【作者】 龚方；

【导师】 池振明；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2008， 博士

【摘要】 菊粉作为植物的一种贮存性多糖,主要存在于如菊芋(或称洋姜,Helianthus tuberosus)、菊苣(Cichorium endivia)、大丽花(Dahlia pinnata)、牛蒡(Arctium lappa)等尚未开发利用的植物根茎中。菊粉酶是一种主要来源于微生物的多糖水解酶,它可以在一定的温度条件下水解菊粉为果糖和低聚果糖等,其水解产物在食品发酵、医药卫生等方面都有广泛的应用。菊粉酶的来源广泛,包括霉菌、酵母菌、细菌和放线菌等,但是对于海洋酵母菌产菊粉酶的情况了解甚少,本实验室获得了一株高产菊粉酶的海洋酵母菌strain 1,通过生物学鉴定方法确定该株海洋酵母菌属于季也蒙毕赤酵母(Pichia. guilliermondii)。本实验首先对季也蒙毕赤酵母(P. guilliermondii)strain 1产菊粉酶的液体发酵培养基和发酵条件进行了优化,发现其产酶的最优培养基为4%菊粉,0.5%酵母粉,初始pH值为8.0,海水配制,培养条件为接种量10%(v/v)(OD600nm=20.0),最适温度28 oC ,170 rpm培养,得到的最高菊粉酶活力达到了61.5U/mL。同时,对strain 1菊粉酶粗酶水解底物菊粉水解菊粉的产物进行了薄层层析检验,发现该酶能够充分水解菊粉,得到的终产物以单糖为主,证明了strain 1菊粉酶具有较高的外切菊粉酶活力。在最适产酶条件下培养,将获得的季也蒙毕赤酵母(P. guilliermondii)strain 1胞外菊粉酶粗酶进行超滤浓缩,通过分子筛SephadexTM G-75和阴离子交换层析柱DEAE Fast Flow分离纯化,纯化后蛋白酶SDS- PAGE凝胶电泳显示分子量约为50kDa。对纯化后的菊粉酶进行了一系列酶学性质的研究,发现纯化后的菊粉酶最适反应pH和最适反应温度分别为6.0和60oC,该酶对温度不敏感,并具有热稳定性,对pH较敏感,只在5-7范围内活力稳定。另外,很多金属离子对纯酶活力有一定程度的激活作用;当存在Hg2+、Mg2+、Ag+时,菊粉酶活明显降低。蛋白抑制剂PMSF和碘乙酸能够强烈抑制菊粉酶活力。纯化后的菊粉酶对底物菊粉的动力学常数Km值和Vmax分别是21.1 mg/mL和0.082 mg min-1蛋白,说明其与底物亲和力较高。纯化菊粉酶对底物菊粉也具有很高的外切菊粉酶活力,水解底物层析发现产物只有单糖。克隆得到了季也蒙毕赤酵母(P. guilliermondii)strain 1胞外菊粉酶基因INU的全长(登陆号EU195799),共1542bp,无内含子,根据DNA序列推导出该了菊粉酶前体肽的氨基酸序列,共514个氨基酸,推导分子量为58042Da。利用生物学软件分析菊粉酶基因INU及其上下游碱基序列,推导出其启动子和终止子位置及结构特点等。分析菊粉酶前体肽氨基酸序列,推测出N端有18个氨基酸残基的信号肽,并含有10个可能的N端糖基化位点,对菊粉酶前体肽氨基酸序列的保守区进行了分析,推测出约在20-300个氨基酸处具有糖苷酶家族N端特性。同时构建pPICZαAINU表达载体,将克隆得到的strain 1菊粉酶基因INU在巴斯德毕赤酵母真核表达体系中进行了功能表达。对重组酶进行了进行SDS-PAGE检测和Western blotting分析,得到了大小为60kD的重组酶条带,并通过免疫印迹证实了菊粉酶基因在真核表达系统中的表达。诱导48h的重组菊粉酶粗酶液酶活为30.7±0.12 U/ml,使用薄层层析方法检测重组酶对底物菊粉的水解作用,证明了重组酶对菊粉有一定的水解作用。同时还利用响表面方法(RSM)对海洋季也蒙毕赤酵母strain 1的高产菊粉酶突变株M-30的固体发酵条件进行了优化,得到最终的最优条件为初始湿度60.5%,接种量2.5%,麸皮:稻糠=0.42,30 oC培养,初始pH6.5。在最优条件下,突变株M-30固体发酵酶活达到455.9 U/gds,明显高于原始菌株(291.0 U/gds),说明通过诱变方法确实使突变株的酶活力明显得到了提高,具有稳定的较高的外切菊粉酶活力。综上所述,本实验对于海洋季也蒙毕赤酵母(P. guilliermondii)strain 1菊粉酶的研究较全面系统,对于开发利用海洋酵母菌菊粉酶具有积极意义。更多还原

