Preparation Of Crude Enzyme From Recombinant Escherichia Coli And Study Of Alginate Hydrolysis Condition

Alginate is the most abundant polysaccharose in brown algae, it could be degraded to oligosaccharide and then be used to product ethanol by microbe in theory, in addition, alginate oligosaccharide has many biological activities. Alginate oligosaccharide will have good application prospects in food, medical, agriculture etc. field. However, alginate lyase yield and activity of alginate lyase producing strains now found are low, therefore, alginate oligosaccharide production can't be industrialized. It is key to enhance the alginate lyase yield and activity for preparation of alginate oligosaccharide. It's significance to enhance the alginate lyase yield and activity in many fields.With the development of genetic engineering, cloning alginate lyase gene to industrialize host cell like Escherichia coli become an important way to obtain high-yield alginate lyase. Though there are more than 20 kinds alginate lyase gene have been cloning and sequencing, and some recombinant genetic engineering bacterias have been constructed, their expression level are low. It's necessary to research the fermentation process of recombinant genetic engineering bacteria for enhancing the alginate lyase yield.The batch fermentation kinetics of alginate lyase recombinant Escherichia coli TOP10 was first researched. Analysed the effect of different initial glucose concentration and inoculate quantity on fermentation of recombinant Escherichia coli, and batch fermentation process of recombinant Escherichia coli in 5L fermenter. Determine the cell concentration, residual glucose concentration and enzyme activity of fermentation liquid with time change. According to relevant kinetic equation fitted experimental data, set up recombinant Escherichia coli growth, product synthesis and substrate consumption models by Origin software.The results showed the optimal initial glucose concentration is 10g/L, optimal inoculate quantity is 5%, the synthesis of alginate lyase is part relate to cell growth, the fermentation type is mixed type. Set up recombinant Escherichia coli growth model, product synthesis model and substrate consumption model as follow: , ,These models can reveal the dynamic character of Escherichia coli fermentation process, the results have certain instructive significance to fermentation scale-up and fermentation process optimal control of recombinant Escherichia coli. ddXt + 0.2X? ddSt = 0.15 1ddSt +0.095X The alginate lyase product from recombinant Escherichia coli TOP10 is endoenzyme, it's necessary to break the cells by appropriate way. Ultrasonic wave disruption method is easy to operate and it's suitable for gene engineering bacterium and suitable for laboratory scale production. However, high intensity ultrasonic wave will product thermal effect, besides, high temperature and high pressure will be produced when cavitation bubble fracture, these effects can inactivate alginate lyase. So it's necessary to study the conditions of ultrasonic wave disruption for obtaining high-activity alginate enzyme.The effects of disruption intensity, total working time and NaCl concentration on extraction of alginate enzyme were studied. According to enzyme activity determine the optimal conditions of ultrasonic wave disruption by single factor experiment and orthogonal test. The optimal conditions are as follows, sample concentration 40mg/ml, volume 50ml, working / interval time 4s / 8s, disruption intensity 50%, total working time 6min, NaCl concentration 0.15mol/L. Enzyme activity reached 21U/ml under the optimal conditions. The influence of NaCl concentration on enzyme activity is greater than others, but it has little effect on cell disruption and protein release.For preparing more alginate oligosaccharide, combined with physics degradation and enzyme degradation method to degraded alginate. Studied the effects of heat-pressure (autoclaving), ultrasonic radiation and crude enzyme on degradation of alginate; compared their effects according to the reducing sugar of hydrolysate. And then the effects of hydrolysis time, substrate concentration, enzyme additive amount, hydrolysis temperature and pH value were studied by single factor experiment and orthogonal test to determine the optimal conditions of degradation of alginate.The results show the activity crude enzyme prepared is relatively low; the effect of crude enzyme on degradation of alginate is not as good as the effect of autoclaving; and the effect of ultrasonic radiation is not obvious. How to enhance the alginate lyase activity is very important, it needs more deep researches.The optimal conditions of degradation are as follow, sodium alginate concentration 1.5%, pH value 7.0, the sodium alginate solution is autoclaved 20min on 121℃in advance, enzyme additive amount 35%(v/v), reaction temperature 35℃and hydrolysis time 5h. The reducing sugar concentration of hydrolysate attained to 670.104μg/ml.