【作者】 戴悦；

【导师】 吴丹； 葛向阳；

【作者基本信息】 江南大学 ， 生物工程（专业学位）， 2022， 硕士

【摘要】 交替糖蔗糖酶（alternansucrase,EC 2.4.1.140,ASR）是GH70家族的四大经典葡聚糖蔗糖酶之一,它可以进行葡萄糖基转移,催化形成具有α-1,3糖苷键和α-1,6糖苷键交替连接的交替糖。交替糖多糖和交替糖寡糖统称为交替糖,聚合度DP为3-10的交替糖称为交替糖寡糖,聚合度DP超过10时则称为交替糖多糖。交替糖独特的键型结构,使其具有优异的特性,应用范围主要涉及食品、纳米科技、医药、化妆品等领域。本研究将Leuconostoc mesenteroides NRRL B-1355来源的ASR基因（asr）在Pichia pastoris中异源高效表达,探究了重组ASR的酶学性质,优化了重组ASR制备交替糖寡糖的工艺,并对交替糖寡糖粗品进行了纯化,获得了高纯度的交替糖寡糖。主要研究结果如下:（1）ASR基因（asr）在P.pastoris中的异源高效表达。构建了重组质粒p PICZαA-asr和p PIC9K-asr,并分别转化两种宿主菌P.pastoris GS115和P.pastoris X33,获得重组工程菌GS115/p PICZαA-asr、X33/p PICZαA-asr、GS115/p PIC9K-asr、X33/p PIC9K-asr。将上述重组菌进行摇瓶发酵96 h,其酶活分别为1.53 U×m L^-1、0.96 U×m L^-1、2.93 U×m L^-1和1.81 U×m L^-1。进一步构建了双启动子菌株GS115/p PIC9K-asr-p PICZαA-asr,将重组菌进行摇瓶发酵,其上清ASR的酶活为8.85 U·m L^-1,约是GS115/p PIC9K-asr摇瓶水平的3.0倍。（2）ASR重组菌的高密度发酵。将重组菌GS115/p PIC9K-asr和GS115/p PIC9K-asr-p PICZαA-asr分别在3-L发酵罐中进行高密度发酵。重组菌GS115/p PIC9K-asr发酵72 h后,其产酶能力达到最大值,酶活为34.5 U×m L^-1,约是其摇瓶水平的11.8倍;重组菌GS115/p PIC9K-asr-p PICZαA-asr发酵105 h后,其产酶能力达到最大值,酶活为145.6 U·m L^-1,约是其摇瓶水平的16.5倍。（3）重组ASR酶学性质的研究。研究重组菌GS115/p PIC9K-asr表达的重组ASR的酶学性质,测得其最适p H为6.0,最适温度为50℃;重组ASR在p H 4.5-8.0的范围内放置7 d后仍能保留90%以上的酶活;重组ASR在40℃下,孵育10 h仍然可以维持50%以上的酶活;重组ASR的K_m和k_cat分别是31.97 m M,0.36 s^-1。（4）ASR制备交替糖寡糖最佳受体分子的研究。分别以麦芽糖、葡萄糖、乳糖和海藻糖为受体底物,以蔗糖为供体底物,利用重组ASR制备交替糖寡糖。当以麦芽糖为受体时,生成的交替糖多糖较少,大部分为DP 3-8的交替糖寡糖;当以其他小分子糖为受体时,生成的交替糖寡糖含量较少,大部分是交替糖多糖。可知,麦芽糖对ASR的亲和力更高,因此在后续选择麦芽糖作为受体分子制备交替糖寡糖。（5）优化了重组ASR制备交替糖寡糖的工艺。优化结果如下:在以蔗糖和麦芽糖为底物,控制总量为300 g×L^-1（蔗糖和麦芽糖的摩尔比为4:1）、在40℃、p H 5.5、加酶量3U×g^-1条件下反应48 h,可生成交替糖寡糖175.96 g×L^-1,转化率达到最大值58.65%。（6）对交替糖溶液粗品进行纯化。采用微生物发酵法对交替糖溶液粗品进行纯化,从10株酵母菌株中,筛选到1株S.cerevisiae菌株,采用菌体接种的方式,在30℃,220r×min^-1条件下,摇瓶24 h发酵,交替糖溶液中寡糖的纯度由58.7%提升到88.3%。在5-L罐中进行交替糖溶液粗品的纯化放大实验,发酵23 h后,经过后处理,获得了23.2 g×L^-1的交替糖多糖粉末、158.8 g×L^-1交替糖寡糖粉末。更多还原

