【作者】 吴昊；

【导师】 张洪斌；

【作者基本信息】 合肥工业大学 ， 制药工程， 2021， 硕士

【摘要】 N-乙酰氨基葡萄糖是一种在制药、医学、食品、化妆品等领域具有广泛应用的多功能单糖,尤其在治疗骨关节炎、骨质疏松方面具有显著的功效。目前,市场上以几丁质为原料制备Glc NAc的方法仍然停留在传统的化学酸解法,该方法缺点明显:污染环境,生产安全隐患大,效率低。而酶解法是一种绿色、环保的以几丁质为原料制备N-乙酰氨基葡萄糖的新方法。本实验室前期已构建好一株重组几丁质酶,可以有效降解胶体几丁质。在此基础上,进行了以下几项工作:（1）进一步研究了实验室已有的几丁质酶（Chi A）的酶学性质及其催化特性:Chi A的最适反应温度为50℃,在50℃以下酶热稳定性良好;最适反应p H为6.0,酶也较为稳定;通过质谱及HPLC结果确定Chi A水解胶体几丁质的产物为（Glc NAc）₂。（2）构建了一株重组β-N-乙酰基己糖胺酶工程菌E.Coli BL21（DE3）/p ET-28a（+）-HJ5N,优化了β-N-乙酰基己糖胺酶HJ5N的产酶条件并研究了其酶学性质。结果表明:在诱导温度为25°C,IPTG诱导浓度为0.4 mmol/L,诱导时间为11 h条件下,HJ5N酶活最高可达440 U/m L;SDS-PAGE显示HJ5N分子量在55k D左右,最适温度为50℃,在40℃以下有较好的稳定性;最适p H在6.0左右,在p H 5.5-6.5范围内具有较好的稳定性。不同金属离子化合物对HJ5N酶活的影响不显著,无明显的促进或者抑制作用;以p NP-Glc NAc为底物测定了HJ5N的动力学常数Km及Vmax:Km值为0.64mmol/L,Vmax值为3.42mol/（min.L）,动力学参数表明HJ5N对p NP-Glc NAc具有较高的亲和力和反应速率,与Chi A相似的酶学性质表明Chi A和HJ5N可以协同催化降解胶体几丁质。（3）以上述双酶的酶学性质研究为基础,成功建立了几丁质酶（Chi A）和β-N-乙酰基己糖胺酶（HJ5N）协同催化降解胶体几丁质制备Glc NAC的新方法,研究了双酶的协同机制。结果表明Chi A存在产物抑制现象,而HJ5N的加入可保证中间产物快速被转化为单糖从而消除这种抑制作用,双酶协同催化效率提高了一倍;最后通过分离纯化获得了纯度极高的Glc NAc,并对其进行了结构表征。（4）采用单因素及正交设计实验对双酶协同催化体系的反应条件进行了优化。结果表明,反应最适反应温度为30℃,最适p H为6.0,最优底物浓度为9%,Chi A:HJ5N添加比例为2:1,总添加量为52.5 U/m,在上述最优反应条件下反应12 h可达到最大Glc NAc浓度18 mg/m L,转化率为60%,研究结果为酶法制备N-乙酰氨基葡萄糖的工业应用提供了一种有效策略。更多还原

【Abstract】 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.更多还原