【Abstract】 Inulin is a fructose polymer that occurs as a reserve carbohydrate in Jerusalem artichoke, dahlia tubers and chicory root. This polymer is a recognized source for the production of either ultra-high fructose syrups, with D-fructose content over 95% or for production of oligofructose syrups. Inulinase (β-2,1-D-fructan fructanohydrolase, EC 3.2. 1.7) targets on theβ-2,1 linkage of inulin and hydrolyzes it into fructose. Inulinase is produced by many microorganisms, including fungi, yeasts and bacterias. But little is known about inulinase produced by marine yeasts. Marine yeast strain 1 which was found to secrete a large amount of inulinase into the medium was selected from the collection culture of marine yeasts available in this laboratory. This marine yeast strain was identified to be a strain of Pichia guilliermondii according to the results of routine yeast identification and molecular methods.The optimal medium and cultivation conditions for inulinase production were determined first. Seawater containing 4.0% (w/v) inulin and 0.5% (w/v) yeast extract , cultivated under the optimal condition of pH 8.0, 28 oC and 170 rpm. Over 60 U ml-1 of inulinase activity was produced within 48 h of fermentation at shake flask level. A large amount of monosaccharides and a trace amount of oligosaccharides were detected after the hydrolysis, indicating that the crude inulinase had a high exoinulinase activity.The extracellular inulinase in the supernatant of cell culture of the marine yeast Pichia guilliermondii strain 1 was purified to homogeneity with a 7.2-fold increase in inulinase activity as compared to that in the supernatant by ultrafiltration, concentration, gel filtration chromatography (SephadexTM G-75) and anion-exchange chromatography (DEAE-Sepharose Fast Flow Anion-Exchange). The molecular mass of the purified enzyme was estimated to be 50.0 kDa. The optimal pH and temperature of the purified enzyme were 6.0 and 60 (C, respectively. The enzyme was activated by Mn2+, Ca2+, K+, Li+, Na+, Fe3+, Fe2+, Cu2+, and Co2+. However, Mg2+, Hg2+ and Ag+ acted as inhibitors in decreasing activity of the purified inulinase. The enzyme was strongly inhibited by Phenylmethanesulphonyl fluoride (PMSF), iodoacetic acid. The Km and Vmax values of the purified enzyme for inulin were 21.1 mg ml-1 and 0.082 mg min-1, respectively. A large amount of monosaccharides were detected after the hydrolysis of inulin.The inulinase structural gene was isolated from genomic DNA of P. guilliermondii strain 1. The gene had an open reading frame of 1542 bp long encoding an inulinase (accession number EU195799). The coding region of the gene had no intron. It encoded 514 amino acid residues of a protein with a putative signal peptide of 18 amino acids. The protein sequence deduced from the inulinase structural gene contained the consensus sequence of glycosyl hydrolases family 32 N terminal and ten conserved putative N-glycosylation sites. The inulinase encoding DNA was subcloned into pPICZαA expression vector and the resulting recombinant plasmid was named pPICZαAINU. The recombinant plasmid was expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum activity of 30.71 U/mL was obtained from supernatant of P. pastoris X-33 harboring pPICZαAINU. The crude recombinant inulinase had hydrolytic activity towards inulin.One mutant (M-30) with enhanced inulinase production was obtained by treating P. guilliermondii strain 1 with UV light and LiCl2. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant in solid-state fermentation. The initial moisture, inoculum, the amount ratio of wheat bran to rice bran, temperature, pH for maximum inulinase production by mutant M-30 were found to be 60.5%, 2.5%, 0.42, 30 oC and 6.50, respectively. Under the optimized conditions, 455.9 U/gram of dry substrate (gds) of inulinase activity was reached in the solid state fermentation culture of mutant M-30 whereas the predicted maximum inulinase activity of 459.2 U/gds was derived from RSM regression. Under the same conditions, its wild-type only produced 291.0 U/ gds of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far.更多还原