【作者】 韩亮；

【导师】 杨瑞金；

【作者基本信息】 江南大学 ， 食品科学与工程， 2016， 硕士

【摘要】 乳果糖是一种功能性低聚糖,具有热值低、安全性高、不发生美拉德反应等多种特性,在医药、食品保健品和动物饲料领域有着广泛的应用。传统酶法制备乳果糖是利用酶的转糖苷作用将果糖连接到乳糖的半乳糖基上,转化率低,目前的产量无法满足工业化生产需要。来源于Caldicellulosiruptor saccharolyticus的纤维二糖差向异构酶(Cs CE),能够催化单一底物乳糖为乳果糖,是目前最高效的乳果糖制备用酶。本课题组在前期研究中将Cs CE在大肠杆菌胞内表达,并通过随机突变筛选到突变体C4-C5,其比酶活为野生型Cs CE的2.8倍。本研究首次将Cs CE在毕赤酵母(Pichia pastoris)表达系统中异源表达,并采用密码子优化以及发酵条件优化等方法,提高重组菌株产酶能力;构建突变体重组菌株,获得高酶活的毕赤酵母重组菌;进一步研究了野生型Cs CE(Cs CEm)与突变体(Cs CEmt)酶学性质,并对重组酶催化乳糖转化为乳果糖进行初步探讨。主要研究结果如下:首先,根据Cs CE核苷酸序列和毕赤酵母密码子偏好性,合成密码子优化后的基因Cs CEm。核苷酸序列比对结果显示,Cs CEm与野生型基因Cs CE具有78.09%的同源性。将Cs CEm连接到表达载体p PIC9K上,测序正确后转化至毕赤酵母GS115中,然后经G418筛选、微孔板筛选得到具有较高酶活的重组酵母转化菌株GS115/Cs CEm 4-19。经发酵条件优化,在诱导144 h后最高酶活为0.42 U/m L,上清蛋白浓度为204.1±5.67 mg/L。与密码子优化前重组毕赤酵母最高酶活(0.12 U/m L)相比,酶活提高3.5倍。其次,在密码子优化的基础上,对R5M,I52V,A12S,K328I和F231L 5个氨基酸位点进行突变,突变后的基因Cs CEmt与表达载体p PIC9K连接、转化至毕赤酵母GS115中,筛选到具有较高酶活的重组酵母转化菌株GS115/Cs CEm 7-14。诱导144 h后,最高酶活为2.40 U/m L,为目前已报道的Cs CE最高酶活,上清蛋白浓度达到368.73±6.79mg/L。与定点突变前重组毕赤酵母最高表达酶活(0.42 U/m L)相比,酶活提高5.7倍。然后,采用硫酸铵盐析与Q-Sepharose F.F阴离子柱层析两步纯化野生型Cs CE与突变体,得到电泳纯的重组酶,其表观分子量为45 k Da,与理论值47.3 k Da相近。最终纯化得到的Cs CEm比酶活由2.43 U/mg提高到5.6 U/mg,Cs CEmt比酶活由5.52 U/mg提高到14.75 U/mg。与野生型Cs CE相比,突变体比酶活提高1.64倍。最后,重组酶酶学性质研究表明:与野生型Cs CE相比,突变体最适作用温度范围更广、热稳定性更高。米氏常数Km由120.27 mmol/L减小到74.24 mmol/L,Kcat/Km增加,表明突变后对底物乳糖的催化效率提高。金属离子Zn2+、Cu2+、Ni2+、Co2+和Fe3+对野生型Cs CE与突变体具有强烈抑制作用,残留酶活均在5%以下,其他金属离子和EDTA对酶活影响不明显。采用500 g/L乳糖溶液,重组酶添加量为20 U/m L,80℃反应4.5 h,突变体催化乳糖合成乳果糖的转化率达到63.7%,比野生型Cs CE提高7.5%,具有更好的工业化应用潜力。更多还原

