【作者】 李兆丰；

【导师】 陈坚；

【作者基本信息】 江南大学 ， 发酵工程， 2009， 博士

【摘要】 环糊精葡萄糖基转移酶(简称CGT酶,EC 2.4.1.19)是一种能够通过分子内转糖基化反应转化淀粉及相关基质合成环糊精的胞外酶。随着环糊精在食品、医药、化妆品、农业等领域的应用越来越广,CGT酶已经成为当今研究的热点。为了克服天然菌株的低CGT酶生产能力,cgt基因在大肠杆菌(Escherichia coli)中过量表达被认为是最有效途径之一,然而,以前的报道表明,CGT酶通常在E. coli中形成不溶性包涵体或积累于周质空间,限制了其工业应用,因此,实现重组CGT酶的胞外生产是迫切需要的;另外,用CGT酶生产环糊精最不利的条件之一是产物特异性差,所有已知的野生CGT酶生产的均是α-、β-和γ-环糊精的混合物,不利于下游操作。本论文主要将来源于软化类芽孢杆菌(Paenibacillus macerans) JFB05-01的α-CGT酶基因表达于E. coli中,并通过采用一些培养策略,实现了重组酶的胞外过量生产,同时对该酶的产物特异性进行了一定的分析与改善。主要研究结果如下:1.将来源于P. macerans JFB05-01的α-CGT酶基因分别插入质粒pET-20b(+)和pET-22b(+)的pelB信号肽序列下游,构建了表达载体pET-20b(+)/cgt和pET-22b(+)/cgt,并将它们转化宿主E. coli BL21(DE3)。对E. coli BL21(DE3)(pET-20b(+)/cgt)进行摇瓶发酵,确定其胞外生产重组α-CGT酶的较优条件为:发酵培养基为TB培养基,诱导剂异丙基-β-D-硫代半乳糖苷(IPTG)浓度为0.01 mM,诱导温度为25°C。在该条件下诱导90 h后,培养基中酶活达到22.5 U/mL,与较优发酵条件下天然菌株P. macerans JFB05-01所产的胞外酶活相比,提高了大约42倍。E. coli BL21(DE3)(pET-22b(+)/cgt)需要更高的IPTG浓度(0.2 mM)进行诱导,且诱导90 h后的胞外酶活仅为17.5 U/mL,因此,E. coli BL21(DE3)(pET-20b(+)/cgt)更适于胞外生产重组α-CGT酶。这是国内外首次报道α-CGT酶在E. coli中的胞外生产。2.当诱导温度恒定时,25°C最适合重组α-CGT酶的胞外生产,然而,诱导的前50 h重组酶主要积累于周质空间,而只有少量释放到培养基中。较高的诱导温度抑制了重组酶的胞外分泌,可能原因是具有信号肽的前体蛋白合成速率太快,导致它们在细胞内膜内侧聚集而形成大量包涵体,并堵塞了内膜转运通道。在较低温度下诱导一段时间后升高温度有利于重组α-CGT酶的胞外生产,尤其是在25°C下诱导32 h后将温度升高到30°C导致胞外酶活明显增加,诱导90 h后的酶活达到32.5 U/mL,相比于恒定在25°C的情况提高了45%。3.甘氨酸的添加促进了重组α-CGT酶的胞外分泌并能明显缩短发酵时间,特别是甘氨酸浓度为150 mM时,诱导40 h后的胞外酶活达到23.5 U/mL,相比于对照在相同诱导时间的酶活提高了10倍,而且,诱导36 h后的重组酶生产强度最大,达到0.60 U/mL/h,相比于对照诱导80 h后的最大生产强度提高了1.5倍,其潜在机理是甘氨酸导致E. coli细胞膜透性的明显增加。然而,甘氨酸对E. coli细胞的生长有明显的负面影响,这限制了重组酶生产强度的进一步提高。Ca2+能补偿甘氨酸对细胞生长的抑制作用,主要体现在单位体积中的细胞数和活细胞数明显增加、细胞自溶明显减少以及细胞形态修复。当TB培养基中同时添加150 mM甘氨酸和20 mM Ca2+,诱导40 h后的胞外酶活达到35.5 U/mL,诱导36 h后的最大生产强度达到0.90 U/mL/h,相比于只添加甘氨酸的情况均提高了50%。4.重组α-CGT酶在C-末端连有6×His-tag,因此能用镍柱进行一步亲合层析,但该方法纯化酶的回收率很低;重组和天然α-CGT酶能通过阴离子交换和疏水色谱两步纯化,纯化酶的回收率较高。α-CGT酶在溶液中是单聚体;重组α-CGT酶环化反应的最适温度为45°C,在40°C、45°C和50°C下的半衰期分别为8 h、1.25 h和0.5 h,而天然酶有稍高的最适温度(50°C)和热稳定性(t1/2, 50°C=0.8 h);重组和天然酶环化反应的最适pH均为5.5,且分别在pH 6~9.5和6~10之间相对稳定;α-CGT酶的环化活力不依赖于金属辅因子,Hg2+、Ni2+、Fe2+和Co2+对环化活力有抑制作用,而一些二价金属离子能激活环化活力,特别是Ca2+、Ba2+和Zn2+。在酶转化的起始阶段,α-CGT酶生产α-环糊精作为主要产物,随着反应时间的延长,β-环糊精的比例明显增加,最终β-环糊精为主要产物;相比于天然酶,重组酶有更高的α-环糊精特异性。α-CGT酶环化反应的动力学性质适合用Hill方程进行描述,Hill常数高于1暗示酶单聚体的底物结合有正协同作用。5.亚位点?3处的Asp372和Tyr89的突变能改变P. macerans CGT酶的环化活力和环糊精产物比例,说明这两个残基对环糊精产物特异性具有重要作用,也进一步证实亚位点-3对CGT酶产物特异性来讲是关键位点。Asp372突变成赖氨酸和Tyr89突变成精氨酸显著提高了P. macerans CGT酶的α-环糊精特异性,单突变体D372K和Y89R在产物特异性上的改变能累加于双突变D372K/Y89R;相比于野生CGT酶,双突变体D372K/Y89R的α-环糊精产量提高了50%,而β-环糊精产量下降了43%。具有更高α-环糊精特异性的突变体比野生酶更适合于α-环糊精的工业化生产。6.氨基酸残基47是定位于亚位点?3附近的主要残基之一,不同类型的CGT酶之间残基47的氨基酸种类明显不同,暗示残基47的种类可能与CGT酶的产物特异性有关。Lys47的突变能改变P. macerans CGT酶的环化活力并影响环糊精的生产,说明这个残基对环化反应和环糊精产物特异性具有重要作用。Lys47的所有突变降低了P. macerans CGT酶的α-环化活力,暗示Lys47对α-环化反应非常重要,但这些突变能显著增加CGT酶的β-环化活力,尤其是Lys47突变成苏氨酸、丝氨酸或亮氨酸,这三个单突变转化P. macerans CGT酶从α-型到β/α-型,作为结果,所有突变体具有更高的β-环糊精特异性,它们比野生酶更适合于β-环糊精的工业化生产。更多还原

【Abstract】 Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) is an extracellular enzyme capable of converting starch or starch derivates into cyclodextrins through an intramolecular transglycosylation reaction. With cyclodextrin applications expanded in the industries related to food, pharmaceuticals, cosmetic, agriculture, etc, CGTase has become the focus of scientific research nowadays.To overcome the low CGTase productivity of wild strains, the overexpression of cgt gene in Escherichia coli has been expected. However, previous reports showed that the CGTases expressed in E. coli were usually accumulated in the cytosol as inactive inclusion bodies and/or the periplasm as soluble forms, which limited its industrial utilization. Thus, it is highly desirable to extracellular production of the recombinant CGTase in E. coli. In addition, a major disadvantage of cyclodextrin production by CGTase is that all known wild-type