普鲁兰酶的产生菌筛选及其表达与分泌调控

Screening on Producing Strains, Expression and Secretion Regulation of Pullulanase

作者: 专业:发酵工程 导师:徐岩 年度:2013 学位:博士 

关键词
大肠杆菌 克雷伯氏菌 普鲁兰酶 异源分泌表达 自诱导——双阶段温度调控策略

Keywords
auto-induction-two stage temperature control strategy, Escherichia coli, heterologously secretory expression, Klebsiella, pullulanase
        普鲁兰酶(pullulanase, EC3.2.1.41)是一种可以特异地水解普鲁兰多糖、淀粉及相关分支多糖中α-1,6糖苷键的淀粉脱支酶。由于它对α-1,6糖苷键的特异水解功能,普鲁兰酶被广泛地应用于淀粉糖加工工业、医药领域、饲料、啤酒及白酒酿造等工业中。但是,工业用普鲁兰酶由于异源表达菌株的分泌性能较差,产酶效率过低等因素,在我国极大地限制了普鲁兰酶的规模化工业生产,长期依赖进口。为此,本研究针对普鲁兰酶工业化生产为目标,从普鲁兰酶生产菌株的筛选入手,对采自于全国多地的数十个土壤样品进行了普鲁兰酶生产菌株的筛选。之后,利用分子生物学技术对其进行鉴定并克隆其中的普鲁兰酶编码基因,成功地构建了高效表达分泌重组普鲁兰酶的工程菌株。最后,通过自诱导培养、分泌调控等策略将胞外普鲁兰酶的产量提高了270多倍,达到97U mL-1。(1)筛选得到了一株生产胞外普鲁兰酶的微生物菌株。为克服普通筛选方法工作量大、目标不明确、操作繁杂的缺点,本研究采用平板显色法和普鲁兰沉淀法相结合的策略,对32个土壤样品进行了筛选。通过平板显色的方法缩小了筛选目标,共筛选得到60多株具有分解红色普鲁兰能力的微生物菌株。根据透明圈大小对这些菌株进行分类之后,选择透明圈与菌落大小之比大于2的菌株进行验证和液体培养基中的复筛。最终得到一株产酶能力较高的微生物菌株。经过生理生化及分子遗传学鉴定,将其鉴定为克雷伯氏菌SHN-1(Klebsiella sp. SHN-1)。向NCBI提交其16S rDNA序列,获得数据库登记号为:HM037179。通过对野生菌株产酶条件的初步探索,最终将该普鲁兰酶生产菌株的产酶能力提升到10.98U mL-1。(2)构建了一株可以高效生产胞外普鲁兰酶的大肠杆菌的工程菌株。虽然胞外分泌对于普鲁兰酶的规模化生产意义重大,但是,众多研究者构建的普鲁兰酶生产菌株都不具备分泌能力。考虑到大肠杆菌与克雷伯氏菌的亲缘关系及野生菌株生产胞外普鲁兰酶的能力,本研究根据NCBI上公布的克雷伯氏菌属的普鲁兰酶编码基因设计引物,克隆了Klebsiella sp. SHN-1的普鲁兰酶及信号肽的编码序列(NCBI登陆号为JX087429),以pET-28a(+)为载体构建了重组表达质粒pET-28a(+)-pulA,转化大肠杆菌后获得了重组大肠杆菌E. coli BL21(DE3)/pET-28a(+)-pulA。当该重组大肠杆菌在LB培养基中生长时,在IPTG的诱导下,重组普鲁兰酶可以得到有效表达并分泌到细胞外部。该重组菌的这些生产特性展现出了它作为工业用生产菌株的潜力。(3)构建了信号肽编码序列删除的表达质粒pET-28a(+)-pulA-sig-。有研究表明,为了实现重组普鲁兰酶在大肠杆菌中的高效分泌表达,必须同时克隆野生克雷伯氏菌中与普鲁兰酶分泌相关的分泌基因(包括信号肽编码序列)。然而本研究在仅克隆了普鲁兰酶的编码基因及其信号序列的情况下就实现了重组普鲁兰酶在大肠杆菌中的高效分泌表达。为了考察普鲁兰酶编码基因内部与分泌相关的信号序列,本研究构建了信号肽编码基因删除的表达质粒pET-28a(+)-pulA-sig-。当在LB液体培养基中培养该工程菌时,加入诱导物IPTG,重组普鲁兰酶仍然能够得到表达并分泌到发酵液中。随后对重组普鲁兰酶在胞外、胞膜、周质空间和胞内部分分布的考察表明,有高达65.49%重组普鲁兰酶被释放到细胞外部,这说明重组普鲁兰酶在大肠杆菌中的分泌与信号肽的存在关系不大。通过对普鲁兰酶成熟肽序列的分析发现,普鲁兰酶编码基因的内部存在着两个与蛋白分泌相关的序列,这可能是重组普鲁兰酶在没有信号肽的情况下仍然能够被释放到细胞外部的原因。随后,以该工程菌为研究对象,对其在LB培养基中以IPTG为诱导物生产胞外普鲁兰酶的条件进行了研究。最终,本研究将胞外重组普鲁兰酶的产量提高到了10U mL-1左右的水平。接下来,本研究又通过镍柱对重组普鲁兰酶进行纯化并考察了该重组普鲁兰酶的部分酶学性质,确定其最适作用温度和pH分别为55℃和5.0。当以纯酶作用于1%的普鲁兰多糖的溶液时,得到的主要产物为麦芽三糖,再次证明该酶是I型普鲁兰酶。(4)采用涉及可视化筛选、自诱导培养和温度调控的组合策略生产高水平的胞外重组普鲁兰酶。重组蛋白表达水平的下降会导致最终目标蛋白产量的下降,为此,本研究提出了可视化筛选策略来考察各个克隆子的产酶能力。在此基础之上,针对发酵过程中细胞浓度过低和普鲁兰酶胞内积累严重的问题,提出了自诱导——双阶段温度调控策略,使胞外普鲁兰酶产量提高到50U mL-1。另外,细胞渗透物的使用也有效地提升了胞外重组普鲁兰酶的产量,达到68.23U mL-1。最后,在7L NBS发酵罐上进行了重组普鲁兰酶的分批发酵和分批-补料发酵实验,在进一步提高细胞密度的基础上,又将胞外普鲁兰酶的产量提高到97U mL-1。本课题通过开展系统的研究工作,极大地提高胞外普鲁兰酶的产量。为了得到一株高效生产胞外普鲁兰酶的微生物菌株,本研究提出了平板显色与普鲁兰多糖沉淀相结合的策略。通过构建高效生产胞外普鲁兰酶的工程菌株发现了普鲁兰酶在大肠杆菌中的信号肽非依赖型分泌现象,为研究蛋白的高效分泌提供了参考和理论依据。根据重组蛋白的表达与分泌的特点,本研究又提出了高效生产胞外普鲁兰酶的自诱导——双阶段温度调控策略,结合细胞渗透物的使用,极大地提高了工程大肠杆菌生产胞外重组普鲁兰酶的能力。因此,本研究的开展将会为今后重组普鲁兰酶的规模化生产提供重要的理论指导。
    Pullulanase (EC3.2.1.41) is a debranching enzyme, which specifcally cleaves α-1,6glycosidic bonds in pullulan, starch and other related amylaceous polysaccharides. Hence,pullulanase can be employed to break down starch to produce glucose, fructose, maltosesyrups, and amylose, in conjunction with or without α-amylase, β-amylase, glucoamylase. Inaddition, pullulanase could also be used in the industry of pharmaceuticals, feed, brewing etc.to enhance the utilization rate of starch. However, low yield and productivity have been thebottleneck for the large-scale production and widespread application of pullulanase.In order to obtain pullulanase-producing microbes of high efficiency, this study beganwith the screening of pullulanase producers from the soil samples taken from severalprovinces in China. Subsequently, an engineered Escherichia coli was constructed, whichcould produce extracellular pullulanase with high efficiency. For the sake of improved yield, acombined strategy involving auto-induction, temperature regulation, and osmolyte additionwas developed. As a result, its application greatly enhanced the yield of extracellularpullulanase.(1) A pullulanase-producing strain was obtained. In order to overcome the problem oftraditional screening method, such as heavy workload and complex operation, a plate assay,based on red-pullulan, was employed to reduce the scope of screening. Subsequent abilityconfirmation of the isolates was carried out with the traditional screening method.Consequently, more than60isolates were obtained, which presented the ability to de-color thered-pullulan. In order to achieve an excellent strain, the isolates with haloes2times thecolonies were subjected to the second round of screening. Fortunately, an isolate with thehighest capability was obtained, which was identified as Klebsiella sp. SHN-1(GenBankAccession Number: HM037179). Under the optimized fermentation condition, the Klebsiellasp. SHN-1could produce about10.98U mL-1extracellular pullulanase.