| Myrosinase(EC3.2.1.147),which exists in Cruciferae family,can hydrolyze glucosinolates into glucose,sulfat,isothiocyanates,nitrile and thiocyanate.Thereinto,The enzymatic products of isothiocynate have several biological activities.But the isothiocynate is unstale in water,it easily degrade and lose activities.Glucosinolates have a good stabilization,and also myrosinase have a good stabilization by immobilized.But the economic efficiency of withdrawing myrosinase from the plant is low,cannot satisfy the production needed.Therefore,we use transgene technology to construct the appropriate project bacteria,to obtain high purity and activity myrosinase.In this experiment,we attempt to hetorologous expression throug the MYR1 gene cloning to prokaryotic expression vector.In order to highly effective expresses myrosinase,we use the pGEX-4T-1 express system.At first,we withdraw total RNA from the rape seedling,uses the RT-PCR to expand the MYR1 encoded mature peptide gene sequence increases.By sequencing after the clone,contrasting with the NCBI sequence,we know this gene there has 99%homology with NO.60214, the amino acid only one is difference.Again,we use the Salâ… spot to connect with the pGEX-4T-1,constructing recombinant pGEX-4T-1-MYR1.Confirmating by PCR,Salâ… enzyme cutter and EcoRâ… enzyme cutter,sequencing,We screen the correct recombinant.Expression product at OD6000.8,37℃,0.08mmol/LIPTG,and 2.5h,and it expressed highly at 90KDa contrasting with the product of pGEX-4T-1 when run in the SDS-PAGE.But it had not obtained the soluble GST fusion protein,by supposing the GST fusion protein massive expressions gathering and forming inclusion body.After changing conditions,we induced 2.5h at 20℃,OD6000.8,IPTG 0.02mM,after lysised cell by fluid nitrogen and concentrated,then used a little of phosphoric buffer(pH=6).Finally,we obtain a little of dissolve activity GST fusion protein,as SINIGRIN is substrate,The specific activity of the fusion protein is 3.1×10-3U/mg.
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