| Glutaminase(EC 3.5.1.2)can catalyze the hydrolytic degradation of glutamine to produce glutamic acid and ammonia.Glutaminase was mainly used in food industry and cancer treatment research.The glutaminase was used for increasing production of L-glutamic acid in some femented foods and can enhance the taste of cooking.In this study,the four glutaminase genes glsB,ylaM,ybgJ and Mglu were efficiently expressed in E.coli BL21 or B.subtilis 168.The recombinant glutaminases were purified and characterized.The expression and characterization of these recombinant glutaminases were compared and analysed,then one recombinant strain was chosen for fermentation.In order to improve the production of gutaminase,the culture medium and environment conditions were optimized.(1)The four glutaminase genes glsB,ylaM,ybgJ and Mglu were amplified by PCR and six recombinant strains E.coli BL21/pET28a-glsB,E.coli BL21/pET28a-ylaM,E.coli BL21/ pET28a-ybgJ,E.coli BL21/pET28a-Mglu,B.subtilis 168/pMA5-ylaM and B.subtilis 168/pMA5-Mglu were constructed.Then the six recombinant strains were expressed and the glutaminase activities were 58.91±1.34 U·m L-1,16.43±2.47 U·m L-1,41.59±2.71 U·m L-1,30.37±1.03 U·m L-1,7.322±0.75 U·m L-1 and 32.67±2.21 U·m L-1,repectivily and were significantly higher than that of origin strains E.coli BL21 and B.subtilis 168.(2)The glutaminases from E.coli BL21/ pET28a-glsB,E.coli BL21/pET28a-ylaM,E.coli BL21/pET28a-ybgJ and E.coli BL21/pET28a-Mglu were purified and characterised.The specific activity of the four recombinant glutaminases were 2465 U·mg-1,939.5 U·mg-1,1005 U·mg-1 and 1326 U·mg-1,repectivily.The optimum pH of the four recombinant glutaminases was 7.5 and they were stable at pH 7.0-8.0.The optimum temperatures of the four recombinant glutaminases were 55°C,45°C,55°C and 50°C,repectively.E.coli BL21/ pET28a-ylaM and E.coli BL21/pET28a-Mglu glutaminases were stable below 25°C.E.coli BL21/pET28a-glsB and E.coli BL21/pET28a-ybgJ glutaminases were stable below 35°C.The enzyme activity of glutaminases was slightly promoted by partial divalent metal ions.The enzyme activity of glutaminases was inhibited by trivalent mental ions,and it was not affected by valence metal ions and EDTA.E.coli BL21/pET28a-glsB and E.coli BL21/pET28a-ybgJ were inactivated by NaCl above 15%.More than 50.4% and 77.4% of the maximum activity was retained in the presence of 15%-17.5% NaCl with E.coli BL21/pET28a-ylaM and E.coli BL21/pET28a-Mglu glutaminase.The E.coli BL21/pET28a-Mglu glutaminase was more stable in the presence of 15%-17.5% NaCl.(3)Compared to other recombinant strains,the recombinant B.subtilis 168/pMA5-Mglu was choosen for glutaminase production.The culture medium was optimized by one-factor-at-a-time.The optimal medium compositon was determined to be glucose,25 g·L-1;angle yeast,40 g·L-1;NH4Cl,4 g·L-1;L-glutamic acid sodium salt,2 g·L-1;K2HPO4·3H2O,1.875 g·L-1;KH2PO4,1.125 g·L-1;CaCl2 2 g·L-1.The optimal environment conditions were initial pH 7,fermentation temperature of 24°C,rotational speed of 180 r·min-1,and inoculation size of 4%(v/v).The highest enzyme activity of B.subtilis 168/pMA5-Mglu glutaminase can reach 249.24±13.12 U·m L-1.(4)Based on the optimal culture medium and environment conditions,the recombinant B.subtilis 168/pMA5-Mglu was cultivated in a 5 L fermenter with pH control and agitation speed strategy.The strategy of fermentation was as follow: pH 6.8-6.9,the agitation speed was 450 r·min-1,supplied with 3 g·h-1·L-1 glucose and 3 g·h-1·L-1 angel yeast after 18 h.Fermentation results showed that the highest enzyme activity of glutaminase can reach 1052±23 U·m L-1 and the production of glutaminase can reach 0.79 mg·m L-1. |
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