| D-hydroxyphenylglycine is a kind of pharmaceutical intermediates of production of semi-synthetic β-lactam antibiotics,and the market demand has exceeded 10 million tons.Optical pure D-HPG could be synthesized by D-hydantoinase and N-carbamoylase directly with its outstanding stereochemical specificity.To excessive synthesis of D-HPG,we overexpressed the D-hydantoinase and N-carbamoylase,modified the relevant genes and evaluated the effect of modified genes.The expression cassette Paco induced expression of D-hydantoinase（sd1 or hyd）,the expression of N-carbamoylase by PAE（adc）,respectively.The recombinant strains 168/pHCY and 168/pHCS were constructed with plasmid pHP13.The enzyme activity of 168/pHCY was 0.25 U/mL,while the total enzyme activity of 168/pHCS was 0.36 U/m L.The results showed that the relative enzyme activity of sd1 was higher than that of hyd.The acoR and sigL were overexpressed in B.subtilis 168 N.QRT-PCR indicated that the mRNA of acoR and sigL were average 248.2 times and 80.7 times of 168 N.The enzyme activity of WD-3/pHCS was 0.56 U/mL,increasing 80% to 168/pHCS.The results showed that increasing the level of aco R and sigL are necessary to improve the catalytic activity.D-hydantoinase and N-carbamoylase were overexpressed with plasmid pUB110 in WD-3,and the total enzyme activity reached 0.98 U/ml,which increased by 75% compared with WD-3/pHCS.The result showed that the dosage of the two kinds of enzymes could increase the whole cell catalytic activity of engineered strain.The skf and sdp gene were knocked out in WD-3.The amount of spores of WD-5/pUCS was one order of magnitude,the activity of the enzyme was equivalent,and the decay rate of the enzyme activity was significantly decreased.The kinA and kinB gene were knocked out in WD-5.The WD-7/pUCS cannot form a spore,the enzyme activity and the equivalent of the reaction in the enzyme activity of the decay rate is also considerable. |
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