【作者】 王杰；

【导师】 毛国涛；

【作者基本信息】 河南农业大学 ， 微生物学， 2022， 硕士

【摘要】 昆布寡糖是一种具有独特生理活性的低聚糖,在食品、医药、农业等领域有着广泛的应用价值。酶法制备昆布寡糖是一种理想的制备方法,但目前酶法制备昆布寡糖的昆布寡糖磷酸化酶存在活性低、稳定性差、催化效率低等问题。因此开发具有高活性、高催化效率和稳定性好的昆布寡糖磷酸化酶非常重要,对酶法生产昆布寡糖具有一定的理论和现实意义。来自Thermosipho african热稳定的THA＿1941可催化昆布三糖的磷解,THA＿1941是归属于GH161家族的昆布寡糖磷酸化酶。但是,有研究报道THA＿1941归属于GH94家族,具备纤维二糖磷酸酶（cellobiose phosphorylase,CBP）和纤维寡糖磷酸酶（cellodextrin phosphorylase,CDP）的双功能催化活性。为确定THA＿1941的家族归属和催化性质,本课题以THA＿1941为对象展开研究:（1）THA＿1941是归属于GH161家族的昆布寡糖磷酸化酶。本研究从THA＿1941生物信息学入手,对THA＿1941进行了序列保守性和进化分析,进而通过薄层层析、液质联用和核磁碳谱等方法,对THA＿1941底物特异性和催化产物结构进行分析,确定了THA＿1941的归属家族和催化性质。（2）THA＿1941具有优异的酶学性质。在pH 5-6条件下孵育8 h,THA＿1941仍能维持100%的催化活力。在pH 7-10条件下孵育8 h,THA＿1941仍能维持95%以上的催化活力,表明THA＿1941具有优异的pH耐受性。在50℃和60℃的温度范围内孵育24 h,THA＿1941仍能维持80%的催化活力,表明THA＿1941具有优异的热稳定性。合成方向以昆布二糖为底物时,THA＿1941的催化效率可达271.9 s^-1mM^-1,表明THA＿1941能高效的催化昆布寡糖的合成。（3）THA＿1941可以合成不同聚合度的昆布寡糖。当葡萄糖的浓度为1 mM时,THA＿1941合成昆布寡糖的聚合度为10.33,随着葡萄糖浓度升高至5 mM-10 mM合成昆布寡糖的聚合度会下降至5.49-7.58。当以1 mM龙胆二糖为底物时,THA＿1941合成昆布寡糖的聚合度为6.77;当葡萄糖的浓度升高至10 mM时,THA＿1941合成昆布寡糖的聚合度为10.28。因此,通过调整底物的浓度,THA＿1941可以合成不同聚合度的昆布寡糖。（4）以秸秆水解液为原料制备昆布寡糖的多酶催化体系的开发。该体系解决了THA＿1941在催化昆布寡糖反应过程中所需底物葡萄糖-1-磷酸价格昂贵的问题,进一步降低了昆布寡糖的工业化生产成本。更多还原

【Abstract】 Laminarioligosaccharide is a type of oligosaccharide with unique physiological activity.It has a wide range of applications in food,medicine,agriculture and other fields.Enzymatic preparation of laminarioligosaccharides is an ideal preparation method,but at present,the laminarioligosaccharides phosphorylase for enzymatic preparation of laminarioligosaccharides has the problems of low activity,poor stability and low catalytic efficiency.Therefore,it is very important to develop Laminarioligosaccharide phosphorylase with high activity,high catalytic efficiency and good stability,which has certain theoretical and practical significance for the enzymatic production of Laminarioligosaccharide.Thermally stable from Thermosipho african THA₁941 can catalyze the phosphorolysis of laminarin.THA₁941 is a laminarin oligosaccharide phosphorylase belonging to the family GH161.However,studies have reported THA₁941 belongs to the GH94 family and has bifunctional catalytic activities of cellobiose phosphatase（CBP）and cellooligosaccharide phosphatase（CDP）.In order to determine the family affiliation and catalytic properties of THA₁941,this topic is based on THA₁941 as the research object:（1）THA₁941 is a laminarioligosaccharides phosphorylase belonging to GH161 family.This study from THA₁941 bioinformatics,THA₁941 sequence conservation and evolution analysis were carried out,and then tha was analyzed by thin layer chromatography,liquid chromatography-mass spectrometry and nuclear magnetic carbon spectroscopy THA₁941substrate specificity and catalytic product structure were analyzed to determine THA₁941 family and catalytic properties.（2）THA₁941 has excellent enzymatic properties.Incubate at p H 5-6 for 8 h,THA₁941 can still maintain 100% catalytic activity;Incubate at p H 7-10 for 8 h,THA₁941 can still maintain more than 95% catalytic activity.Incubate for 24 h in the temperature range of 50℃and 60℃,THA₁941 can still maintain 80% catalytic activity.With laminarin as substrate,the catalytic activity of 1941 was 13.6 times that of Laminaria oligosaccharide phosphorylase of Paenibacillus polyyxa ATCC 842.（3）THA₁941 can synthesize laminarioligosaccharides with different degrees of polymerization（DP）.When the concentration of glucose is 1 m M,the DP of Laminarioligosaccharide synthesized in THA₁941 is 10.33.With the increase of glucose concentration to 5 m M-10 m M,the DP of Laminarioligosaccharide will decrease to 5.49-7.58.When using 1 m M gentian disaccharide as substrate,the DPn of Laminaria oligosaccharide synthesized in THA₁941 was 6.77;Tha increased when the concentration of glucose increased to 10 m M,the DP of Laminaria oligosaccharide synthesized in THA₁941 was 10.28.（4）Development of multi-enzyme catalytic system for the preparation of Laminari oligosaccharide from straw hydrolysate.The system solves THA₁941 in the expensive substrate G1 P required in the catalytic reaction of laminarioligosaccharides further reduced the industrial production cost of laminarioligosaccharides.更多还原