【作者】 王淑军；

【导师】 陆兆新；

【作者基本信息】 南京农业大学 ， 食品科学， 2008， 博士

【摘要】 α-淀粉酶为α-1,4-葡聚糖-4-葡聚糖水解酶(a-1,4-glucan-4-glucanohydrolase EC.3.2.1.1),是最重要的工业酶制剂之一,已被广泛应用在食品、发酵、纺织、造纸和制药等诸多行业。目前工业用α-淀粉酶主要来自嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)和地衣芽孢杆菌(Bacillus licheniformis),这些酶的最适作用条件为90℃和pH 6,存在的主要问题是热稳定性差,高于100℃和pH低于6.0时易失活,热稳定性依赖Ca2+。超嗜热微生物由于能产生在高温下活性高,且具有很好热稳定性的超嗜热酶,而成为研究的热点。本文主要对一株分离自深海热液口的超嗜热古菌HJ21进行生物学特性和分子鉴定、高温酸性α-淀粉酶发酵条件和酶学性质、α-淀粉酶基因的克隆和序列分析、αC-淀粉酶基因在大肠杆菌中的表达和重组酶性质以及重组大肠杆菌高密度高表达培养条件的研究,主要研究结果如下：1.对一株分离自深海热液口的极端厌氧的超嗜热古菌HJ21进行了生长特性研究和分子鉴定的研究。菌株HJ21直径为1-1.2μm,菌株生长温度范围60-94℃,菌株最适生长温度88℃；菌株生长pH范围为5.0-9.0,其最适生长pH范围为6.5；生长NaCl浓度范围为10-50g·L-1,最适NaCl浓度为20 g·L-1。该菌株在酵母粉、蛋白胨、胰蛋白胨和酪蛋白等培养基中生长良好；淀粉、麦芽糖、葡萄糖、蔗糖、纤维二糖、乳糖、糖原、明胶促进生长。菌株HJ21生长不需要硫,但没有硫菌株生长缓慢,而在培养基中添加硫会促进菌株生长,振荡培养比静止培养可显著减少硫的添加量。根据其形态特征、生理生化特性以及16S rRNA基因序列分析结果,鉴定HJ21菌株为Thermococcus siculi。目前国内外尚无Thermococcus siculi产高温α-淀粉酶的研究报道。2.对Thermococcus siculi产α-淀粉酶发酵条件和酶学性质研究。该菌株发酵9h后到达产酶高峰,产酶温度范围为60-90℃,其最适产酶温度为80℃；产酶pH范围为5.0-9.0,最适产酶pH为7.5；产酶NaCl浓度范围为5-40 g·L-1,最适NaCl浓度为25 g·L-1；菌株接种量5%(v·v-1)。糖原、淀粉、麦芽糖、酵母膏和蛋白胨促进产酶。该菌株产生的α-淀粉酶的分子量为51.4 KDa,最适作用温度为95℃,在100℃仍有60%的酶活力。酶在90℃的半衰期为5h,在100℃2h仍有40%的残余酶活,酶的热稳定性不依赖Ca2+。酶的最适作用pH 5.0,在pH 4.5仍有80%的酶活力,pH在5.0-7.0较稳定(80℃2h)。金属离子Mg2+、Sr2+、K+、Na+对酶有激活作用,Cu2+、Pb2+、Hg2+、Zn2+、Al3+对该酶均有抑制作用。1mmol·L-1和5mmol·L-1 N-溴代丁二酰亚胺对酶的活性具有较强抑制作用。3.克隆了T. siculi HJ21 a-淀粉酶基因并进行了序列分析。根据NCBI中已报道的超嗜热古菌α-淀粉酶的基因序列设计引物,通过聚合酶链式反应(PCR)的方法获得了T. siculi HJ21 a-淀粉酶基因的保守序列,采用SiteFinding-PCR技术扩增获得T. siculi HJ21 a-淀粉酶基因两端的序列。通过序列分析和拼接获得了1374bp完整的T. siculi HJ21 a-淀粉酶基因。经生物信息学软件分析该基因与T. hydrothermalis和T. sp. OGL-20P的耐高温α-淀粉酶亲缘关系最近,相似度达到95%以上。该α-淀粉酶基因片段包含与4个其他超嗜热古菌的α-淀粉酶相同的保守区域。将该α-淀粉酶氨基酸序列提交http://swissmodel.expasy.org蛋白质在线分析服务器。表明T. siculi HJ21 a-淀粉酶具有典型的(α/p)8桶状结构形成一个紧密的球体结构。通过分析密码子的使用情况得知,该α-淀粉酶存在着密码子的兼并性和使用的偏向性问题。4. T. siculi HJ21 a-淀粉酶基因在大肠杆菌中得到表达并研究了重组酶的性质。构建了在N端添加His6标签的表达载体pEt-28a- His6-amy,获得了重组大肠杆菌。重组酶经过加热处理、DEAE离子交换层析、Ni亲和层析后和胶回收的方法得到纯化。对重组酶酶学性质研究发现,该酶的分子量约为50 KD。重组α-淀粉酶的最适作用温度90℃,在95℃和100℃分别有90%和70%的酶活力。重组α-淀粉酶热稳定性不依赖于Ca2+,酶在80、90、100℃分别保温5h后,其残余酶活分别为82%、77%和69%。重组酶的作用pH范围为pH 4-9,最适作用pH为5.0-5.5,在pH 4.5有81%残余酶活。该酶在100℃1 h,pH 5-7.5的范围内有较好的热稳定性。金属离子K+、Na+对酶有激活作用,Hg2+、Pb2+、Al3+、Cu2+、Fe3+和Zn2+等能够强烈的抑制酶活性。1 mmol·L-1和5 mmol·L-1 N-溴代丁二酰亚胺对酶的活性具有较强抑制作用。该酶的Km为45mg·mL-1, Vmax为9 mg·ml-1·min-1。该重组酶分解马铃薯淀粉的主要产物是麦芽糖和麦芽三糖。5.研究了在摇瓶条件下重组大肠杆菌高密度高表达培养条件：接种量1%,装液量为20%(v·v-1),pH 7.0,温度37℃,培养菌液的OD600达到1.0(约4h),再加入浓度为1 mmol·L-1的诱导剂IPTG,诱导时间4 h。葡萄糖在0.5 g·L-1,甘油浓度在0.5 g·L-1,蛋白胨的浓度在10 g·L-1,酵母粉浓度在5 g·L-1,磷酸盐浓度在10mmol·L-1,硫酸镁浓度为0.6 g·L-1。利用响应面方法对重组菌产α-淀粉酶的发酵条件进行优化。选择培养基装液量、pH、诱导时机和诱导时间4因素,分别在3个水平上进行考察,优化的发酵条件为：培养基装液量为23.57%,pH为6.4,培养4.03 h后进行IPTG诱导并持续4.16 h。重组菌酶比活力为0.4 U·mg-1,较优化前提高了1.56倍,目的蛋白表达量为8.75%,较优化前提高了1.64倍。更多还原

【Abstract】 a-Amylase(EC 3.2.1.1), endo-acting enzyme that hydrolyzes starch by cleaving-1,4-glucosidic linkages at random sites, is one of the most important commercial enzymes widely used in starch-processing, brewing, alcohol production, textile, and other industries. The most thermostable a-amylase in industry is produced from Bacillus licheniformis. This enzyme operates optimally at 90℃and pH 6, and it requires addition of calcium ion (Ca2+) for its thermostability. Hyperthermophiles that optimally grow at temperatures above 80℃, have attracted many researchers’ attention as they are a source of enzymes with thermoactive