| The alginic contains Fucoidan and mannitol. Algae and mannitol could be got bydispose of alginic. Algae and mannitol could be used to product ethanol by microbe intheory. In addition,alginate oligosaccharide has many biological activities. Alginateoligosaccharide will have good application prospects in food,medical,agriculture etcfield. Enzymatic method because of its mild reaction conditions,easy to control,product uniformity of the advantages of being widely used in the hydrolysis ofpolysaccharides. Alginate lyase yield and activity of alginate lyase producing strainsare low. Although,with the development of genetic engineering, more than20kindsof alginate lyase gene to be cloned and sequenced,and to construct a variety ofrecombinant alginate lyase gene engineering bacteria,but the expression of therecombinant alginate lyase gene engineeringthe level is still relatively low,far can notmeet the demand of the industrialized preparation of brown algae. The high activity ofalginate lyase become a key step in the preparation of alginate oligosaccharides. If wecan increase the production of alginate lyase enzyme,alginate oligosaccharidesprepared in many ways are of great significance.In order to improve the yield of the of recombinant bacteria alginate lyase,thehigh density fermentation of genetically engineered microorganism is necessary.Much more alginate lyase was producted by fermentation technology. In this study,laboratory built the alginate lyase genetically engineered bacteria as bacteria,alginatelyase genetically engineered bacteria cell density by changing the feeding strategy ofhigh density fermentation process,thus providing the alginate lyase expressionamount to improve the activity of the alginate lyase. Brown algae and prepared byfermentation of alginate lyase preparation to study the role of brown algae,opened upnew ways to improve the effective utilization of the brown algae.1. In order to find the optimized condition to produce alginate lyase,usingfed-batch culture technology,the engineered E.coli BL21was cultured in5Lfermented at high density. The use of single-factor test to optimize the concentration of carbon source in the feed medium,single factor tests and simplex optimizationmethod to optimize the induction time and IPTG concentration,to obtain optimalhigh-density fermentation conditions：fermentation medium glucose10g/L,yeastextract5g/L,peptone20g/L. Feed medium was glucose150g/L,peptone20g/L,yeastextract10g/L. The stream acceleration rate was set to100mL/h form4hours to10hours,he stream acceleration rate was set to200mL/h form10hours to16hours.Induction time was at4.5h,and the IPTG was final concentration to0.60mmol/L.Dissolved oxygen in the fermentation process wad controlled in the30%to40%,andpH was controlled at7.0~7.2.The results showed that in un-induced condition,theOD600was0.696when final fermentation liquid were diluted200times. Theengineered E.coli BL21concentration was65.38g/L. After induce with IPTG,thevalue of OD600was0.457when fermentation liquid were diluted200times. theconcentration of the engineered E.coli BL21was60.15g/L,which was8.43timecompared with the value from the batch fermentation. Finally,the crude enzyme wasextracted from the high density fermentation strain by ultrasonication. The enzymeactivity was26.37U/mL, which was up to5.48time compared with the batchfermentation.2. Affect the the alginate efficiency of alginate lyase hydrolysis of five factors：the substrate concentration,enzyme dosage,reaction time, anti temperature and pHvalue of a single factor and orthogonal experiment to determine the optimalconditions：pH value of6.0hydrolysis temperature50°C,hydrolysis time4h,45%of the amount of the enzyme,the substrate concentration of0.4%. Optimal hydrolysisconditions,the use of alginate lyase hydrolyzed alginate preparation of brown algae,brown algae yield0.541g/g sodium alginate. The brown algae proliferation ofbifidobacteria study,the results showed that the proliferation effect is remarkable,brown algae as a carbon source modified MRS medium balance of bifidobacteriabody concentration of3.09×10~8cfu/mL than glucose MRS medium resulting cellconcentration of the carbon source is increased by3.68times.3. Candida tropicalis fermentation of mannitol production of ethanol,singlefactor five factors affecting fermentation: fermentation temperature,fermentationtime,liquid volume,inoculum size,initial pH and select relevant factor orthogonalexperiment to determine the best conditions for ethanol production： mannitolfermentation concentration of60g/L,8%of the inoculum size,initial pH5.0,fermentation temperature36°C, the fermentation broth volume of120mL. Fermentation optimum fermentation conditions for72h,ethanol generates2.423%. |