【Abstract】 Alternansucrase（EC 2.4.1.140,ASR）is one of the four classical glucansucrases of GH70family.It can catalytic glycosidic transfer reaction to form alternan,which is a kind of glucan withα-1,3 andα-1,6 glycosidic linkages linked alternately.Oligoalternan and polyalternan are called by a joint name alternan,when the DP is between 3 and 10,it is called oligoalternan,when the DP exceeds 10,it is called polyalternan.Alternan has unique glycosidic linkage structure,which makes it have excellent characteristics.Its application scope mainly involves food,nanotechnology,medicine,cosmetics and so on.In this study,the alternansucrase gene（asr）from Leuconostoc mesenteroides NRRL B-1355 was high-level heterologously expressed in Pichia pastoris and explored the enzymatic properties of the recombinant ASR.In addition,the process of preparing oligoalternan by the recombinant ASR was optimized.Last but not least,the crude product of oligoalternan was purified and high-purity oligoalternan was obtained.The main results are as follows:（1）ASR gene（asr）was high-level heterologously expressed in P.pastoris.The recombinant plasmid p PICZαA-asr and p PIC9K-asr was constructed and transformed into P.pastoris X33 and P.pastoris GS115,respectively,to obtained recombinant strains GS115/p PICZαA-asr、X33/p PICZαA-asr、GS115/p PIC9K-asr、X33/p PIC9K-asr.The extracellular enzyme activities of the recombinant strain were 1.53 U·m L^-1,0.96 U·m L^-1,2.93U·m L^-1and 1.81 U·m L^-1,respectively.Then the double promoter strain GS115/p PIC9K-asr-p PICZαA-asr was constructed,the extracellular enzyme activity of the recombinant strain was 8.85 U·m L^-1,which was 3.0 times of GS115/p PIC9K-asr in shake flasks level.（2）High density fermentation of ASR recombinant strain.The high-density culture of GS115/p PIC9K-asr and GS115/p PIC9K-asr-p PICZαA-asr were carried out in 3-L fermenter,respectively.After 72 h of fermentation,the enzyme activity of GS115/p PIC9K-asr reached the maximum value of 34.5 U·m L^-1,which was 11.8 times than that of shake flask level.After105 h of fermentation,the enzyme activity of GS115/p PIC9K-asr-p PICZαA-asr reached the maximum value of 145.6 U·m L^-1,which was 16.5 times than that of its shake flask level.（3）Study on enzymatic properties of recombinant ASR.The enzymatic properties of the recombinant enzyme expressed by recombinant strain GS115/p PIC9K-asr were studied.The optimum p H was 6.0 and the optimum temperature was 50℃.The recombinant ASR could still retain more than 90%of enzyme activity after 7 days incubation in the range of p H4.5-8.0 and more than 50%of enzyme activity after 10 h of incubation at 40℃.The K_mand k_catof recombinant ASR were 31.97 m M and 0.36 s^-1respectively.（4）Study on the best receptor for the preparation of oligoalternan.Oligoalternan were prepared by recombinant ASR with maltose,glucose,lactose and trehalose as receptor substrates,sucrose as donor substrate.When maltose was used as receptor,the product contained a small amount of polyalternan,most of which were DP 3-8 oligoalternan;When taking other small molecule carbohydrate as receptors,the content of oligoalternan in the product is less,and most of them are polyalternan.It showed that maltose has higher affinity for ASR.Hence,we selected maltose as the receptor substrate in the further study.（5）The preparation process of oligoalternan by recombinant ASR was optimized.The optimum preparation conditions are as follows:the total amount of sucrose and maltose is controlled to 300 g×L^-1（the molar ratio of sucrose to maltose is 4:1）,the reaction temperature is 40℃,p H is 5.5,the amount of enzyme is 3 U×g^-1and the reaction time is 48 h.After reaction,the maximum conversion can reach 58.65%,the yield of oligoalternan was 175.96g×L^-1.（6）The crude product of alternan solution was purified by fermentation.One strain of S.cerevisiae was screened from ten kinds of yeast.The purity of oligosaccharides in alternan solution was increased from 56.7%to 88.3%by shaking flask fermentation at 30℃and 220r×min^-1.Then,the crude product of alternan solution was purified in 5-L fermentor.After 23 h fermentation,the purified alternan solution was treated to obtain 23.2 g×L^-1polyalternan powder and 158.8 g×L^-1oligoalternan powder.更多还原