and outstanding thermostability. In this paper, we report the biological characteristics and molecular identification of a hyperthermophilic archaeon (strain HJ21), the production and characterization of an thermoactive a-amylase produced by this strain, the cloning and sequence analysis of a-amylase gene, the expression in E. coli and the characterization of recombinant a-amylase, and cultivation optimization of high cell density and high expression for the recombinant E. coli. Main study results as following:1. The identification and characterization of extremely anaerobic hyperthermophilic archaeon strains HJ21, isolated from a deep-sea hydrothermal vent, has been studied. The cells were round to slightly irregular cocci,1 to 1.2μm in diameter. The isolate grew between 60 and 94℃with an optimum around 88℃. The pH range for growth was 5.0 to 9.0, with an optimum around 6.5～7.0. The NaCl range for growth was 10 to 50 g·L-1, with an optimum around 20 g·L-1. Strain HJ21 grew much better in the medium with yeast extract, peptone, tryptone, casein and HJ21 was capable of growing on starch, maltose, glucose, sucrose, cellobiose, lactose, glycogen. The growth of strain HJ21 could be enhanced by elemental sulfur. Cells were also grown in the absence of elemental sulfur; however, the growth was very slow. The growth of strain HJ21 could be enhanced by elemental sulfur. The amount of elemental sulfur added in medium in shaken cultures was less than the medium in stable cultures. On the basis of 16S rDNA sequence comparisons, in combination with morphology, physiological characteristics, it is identified as Thermococcus siculi. At present the research about thermoactive a-amylase from Thermococcus siculi have not reported.2. The production and characterization of a-amylase from T. siculi HJ21 has been studied. Maximum enzyme production was achieved after 9 h cultivation. The temperature of a-amylase production were between 60 and 90℃with an optimum around 80℃. The pH range for production of amylase was 5.0 to 9.0, with an optimum around 7.5. The NaCl range for production of amylase was 10 to 50 g·L-1, with an optimum around 2.5 g·L-1. Inoculation amount of the strain was 5%(v·v-1). Addition of yeast extract, peptone, glycogen, starch and maltose enhanced enzyme production. The strain HJ21 produced extracellular thermostable and acid-stable a-amylase. The molecular weight of the enzyme was estimated to be 51.4 KDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The a-amylase exhibited maximal activity at 95℃. The half-life of the enzyme was 5 h at 90℃, more than 40 of the enzyme activity remained after 2h of incubation at 100℃. The enzyme did not require Ca2+ for thermostability. The optimal pH of a-amylase was observed at 5.0, and more than 80% amylase activity of the maximum enzyme activity was detected at pH 4.5. The enzyme was shown to be stable in the broad range of pH 5.0 to 7.0 (at 80℃2h). The enzyme was activated by Mg2+, Sr2+, K+, Na+. It was inhibited by Cu2+、Pb2+、Hg2+、Zn2+、Al3+. N-Bromosuccinimide (at 1 mmol·L-1 and 5 mmol·L-1) showed a significant repression to enzyme activity.3. Thisα-amylase gene was cloned and its nucleotide sequence analysed The gene sequence between the conserved region was acquired by PCR using the primers designed based on the sequence