| Chitosanase is a kind of specific enzyme to hydrolysis chitosan, the product from hydrolysis of chitosan by chitosanase is oligosaccharides (Chitooligosaccharides, referred to as COS),which is a kind of new products with good solubility in water, functions and high biological activity, in the field of medicine, food, agriculture, the biological veterinary drugs and feed additives are tempting prospects. Oligosaccharides from hydrolysis of chitosan by chitosanase is mild reaction conditions, oligosaccharide activity and yield highly, so the efficient production of chitosanase has become a research hotspot in recent years in this field. Now the commercial chitosanase is less,and chitosanase activity is low, and can not meet the demand of the industrial production of Oligochitosan. There is a Pichia strain Stored in our laboratory who can product chitosanase, In this paper,the target product is the expression of chitosanase of this strain, to improve amount of chitosanase expression through the fermentation process optimization and establish the fermentation process of producting chitosanase. The main contents are as follows:1,Growth characteristics. As the main indicator of fermentation broth OD600nm researching the the first order seed, on YPG medium, and find that the first order seed is 28h growth broth,and the second order seed is 20h growth broth; the chitosanase activity of fermentation broth as the main indicator to study the strain tolerance of methanol, the result is that the optimum concentration of methanol is 0.5%, The expression of chitosanase will be checked when the methanol concentration is more than 1%; The chitosanase activity of fermentation broth as the main indicator, when the induction temperature is lower from 30 ° C to 20 ° C after amount of Chitosanase expression the expression of chitosanase is increased to 1.24 times.2, High-density fermentation process optimization. To compare the constant dissolved oxygen with exponential flow plus two flow glycerol, the results show that exponential feeding glycerol bacterial growing faster, time-consuming shorter, and bacterial glycerol yield, Yx/s reached 0.615g/g; when improving the wet weight from 220g/L to 300g/L on starting to the methanol induction, the enzyme activity based on the original is improved tol.33-fold, reaching 562.4U/ml; when the control mode of methanol stream becomes methanol electrode control from DO control,the enzyme activity before based on increased 1.62 times, reach 913.0U/ml; the induced temperature reduced by 30 ° C to 20 ° C, enzyme activity increased 1.35 times before on the basis of to reach 1231.1U/ml, protein concentration of 6.86 g of/1. Fermentation broth total enzyme activity increased to 2.92 times.3,The spray-drying process optimization. Equal protection agent is added to the solution of chitosanase, the indicator is relative residual enzyme activity after 20min in water bath at 55 ° C; the starch was to be the best protective agent, dextrin and trehalose were followed,then the inlet air temperature, spray speed and the amount of the protective agent added were to be the indexes, three factors and three levels orthogonal experiment were done, The best spray drying conditions of the chitosanase solution was 2% of the doses of protection,0.5L/h of atomization speed,120°C of inlet air temperature. Under these conditions, The chitosanase residual enzyme activity was 69.6% after spray drying.4 Chitosanase storability. Enzyme solution were placed at room temperature (25 ° C),at 4 ° C and-20 ° C meanwhile its enzyme activity was measured every week. Enzyme powder were placed at room temperature (25° C) and 4 ° C meanwhile its enzyme activity was measured every week. The enzyme activity was 52.1% when enzyme solution were placed at room temperature 2 Week, the enzyme activity was 61.3% when enzyme solution were placed at 4 ° C for 5 week, the enzyme activity was 32.8% when enzyme solution were placed at-20 ° C for 5 week, enzyme activity was 89.6% when enzyme powder were placed at at room temperature (25 ° C) for 5 week, enzyme activity was 90.5% when enzyme powder were placed at at 4° C for 5 week. |
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