Molecular Cloning,Over-Expression And Enzymatic Characterization Of Laminarinases LamA1 And LamA2 From Microbulbifer Sp.ALW1

The bioavailability and biological activity of laminarin can be improved after enzymatic degradation.Expression of laminarinase and characterization of enzymatic properties are important for the preparation of oligosaccharides and the industrial production of laminarinase.Based on the sequencing of the whole genome sequence of marine bacterium Microbulbifer sp.ALW1,two laminarinase genes of lamAl and lamA2 were cloned by PCR.Firstly,a series of bioinformatics analysis of the two laminarinase genes were carried out.Then,the two genes were cloned,expressed and purified to obtain the laminarinases of LamA1 and LamA2.In addition,the enzymatic properties of the two laminarinases and the antioxidant activities of the enzymatic hydrolysates were studied.The main conclusions were as follows:Two laminarinase genes of lamA1 and lamA2 were cloned from Microbulbifer sp.ALW1 by PCR.The lamA1 gene contained 1545 bp and encoded a 514-amino acid protein classified into the GH128 family.The lamA2 gene contained 1545 bp and encoded a 514-amino acid protein classified into the GH55 family.The lamA1 and lamA2 genes were expressed in Pichia pastoris,and the recombinant laminarinases were partially purified.The enzymatic properties of laminarinase LamAl were studied using laminarin as the substrate.The optimal reaction temperature and pH of laminarinase LamAl were 45℃ and pH 5.5,respectively,and the enzyme was stable below 45℃.1 mM K+had a weak promotion of enzyme activity.10 mM Ca2+ promoted the enzyme activity.Na+,Mg2+ and Ba2+ promoted the enzyme activity in different degrees.Cu2+,Zn2+,Cd3+,Al3+ and Fe3+ had different degrees of inhibition on enzyme activity.EDTA had little effect on enzyme activity.DTT promoted the enzyme activity.In addition to SDS and CTAB,LamAl had certain resistance to other detergents tested.LamAl retained more than 60%of residual activity after treatment with 2%Tween 20 and Tween 80 for 1 h at room temperature.LamAl had certain resistance to urea,and it maintained 50.4%of residual activity after treatmet with 5 M urea for 1 h at room temperature.The results of antioxidant research showed that the anti-oxidation activity of laminarin was enhanced after being degraded by LamAl.The IC50 values of the enzymatic hydrolysates towards DPPH,ABTS and OH·radicals were 7.3 mg/mL,1.1 mg/mL and 1.9 mg/mL,respectively.The enzymatic properties of laminarinase LamA2 were studied using Laminarin as the substrate.The optimal reaction temperature and pH of LamA2 were 45℃ and pH 5.0,respectively.10 mM Ba2+ promoted the enzyme activity.Na+and Ca2+had promotion effects on the enzyme activity,while Mg2+and K+had no effect on the enzyme activity.Cu2+,Zn2+,Cd3+,Al3+ and Fe3+had different inhibitory effects on LamA2.When treated with 10 mM Fe3+,the enzyme activity was lost.β-ME had no effect on the enzyme activity,and DTT had a significant promoting effect on the enzymatic activity of laminarinase LamA2.2 mM EDTA had no effect on the enzyme activity,and 10 mM EDTA had moderate inhibition on the enzyme activity.In addition to CTAB and SDS,LamA2 had some degrees of resistance to other detergents tested.After LamA2 was treated with 2%Triton X-100,Tween 20 and Tween 80 for 1 h at room temperature,it retained more than 45%of residual activity.LamA2 had certain resistance to urea.After treatment with 5 M urea for 1 h at room temperature,LamA2 retained 49.2%of residual activity.The results of antioxidant research showed that the anti-oxidation activity of laminarin was enhanced after being degraded by LamA2.The IC50 values of the enzymatic hydrolysate towards DPPH,ABTS and OH·radicals were 6.8 mg/mL,1.5 mg/mL and 1.8 mg/mL,respectively.