CGTases produce a mixture ofα-,β-, andγ-cyclodextrins, which were not favorable to its downstream processing. In the present study, the gene encodingα-CGTase from Paenibacillus macerans JFB05-01 was expressed in E. coli and the recombinant enzymes were targeted into the culture medium of E. coli through some culture strategies. The product specificity of CGTase was also analyzed and improved. The main results are listed as follows: 1. The cgt gene encodingα-CGTase from P. macerans JFB05-01 was cloned into the downstream of pelB signal sequence in a vector pET-20b(+) and pET-22b(+), respectively. Thus, the expression plasmids pET-20b(+)/cgt and pET-22b(+)/cgt were constructed and transformed into the host E. coli BL21(DE3).The culture conditions for extracellular production of the recombinantα-CGTase in E. coli BL21(DE3)(pET-20b(+)/cgt) were optimized in shaking flasks. The E. coli cells were cultured in TB medium at 25°C. After induction with 0.01 mM isopropylβ-D-thiogalactoside (IPTG) for 90 h, the activity ofα-CGTase in the culture medium achieved 22.5 U/ml, which was about 43-fold higher than that of the parent strain, P. macerans JFB05-01. In E. coli BL21(DE3)(pET-22b(+)/cgt), higher IPTG concentration (0.2 mM) was needed to induce the expression ofα-CGTase and, after 90h of induction, the activity ofα-CGTase achieved only 17.5 U/ml. Thus, E. coli BL21(DE3)(pET-20b(+)/cgt) was more suitable for the extracellular production of the recombinantα-CGTase. To the best of our knowledge, this is the first report on the extracellular production ofα-CGTase in E. coli.2. The optimum temperature for extracellular production of the recombinantα-CGTase in E. coli was 25°C. However, at 25°C, only very few recombinantα-CGTase was secreted into the culture medium within first 50 h, while most were accumulated in the periplasmic space.The extracellular secretion of the recombinant enzyme was suppressed at higher temperatures. The possible reason was that the precursor protein with a signal peptide aggregated as inclusion bodies in the inner side of inner membrane due to too high synthetic rate, which might subsequently block protein translocation across the inner membrane. Extracellular production of the recombinantα-CGTase could be stimulated by raising the induction temperature at the later stage of culture. The maximum extracellular production was achieved when the temperature was shifted to 30°C after 32 h of induction at 25°C. After 90 h, the activity was 32.5 U/ml, which is 1.45-fold higher than that at 25°C constant.3. The addition of glycine enhanced the extracellular secretion of the recombinantα-CGTase and markedly shortened the culture time. Especially in the culture with 150 mM glycine, theα-CGTase activity in the culture medium reached 23.5 U/ml at 40 h, which was 11-fold higher than that of the control culture at the same time. The productivity of the recombinant enzyme also reached the maximum value of approximately 0.60 U/ml/h at 36 h, which was 2.5-fold higher than that of the control culture at 80 h. The potential mechanism is considered to be the significantly increased membrane permeabilities of E. coli cells.However, glycine inhibited the growth of E. coli cells, which prevented further improvement in overall enzyme extracellular productivity. Ca2+ could remedy cell growth inhibition induced by glycine as demonstrated by significantly increased cell number and viability, reduced cell autolysis, and repaired cell morphology. In the culture with 150 mM glycine and 20 mM Ca2+, theα-CGTase activity in the culture medium reached 35.5 U/ml at 40 h of culture, which was 1.5-fold higher than that in the culture with glycine alone. The productivity reached the