【作者】 刘金山；

【导师】 毛忠贵；

【作者基本信息】 江南大学 ， 发酵工程， 2005， 硕士

【摘要】 L-谷氨酰胺是人体内普遍存在的,不可缺少的条件性必需氨基酸,也是构成蛋白质的重要氨基酸之一。L-谷氨酰胺在生命活动中起着关键作用,对保持肌肉新陈代谢、细胞分化和生长、组织创伤的修复、体内毒素的中和有重要影响。因此,L-谷氨酰胺已广泛应用于营养补给、抗疲劳、胃肠道功能修复和提高人体免疫等方面,其市场需求量日渐增长。目前,L-谷氨酰胺主要以发酵法生产,酶法生产却很少有研究。本文着重从L-谷氨酰胺的酶法合成能量偶联体系的建立,高效L-谷氨酰胺合成工艺的确定及酶法合成液中L-谷氨酰胺的提取三方面进行了研究。首先,对谷氨酰胺合成酶与酵母酒精发酵能量偶联的谷氨酰胺合成体系进行了研究。确定了气流干燥是对酵母细胞通透性处理的最适方法,并从不同酵母菌种中确定了安琪酵母是最适的菌种;考察了ATP、NAD、AMP及两种金属离子Mg²⁺、Mn²⁺对偶联体系的影响,最适Mg²⁺、Mn²⁺分别都为15mM,不大于5mM的启动ATP对偶联体系有促进作用。其次,从谷氨酰胺合成酶和酵母的底物对偶联体系谷氨酰胺转化的影响进行研究,并通过正交试验进一步优化,经方差分析,发现酶底物谷氨酸对偶联体系谷氨酰胺转化影响最大,在优化条件下,谷氨酰胺产量达17.8g/l,转化率为71.2%。为了进一步提高产量,确定了葡萄糖、谷氨酸的分批三次补料转化工艺,得到谷氨酰胺较好产量为33.8g/L,转化率为67.6%。最后,根据谷氨酸和谷氨酰胺溶液中离子分布特点,确定了201×4树脂处理成Cl-型,在中性条件下分离谷氨酰胺,确定了合适的提取工艺流程及条件,最终谷氨酰胺收率为66.6%,谷氨酸去除率为95%,产品纯度为97.2%。更多还原

【Abstract】 L-glutamine（L-Gln）, one of important amino acids to construct protein, existinguniversally in human body, is an important conditionally necessary amino acid. L-Gln plays akey role in life, which has important effect on metabolism of muscle, cleavage and growth ofcell, rehabilitation of tissue wounded and neutralization of toxin in body. So, L-Gln is appliedwidely for nutrition supply, resistance tiredness, rehabilitation of gastric and enteric function,enhancement organ immunity and so on,and its market demand is going up increasingly. Atpresent, L-Gln is produced mainly by fermentation, and research on L-glutamine productionby the enzyme method was reported occasionally. In this paper, the foundation of the energycoupling system producing L-Gln by L-glutamine synthetase（GS） and highly efficient methodto produce L-Gln and to extract L-Gln from enzyme broth were built.Firstly, L-Gln produced by GS coupling with yeast alcoholic fermentation was studied. Itis determined that the airflow dry is the best method of the permeable treatment to yeast andAngel yeast is optimum strain comparing to the others. The influence of ATP, AMP, NAD,Mg²⁺, Mn²⁺ on the energy coupleing system was researched. The results showed that theoptimum concentration of Mg²⁺,Mn²⁺ both were 15mM and the optimum concentration forstart-up ATP was 5mM.Secondly, the effect of substrate of GS and yeast on the conversion of L-Gln in thecoupling system was studied and found that glutamate（Glu） affected mostly the conversion ofL-Gln through Orthogonal experiment and analyst. Under the optimum condition of reactive factor, theyield of L-Gln is 17.8g/L and the conversion rate is 71.2%. To get higher yield of L-Gln, feed-batch ofglucose and glutamate was determined. and the final yield of L-Gln is 33.8g/L and the conversion rateis67.6%.Finally, using Cl-resin 201×4 according to the distributional characters of Gln and Gluin the solution to separate L-gln was studied. In the optimum condition, the final recovery rateand the content of L-Gln is 66.6% and 97.2% respectively and the removal rate of Glu is 95%.更多还原