| Alternan includes polyalternan and oligoalternan,which contains alternately linked?-1,6and?-1,3 glycosidic linkages,and its unique structure gives it a wide range of applications in the pharmaceutical,cosmetic,feed,health,food and printing industries.Alternansucrase?EC2.4.1.140?has the activity of?-transglycoside and can catalyze the synthesis of alternan.In this study,the alternansucrase gene?asr?derived from Leuconostoc citreum CICC23234 was heterologously expressed in Escherichia coli BL21 and Bacillus subtilis WB600,respectively.The enzymatic properties of the recombinant enzymes expressed in the two hosts were explored,and the fermentation technology of B.subtilis/pHY300PLK-asr recombinant strain was studied.In addition,the process of preparing polyalternan and oligoalternan by recombinant enzyme was explored.The main findings are as follows:?1?The recombinant strains E.coli/pET24a-asr and B.subtilis/pHY300PLK-asr which contained the asr gene deriving from L.citreum CICC23234 were constructed.The extracellular enzyme activities were 3.77 U mL-1 and 0.43 U mL-1,respectively.Further investigation of the enzymatic properties of the two recombinant enzymes showed that the optimum pH and temperature were 6.0 and 45?respectively.After 7 days in the range of pH4.5-8.0,the residual enzyme activities were both maintained above 70%.In addition,the half-time of recombinant enzymes at 40?were both above 100 h.Since B.subtilis is a food-safe strain,B.subtilis/pHY300PLK-asr recombinant strain was selected for further study.?2?The recombinant B.subtilis/pHY300PLK-asr was optimized in shake flask.The results showed that the optimum medium composition of the recombinant strain was 25 g L-1soy peptone and 25 g L-1 corn syrup as composite nitrogen,5 g L-1 glycerol as a carbon source,and the culture temperature was 30?.Under the above conditions,the recombinant alternansucrase activity reached 0.9 U mL-1.Then,the high-density culture was carried out in a 3-L fermenter by fed-batch fermentation.After 82 h of fermentation,the enzyme-producing ability of the recombinant B.subtilis/pHY300PLK-asr reached a maximum value of 3.9U mL-1.It is 4.3 times of the enzyme activity of shake flask fermentation optimization.?3?The enzymatic products of the recombinant enzyme were identified by gel permeation chromatography,nuclear magnetic resonance and mass spectrometry.When the sucrose was the sole substrate,the average molecular weight of the polyalternan was1.9×106 Da,the degree of polymerization was 1.1×104,and the content of?-1,6 glycosidic bond and?-1,3 glycosidic bond in the product structure were respectively 69%and 31%.When maltose and kojibiose were used as receptors,the products were only oligoalternan.When glucose and trehalose were used as receptors,the products contained not only oligoalternan,but also a large number of polyalternan.In the study of the preparation of oligoalternan conditions,the maltose was selected as a receptor molecule to effectively increase the yield of oligoalternan and improve production efficiency.?4?The effects of different conditions on the preparation of polyalternan and oligoalternan by recombinant alternansucrase were investigated.The optimum preparation conditions were as follows:150 g L-1 sucrose as substrate and the amount of enzyme was 2U g-1,the reaction temperature was 40?,the reaction pH was 5.5,the reaction time was 20 h,under the above conditions,the conversion rate can reach 46.5%,and the yield of polyalternan was 69.8 g L-1;225 g L-1 sucrose and 75 g L-1 maltose as substrate,the enzyme amount was 2.5 U g-1,the reaction temperature was 40?,the reaction pH was 5.5,and the reaction time was 24 h,under the above conditions,the conversion rate can reach 59.6%,and the yield of oligoalternan was 178.8 g L-1. |
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