maximum value of approximately 0.90 U/ml/h at 36 h, which also was 1.5-fold higher than that in the culture with glycine alone.4. The recombinantα-CGTase with a C-terminal His-tag could be purified to homogeneity through a nickel affinity chromatography, but the typical yield of purified enzyme was very low. The recombinant and native enzymes were purified by a combination of ion-exchange and hydrophobic interaction chromatography and the relative high yield was obtained.The purifiedα-CGTase was a monomer in solution. The optimum cyclization reaction temperature of the recombinantα-CGTase was 45°C. It retained 50% of its initial cyclization activity after incubation for 8 h at 40°C, 1.25 h at 45°C, and 0.5 h at 50°C. The nativeα-CGTase had higher optimum temperature (50°C) and thermostability (t1/2, 50°C=0.8 h). The recombinant and native enzymes both showed the highest cyclization activity at pH 5.5 and were quite stable in the pH ranging from 6 to 9.5 and 6 to 10, respectively. The metal cofactor was not required for the function ofα-CGTase. However, the cyclization activity ofα-CGTase was inhibited by Hg2+, Ni2+, Fe2+ or Co2+, while the enzyme could be activated by some bivalent metal ions, especially Ca2+, Ba2+ or Zn2+.At the initial stage of enzyme conversion, the recombinant and nativeα-CGTases producedα-cyclodextrin as a main product. At the later stages, the proportion ofβ-cyclodextrin increased. Finally,β-cyclodextrin was the main product. The recombinant enzyme had a higher preference forα-cyclodextrin production than the native enzyme.The kinetics of theα-CGTase-catalyzed cyclization reaction can be fairly well described by the Hill equation and the Hill coefficient was higher than one, indicating the positive cooperativity of substrate binding.5. The mutations of Asp372 and Tyr89 at subsite ?3 in P. macerans CGTase could change cyclization activities and cyclodextrin product ratios, which indicated that the two residues at subsite ?3 played important roles in cyclodextrin product specificity and subsites ?3 was a key site for cyclodextrin product specificity.The replacement of Asp372 by lysine and Tyr89 by arginine enhanced significantlyα-cyclodextrin specificity of CGTase. Furthermore, the changes in cyclodextrin product specificity for the single mutants D372K and Y89R could be combined in the double mutant D372K/Y89R, which displayed a 1.5-fold increase in the production ofα-cyclodextrin, with a concomitant 43% decrease in the production ofβ-cyclodextrin when compared to the wild-type CGTase.These mutants with higherα-cyclodextrin specificity were more suitable for the industrial production ofα-cyclodextrin than the wild-type enzyme.6. Amino acid residue is one of the main residues located near subsites ?3. The nature of residue 47 has a clear discrimination between the different groups of CGTase, suggesting that the identity of residue 47 may affect cyclodextrin product specificity.The mutations of Lys47 in P. macerans CGTase could change cyclization activities and cyclodextrin productions, indicating the residue had important roles in cyclization reaction and cyclodextrin product specificity.All the mutations reducedα-cyclodextrin forming activity of CGTase, suggesting that Lys47 was very important forα-cyclization reaction, while the increase inβ-cyclodextrin forming activity could be achieved. Especially, the mutations of Lys47 into threonine, serine, or leucine converted P. macerans CGTase fromα-type intoβ/α-type. As a result, all the mutants displayed a shift in product specificity towards the production ofβ-cyclodextrin. Thus, these mutants were more suitable for the industrial production ofβ-cyclodextrin than the wild-type enzyme.更多还原