(2) An engineered E. coli BL21(DE3) with the ability to produce extracellularpullulanase was constructed. Although extracellular production is important for the large-scaleproduction of pullulanase, most constructed engineered strains produced pullulanaseintracellularly. Considering the close relationship between E. coli and Klebsiella sp. SHN-1, aprimer pair was designed based on the published pullulanase sequence to clone thepullulanase encoding gene. As a result, a DNA fragment of3000bp was obtained, whichshared99%homogeneity with that from K. variicola At-2and K. pneumoniae342.Subsequently, the sequence was subjected to GenBank (Accession Number: JX087429).When the DNA fragment was cloned into E. coli BL21(DE3) with the plasmid of pET-28a(+)as expression vector, recombinant pullulanase was detected in the supernatant of culture brothin the presence of IPTG.(3) An expression vector without signal peptide sequence was constructed. Numerousstudies suggested that the secretion of pullulanase from Klebsiella in E. coli required theexistence of secretion-related genes, including the signal peptide sequence. In order to explorethe reasons of pullulanase secretion in E. coli, a signal peptide-deleted plasmid was constructed. However, the engineered E. coli BL21(DE) carrying the expression vector couldstill express the gene encoding pullulanase and release the product into surroundingenvironment. The distribution of recombinant pullulanase in subcellular fractions, such as cellmembrane, cytoplasm, petriplasmic space, cell-associated fraction was also investigated. Theresult showed that up to65.49%recombinant pullulanase was secreted, indicating the releaseof recombinant pullulanase was not close to the existence of the signal peptide sequence.Analysis on the sequence of mature pullulanase suggested two inner regions might beresponsible for the secretion of pullulanase. Subsequently, the fermentation conditions of thisstrain in LB medium were optimized to enhance the production of extracellular pullulanaseand10U mL-1extracellular pullulanase was obtained. The recombinant pullulanase was alsopurified by one-step affinity chromatography. Investigation on the purified pullulanaseshowed its optimum reaction pH and temperature were5.0and55oC, respectively. When itwas employed to hydrolyze1%pullulan, maltotriose as the main end products was obtained,demonstrating the property of type I pullulanase.(4) The yield of extracellular pullulanase was enhanced by using a combined strategyinvolving the visible screening method, auto-induction, temperature control strategy. In orderto ensure the stability of the engineered strain, colonies used to carry out the production ofpullulanase were evaluated with the visible screening method. Subsequently, auto-inductionmethod was employed to simplify the production of pullulanase and increase the cell densityof culture. Results showed that the yield of extracellular pullulanase was improved to50U mL-1. Additionally, the ability of the engineered strain was further improved to68.23U mL-1by applying various natural osmolytes. Finally, pullulanase production was performedin a7L fermentor with batch and fed-batch strategy. As a result, the yield of extracellularpullulanase was elevated to97U mL-1.The yield of extracellular pullulanase was greatly improved by performing systematicresearch work. An isolate producing extracellular pullulanase was obtained with color platemethod and pullulan precipitation method. During the construction of an engineered E. coliproducing extracellular pullulanase, the signal peptide-independent secretion of pullulanase inE. coli was discovered. This observation provided the study of effective proteins secretionwith reference and theoretical basis. According to the property of recombinant proteinexpression and secretion, auto-induction-two stage temperature control strategy wasdeveloped. By combination with osmolytes, the yield of extracellular pullulanase was greatlyincreased. Therefore, the study presented theoretical guideline for the large-scale productionof pullulanase in the future.
        

