【作者】 贾慧娟；

【导师】 马莺；

【作者基本信息】 东北农业大学 ， 食品科学， 2002， 硕士

【摘要】 海藻糖是由两个葡萄糖分子通过α α，1→1键结合而成的非还原性双糖。当生物细胞处于饥饿、干燥、高温、低温冷冻、辐射、高渗透压及有毒试剂等各种胁迫环境时，胞内海藻糖含量迅速上升，对多种大分子都具有保护作用，从而保护生命本身。外源性的海藻糖同样对生物体和生物大分子有良好的非特异性的保护作用，使其在分子生物学、医学、食品工业、化妆品工业、农业等领域有广阔的应用前景。 目前国际上主要是通过低成本、高收率的淀粉转化法生成海藻糖，本课题立足于以淀粉为原料，通过微生物酶的分子内转糖基作用合成海藻糖；并对产酶菌株及其培养特性、MTSase和MTHase特性以及海藻糖合成工艺进行了系统的研究。 本实验首先通过对选定的实验菌株进行紫外线诱变选育，采用将菌株直接接种在固体培养基上，立刻进行紫外线照射的方法，筛选出性能优良菌株SP，其生产性能比出发菌株提高了7倍，而且能够保持一定的稳定性。通过对该菌株进行的形态特征鉴定、化学分类鉴定和生理生化特征鉴定，表明该菌为藤黄灰链霉菌，简称ST412。 对菌株ST412培养特性的研究结果表明：在菌株ST412的产酶过程中，麦芽糖为最佳碳源：蛋白胨和牛肉膏为最佳氮源；K2HPO₄和MgSO₄ 7H₂O为最佳无机盐：通过正交试验确定的最佳诱导培养基组合配方为麦芽糖1％、蛋白胨0.5％、牛肉膏0.2％、K₂HPO₄0.2％、MgSO₄ 7H₂O0.05％。产酶的最佳培养条件为：温度为31℃，pH值为7.0，该菌株在供氧充分条件下酶活力较高，所以选择500ml三角瓶中装100ml液体培养基为最佳装液量，培养终点为培养后144小时。 对MTSase和MWHase系统研究表明：在淀粉液化液浓度为10％、液化30min的条件下，菌株ST412产生的MTSase和MTHase最适作用温度为40℃，耐热性较差：最适作用pH值为7.0；Ca²⁺和Mn²⁺对酶略有激活作用，Ba²⁺和Zn²⁺抑制酶活明显，最严重抑制酶活性的是Cu²⁺、EDTA、Fe³⁺、Fe²⁺，分别抑制了酶活性的96％、69％、77％、和79％。通过对MTSase和MTHase底物特异性的研究，排除了以葡萄糖和麦芽糖为底物的可能性，确定在MTSase和MTHase的作用下，以淀粉为底物将低聚糖转变为海藻糖的合成过程。粗酶液4℃时的保存稳定性很差，不适宜在4℃长期贮存。 本实验中，用平板式超滤膜对酶液进行浓缩，选用截留分子量1万的再生纤维素超滤膜，温度控制温度在30℃，压力控制在0.04Mpa以下对酶进行浓缩，酶液浓缩收率达到60％以上。 实验中对海藻糖的合成工艺进行了系统的研究。首先采用单因素确定淀粉液化液浓度为10%、液化30min，然后对其它五个影响因素采用五因素四水平正交试验设计进行工艺优化。确定的最佳反应工艺为：反应温度40℃，反应时间36h，缓冲液浓度0.1M、反应pH值7.0和酶加入量27U／g淀粉。在此最优工艺条件下，海藻糖的得率为28.14％。更多还原

【Abstract】 Trehalose is a non-reducing disaccharide by a a,l-1 glucosidic linkage that bindsthe two glucose molecules. Trehalose inside orpanism cell will rise promptly to protect itSelfwhen it is at different constrained conditions as hunger,dryhightemperatUr,freezing,radiation,h Permeation pressure and POisonous reagent.Thus trehaloseis paid c1ose attention.Trehalose eXterior organism can also act on oI’ganism and biologicmolecule to protect them distinctively .Therefore trehalose is widely used inbi o l ogy pharn aeeuti cals, fbodstuff cosm eti c and argh cu ltUre. A t presenL it is prim ari l y used tOsynthesize trehalose that tranSform amylose with low coSt of manufacture and high ovetallrecovery rate .This stUdy mainly discussed synthesis of halose from amylose throughcombined action of microoganism by intramloecular tranSglycosylation.Scain that producederwme and itS cultUred proPethes,character ofthehalose creataSe and production oftrehalosewere detailed StUdied.The start strain was treded with UV-ray irradiation to obndn a strain of ST4l2 with highyield trehalose.Staln was inoculated dbectly on solid medium and irradiated imrnediatelyunder ultraviolet lamp.Producing CaPaCityits of ST4l2 was increased 7tAnes higher than theoriginal sthen with stability. Modality charaCter, chendcal classificationy, physiology andbiochemiStry characteristic of ST4l2-strain were identified.The result showed that this mein isStraptomeyCes lu!egheus.The cultured propertier of ST4l2-Strain were discussed.The resultS shOwed that: (l )Maltose was the oPtjmum wtn source,pePone and be f endon were the oPtimumnitrOgen source, K2HPO and MgSO4 7H2O were the oPtiInum inowic. (2 ) The formula ofculwt medium boUgh ortboonal exPeriInent Asgh was ope as:the 1 -- .5 %,be f ndOn0Th.K2HPO0 .2% and MgSO4 7H2oc. 05 %. (3 ) TheOPboam twon conditions werempreture3l C .PH7.0,CaPaCity 100ml per 50OrnltriWlar bottle .boention time 144 home.The proPerties oftrehalose cds were considerd SyStematically.The resultS indicaed2002^6^that:If the concentration of liquefied solution of amylose and liquefied time were 10% and SOminutes relatively, ( 1 ) the enzyme exhibited an optimum tempreture of 40*C and the optimum pH value of 7.0 . (2) the enzyme activity was increased by addition of Ca2+ and Mn2+ ,but was inhibited 96%,69%,77% and 79% enzyme activity by Cu2+,EDTA,Fe3+ and Fe2+ , relatively. ( 3 ) Substrate specifixity of trehalose creatase had been verified by experiment, which eliminated possibility of glucose and maltose as its substrate. (4) the conserving stability of crude enzyme solution was inferior.lt is unsuitble to conserve enzyme solution for long time at4"C.It was investigated of the possibility of using ultrafiltration technique in the concentration process of trehalose-creatase.The regenerated cellulose ultrafiltration membrane of one ten thousand dalton were selected to concentrate trehalose-creatase liquor.The time of concentration,yield of concentration and ratio of rejection were determined.And observe the flux,too.The experimental results indicated that molecular weight 10,000d is feasible under the optimum conditions (pressure of 0.04Mpa,tempreture of 30?0 "C) , furthermore yield of concentration could surpass 60%.It was found that using ultrafiltration not only would protect the activities of enzyme.but also could remove low molecular impureties partly effectively.Studies on reaction condition for production of trehalose were made systematically. Above all,it was determined that concentration of liquefied solution of amylose and liquefied time were 1 0% and 30minutes relatively by single factor analyse.lt was optimized by orthogonal experiment design for another five influent factors such as tempreture,time,concentration of buffer and pH and the mount of enzyme.The experimental results indicated that the optium conditions were tempreture 40 癈 , time 36hours,concentration of bufferO.lM, pH 7.0 and the mount of enzyme27 U/g amylose followed with 28. 1 4% tr更多还原