Enzymatic properties of alginate lyase MAAL1 from Microbulbifer sp.QZHA1
To investigate the enzymatic properties of MAAL1,an alginate lyase from Microbulbifer sp.QZHA1,the alginate lyase gene maal1 was constructed into the pET-28a expression vector and heterologously expressed using Escherichia coli BL21(DE3).The recombinant enzyme MAAL1 has the highest homology of 93.69％with the algi-nate lyase(WP_23625014.1)from Microbulbifer sp.ALW1 and is clustered in the Polysaccharide Lyase family 7(PL7).The optimum temperature for the recombinant enzyme MAAL1 is 35 ℃ and the optimum pH is 7.5,and it maintains more than 60％of its enzymatic activity when stored at pH 5.5 to 10.5 for 24 hours.MAAL1 has good re-sistance to organic solvents.Among the nine organic solvents tested,the enzyme activity remained above 59％after the addition of 30％(v/v)of all organic solvents except isopropyl alcohol.The specific activity of recombinant en-zyme MAAL1 was 4.3 U/mg under the optimum conditions,the Km value of the constant was 1.08 mg/mL,the maximum reaction rate Vmax was 4.75 mg/(mL·min),and the catalytic constant Kcat was 4.52 s-1.The recombi-nant enzyme MAAL1 had a substrate preference for polymannuronic acid(PolyM),and no degradation ability to polyguluronic acid(PolyG).Thin-layer chromatography analysis showed that the main end-products of degradation of sodium alginate by MAAL1 were monosaccharides,disaccharides and trisaccharides,the main end-products of degradation of PolyM were disaccharides and trisaccharides,and the main end-products of degradation of hetero-polymeric MG blocks(PolyMG)were monosaccharides and disaccharides.It suggests that MAAL1 is an endolytic polymannuronic acid specific lyase.The recombinant enzyme MAAL1 has good pH and organic solvent tolerance,is substrate specific for PolyM and does not degrade PolyG.This characteristic alginate lyase was first identified in Mi-crobulbifer,and this enzyme may provide a new candidate for use in the preparation of mannuronate oligosaccha-rides.
marine biologyMicrobulbiferalginate lyasecloning and expressionpolymanuronic acidorganic solvent resistance
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