of that of hyperthermophilic archaeon deposited in the GenBank. The upstream and downstream of T. siculi HJ21α-amylase gene was acquired by siteFind-PCR. After Sequence analysis, the complete T. siculi HJ21α-amylase gene about 1374bp was obtained through the juncture of them. The similarity between theα-amylase of T. siculi HJ21 and that of T. hydrothermalis and T. sp. OGL-20P was over 95%. Four highly conserved regions, possibly forming the active site, have been identified in a-amylases. These regions were also found in T. siculi HJ21 a-amylase gene and showed great similarities with those of other Thermococcales a-amylases secreted by thermophilic organisms. The gene sequence of was submitted to http://swissmodel. expasy. org to predict the structure of the a-amylases. T. siculi HJ21 a-amylase was single and classicα/βstructure. There were problems of degenerate and preference of codon usage in a-amylase through analysis the sequence of amimo acid.4. The gene encoding a-amylase from T. sicili HJ21 was expressed in E. coli and the characterization of the recombinant enzyme was studied. The expression plasmid pEt-28a-His6-amy was constructed. The plasmid was transformed into E. coli BL-21(DE3). The recombinant a-amylase was purified to homogeneity by heat treat, DEAE-sepherose ion exchange chromatography, Ni affinity chromatography and gel recovery. The molecular weight of the enzyme was estimated to be 50 KD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum temperature for the activity was 90℃. More than 90% and 70%α-amylase activity of the maximum enzyme activity was detected at 95℃and 100℃respectively. The enzyme did not require Ca2+ for thermostability.82%、77% and 69% of the enzyme activity remained after 5 h of incubation at 80,90 and 100℃respectively. The amylase exhibited maximal activity at pH 5.0 to 5.5 and was stable in the range of pH 5.0 to 7.5 at 100℃,1 h. The enzyme was activated by K+, Na+. It was inhibited by Hg2+、Pb2+、Al3+、Cu2+、Fe3+、Zn2+. N-Bromosuccinimide (at 1 mmol·L-1 and 5 mmol·L-1) showed a significant repression to enzyme activity. The Km and Vmax vales of the amylase for soluble starch were 45 mg·mL-and 9 mg·ml-1·min-1, respectively. The amylase hydrolyzed soluble starch to produce maltose and maltotriose as the main products.5. Cultivation optimization of high cell density and high expression for the recombinant E. coli in shaking condition. Inoculation amount 1%(v·v-1), liquid filling quantity,20%(v·v-1);pH,7.0;temperature,37℃;inducement opportunity, OD6001.0, amount of IPTG,1 mmol·L-1; inducement time,4 h. The optimal concentration in medium were:glucose,0.5g·L-1; glycerol,0.5g·L-1;peptone,10 g·L-1; yeast extract,5 g·L-1; KH2PO4,10 mmol·L-1;MgSO4,0.6 g·L-1. Response surface design was used to optimize the fermentation condition for a-amylase production by the recombinant E. coli. Facters such as liquid filling quantity, pH, inducement opportunity and inducement time were selected and tested at three levers, respectively. The optimum conditions of the parameters were liquid filling quantity 23.57%, pH 6.4, inducement opportunity 4.03 h and inducement time 4.16 h. After Optimization, specify amylase activity was 10.4 U·mg-1, increases by 1.56 times, and the amount of the protein expression was 8.75%, increases by 1.64 times.更多还原