageenovora ASY5,bioinformatic method was used to analyze these three encoding alginate lyase.The results showed that alg1281 gene was consisted of 1089 bp and encoded 362 amino acids.The molecular weight of the predicted protein was 40.04 kDa and the theoretical isoelectric point was 9.02.The alg2491 gene was consisted of 1182 bp and encodes 393 amino acids.The molecular weight of the predicted protein was 42.34 kDa and the theoretical isoelectric point was 4.48.The total length of alg23 gene was 2223 bp,which encoded 740 amino acids.The molecular weight of the predicted protein was 82.6 kDa,and the theoretical isoelectric point was 6.55.Using the P.carrageenovora ASY5 genome as a template,PCR amplification was performed to obtain the full length sequence of three genes.The alginate lyase genes were cloned into the pET-28 a vector,and then expressed in E coli BL21(DE3)to obtain the three alginate lyases.The molecular weights of the three alginate lyases were verified by SDS-PAGE analysis and shown to be about 40 kDa,42 kDa and 83 kDa,which were consistent with the results of bioinformatics analysis.(2)The enzymatic properties of three alginate lyases were determined,and the results showed that AlgL1281 had a certain activity against polyM and polyG,but a higher degradation activity against polyG.The optimum temperature and pH of AlgL1281 were 50℃ and 8.0.This enzyme had good thermal stability.After incubation at 40℃ for 30 min,more than 80%of the maximum enzyme activity could be retained.The enzyme had the characteristics of salt activation.With the addition of salt,the maximum activity of AlgL 1281 enzyme could be increased to 5.56 times of the activity without metal ions addition.AlgL2491 had good degradation activity towards polyM and polyG.Its optimum temperature and pH were 35℃ and 7.5.AlgL2491 had enzymatic activity in a wide range of temperatures,and the enzyme activity could retain more than 50%of the maximum enzyme activity at 4℃.which indicated AlgL2491 was a cryophilic enzyme.This enzyme also had the characteristics of salt activation.Salt addition could increase the enzyme activity of AlgL2491 to 5.33 times of the activity without salt addition.AlgL23 could degrade polyM specifically and had no degradation activity to polyG.The optimum temperature and pH of AlgL23 were 35℃ and 6.0.This enzyme was also a cryophilic enzyme,The enzyme activity could retain about 50%of the maximum enzyme activity at 4℃.The results of enzymatic kinetic experiments showed that the Km of AlgL1281 was the smallest among these three alginate lyases indicating AlgL1281 had the highest substrate affinity.(3)These three alginate lyase were applied for the production of alginate oligosaccharides.The degradation products of these three alginate lyases were analyzed by ESI-MS.The results showed that the main component of the AlgL1281 enzymatic oligosaccharide was DP2.The main components of the AlgL2491 enzymatic oligosaccharides were DP2 and DP3.The main components of the AlgL23 enzymatic oligosaccharides are DP3,DP4 and DP5.All the three kinds of enzymatic oligosaccharides had certain antioxidant ability.Under the same concentration,the antioxidant capacity of AlgL1281 oligosaccharides was slightly stronger than the other two enzymatic oligosaccharides.These three kinds of enzymatic oligosaccharides had obvious growth-promoting effects on Lactobacillus strains,but the promoting effects was limited on the growth of Bacillus subtilis.This phenomenon suggested that the growth-promoting effects of enzymatic hydrolysis were selective.These three enzyme-hydrolyzed oligosaccharides had a significant positive effect on the survival of probiotics in the simulated gastric and intestinal fluid environment.In particular,adding 5 g/L of AlgL2491 enzyme-hydrolyzed oligosaccharides could increase 37.8%of the survival rate of probiotics compared with the control group.