| N-acetylglucosamine is a kind of multifunctional monosaccharide widely used in pharmaceutical,medical,food,cosmetics and other fields,especially in the treatment of osteoarthritis and osteoporosis.At present,the method of preparing Glc NAc from chitin in the market is still in the traditional chemical acid hydrolysis,which has obvious disadvantages:environmental pollution,hidden danger of production safety and low efficiency.Enzymatic hydrolysis is a green and environmental friendly method to prepare N-acetylglucosamine from chitin.A recombinant chitinase has been constructed in our laboratory,which can effectively degrade colloidal chitin.On this basis,the following works are carried out:（1）The enzymatic properties and catalytic properties of chitinase（Chi A）were further studied:The optimal reaction temperature an PH of Chi A was 50℃and 6.0,and Chi A had good thermal stability below 50℃.The hydrolysis product of chitin by Chi A was identified as（Glc NAC）2by HPLC and mass spectrometry。（2）A recombinantβ-N-acetylhexosaminase engineering strain E.coli BL21（DE3）/p ET-28a（+）-HJ5N was constructed.The conditions of producingβ-N-Acetylhexosaminase（HJ5N）were optimized and its enzymatic properties were studied。Results showed that:under the conditions of induction temperature of 25°C,IPTG concentration of 0.4 mmol/L,induction time of 11 h,the highest activity of HJ5N could reach 440 U/ml;SDS-PAGE showed that the molecular weight of HJ5N was about 55 KD,the optimum temperature was 50℃,and it had good stability below40℃;the optimum p H was about 6.0,and it had good stability in the range of p H 5.5-6.5;Different metal ions had no significant effect on the activity of HJ5N;The kinetic constants km and Vmax of HJ5N were determined with p NP-Glc NAc as substrate.The Km value was 0.64mmol/l and Vmax value was 3.42 mol/（min.L）.The kinetic parameters showed that HJ5N had high affinity and reaction rate for p NP-Glc NAc.The enzymatic properties of HJ5N similar to Chi A showed that Chi A and HJ5N could co-catalyze the degradation of colloidal chitin.（3）Based on the study of enzymatic properties of Chi A and HJ5N,a new method for the preparation of Glc NAc from colloidal chitin catalyzed by chitinase（Chi A）andβ-N-acetylhexosaminase（HJ5N）was successfully established.Results showed that Chi A had product inhibition,and the addition of HJ5N could ensure that the intermediate products could be quickly converted into Glc NAc,thus eliminating this inhibition。the efficiency of dual enzymes synergistic catalysis was doubled.Finally,Glc NAc with high purity was obtained by separation and purification,and its structure was characterized.（4）The reaction conditions of the system were optimized by single factor and orthogonal design experiments.The results showed that the optimum reaction temperature was 30℃,the optimum p H was 6.0,the optimum substrate concentration was 9%,the proportion of Chia:HJ5N was 2:1,and the total amount of Chia:HJ5N was about 52.5 U/m L.Under the above optimal reaction conditions,the maximum concentration of Glc NAc was 18 mg/ml,and the conversion rate was 60%.The results provided an effective strategy for the industrial application of enzymatic preparation of N-acetylglucosamine. |