【Abstract】 Lactulose is one kind of functional oligosaccharides, which has many kinds of characteristics, such as low calorific value, high safety, without Maillard reaction and so on. So lactulose is widely used in the pharmaceutical, food and health care products and animal feed industries. The role of traditional enzymatic synthesis for lactulose is to link fructose to galactose based on lactose by transglycosidation. But its conversion rate was low, and the current output can not meet the needs of industrial production. Cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus(CsCE) can catalyze the single substrate lactose into lactulose, and was found to be the most efficient enzyme for the enzymatic synthesis of lactulose. Preliminarily, the research group expressed Cs CE in E.coli. And through random mutation screening, the mutant C4-C5 was gotten,which specific enzyme activity was 2.8 times more than that of the wild type CsCE.In this study, CsCE was firstly expressed in Pichia pastoris. To improve the production of CsCE, codon optimization, site directed mutagenesis and fermentation condition optimization method was used. After expressed in P. pastoris, the enzymatic properties of wide type CsCE(CsCEm) and mutant type CsCE(CsCEmt) were studied, and catalytic action for convering lactose to lactulose by the recombinant enzyme was preliminarily explored. The experimental results obtained were briefly described as follows:Firstly, according to the nucleotide sequence of CsCE and the codon preference of P. pastoris, the gene CsCEm was synthesized. The results of nucleotide sequence alignment showed that CsCEm had 78.09% homology with the wild type gene CsCE. Then the synthesized CsCEm gene was connected with the expression vector pPIC9 K and transformed into P. pastoris GS115 after sequencing.Then by G418-plate and microplate screening,the strain GS115/CsCEm4-19 was screened with the highest enzyme activity of the recombinant yeast. After optimization of fermentation conditions, the highest enzyme activity reached at 0.42 U/mL after induction for 144 h, and the concentration of supernatant protein was 204.1±5.67 mg/L. The enzyme activity was increased 3.5 times than that of the recombinant P. pastoris before optimization(0.12 U/m L).Secondly, based on codon optimization, five amino acid sites of R5 M, I52 V, A12 S, K328 I and F231 L were site-directed, and the mutant gene CsCEmt was connected with expression vector pPIC9 K. Then, the recombinant vector was transformed into P. pastoris GS115 after sequencing, and the GS115/CsCEm 7-14 was screened with highest enzyme activity of recombinant yeast transformed. After optimization of fermentation conditions, the highest enzyme activity reached at 2.40 U/mL after the induction for 144 h, which was the highest activity of Cs CE reported until now. And the concentration of supernatant protein was about 368.73±6.79 mg/L. Compared with the recombinant P. pastoris gene optimizated only, the enzyme activity of site-directed mutagenesis was 5.7 times higher than that(0.42 U/m L).Then, the wide type CsCE and the mutant were purified by ammonium sulfate salting and Q-Sepharose F.F anion column chromatography.And one apparent stripwhich showed on the molecular mass of 45 kDa by SDS-PAGE, which was similar to the theoretical value of 47.3 kDa. The final specific activity of purified CsCEm was increased from 2.43 U/mg to 5.6 U/mg, and the CsCEmt was increased from 5.52 U/mg to 14.75 U/mg. Compared with the wild type CsCE, the activity of mutant was 1.64 times higher than that.Finally, enzyme properties of the recombinant enzyme showed that the most suitable temperature range of the mutant was more extensive and the thermal stability was higher compared with the wild type CsCE. The Michaelis constant Km decreased from 120.27 mmol/L to 74.24 mmol/L and Kcat/Km increased, which indicated that the recombinant enzyme affinity for substrate lactose was enhanced after mutation. The result of metal ions effect for wide type CsCE and mutant showed that Zn2+、Cu2+、Ni2+、Co2+、Fe3 + had a strongly inhibitory effect, which residual enzyme activity was less than 5%. But the other metal ions and EDTA was not obvious for the recombinant enzyme activity. Under the condition of 500 g/L lactose solution, recombinant enzyme of 20 U/mL was added and reacted at 80℃. The conversion rate for lactulose based on lactose was 63.7%, and increased by 7.5% compared with the wild type CsCE. The mutant was more potential for lactulose synthesis in industrial application.更多还原