普鲁兰酶的产生菌筛选及其表达与分泌调控

摘要3-5
Abstract5-6
第一章 绪论11-30
    1.1 普鲁兰及普鲁兰水解酶11-14
        1.1.1 普鲁兰及普鲁兰水解酶11-13
        1.1.2 普鲁兰酶13-14
    1.2 普鲁兰酶的微生物生产及酶学特性14-19
        1.2.1 普鲁兰酶的自然分布14-15
        1.2.2 普鲁兰酶的底物特异性15
        1.2.3 普鲁兰酶与其他淀粉酶脱支酶的差异15-17
        1.2.4 普鲁兰酶的工业应用17-19
    1.3 普鲁兰酶的编码基因及蛋白质分子结构19-21
    1.4 微生物普鲁兰酶的异源表达21-22
    1.5 高效产酶策略22-25
        1.5.1 自诱导培养22-23
        1.5.2 高密度发酵23-25
    1.6 普鲁兰酶研究中存在的科学问题及本研究的意义25-28
        1.6.1 关键科学问题25-27
        1.6.2 本研究的目的和意义27-28
    1.7 研究思路和内容28-30
第二章 普鲁兰酶生产菌株的筛选、鉴定及产酶特性研究30-49
    2.1 前言30
    2.2 材料与方法30-35
        2.2.1 土壤样品30
        2.2.2 试剂及仪器30
        2.2.3 培养基与贮液30-31
        2.2.4 微生物的培养方法及培养条件31-32
        2.2.5 酶样的准备、酶活及细胞生物量的测定32-33
        2.2.6 产酶菌株的鉴定33-34
        2.2.7 菌株产酶条件的探索34-35
    2.3 结果与讨论35-48
        2.3.1 普鲁兰酶生产菌株的初步筛选35-37
        2.3.2 产酶菌株在液体培养基中复筛37-38
        2.3.3 产酶菌株的鉴定38-41
        2.3.4 Klebsiella sp. SHN-1 产酶条件的研究41-48
    2.4 本章小结48-49
第三章 重组普鲁兰酶生产菌株的构建与表达分析49-61
    3.1 前言49
    3.2 材料与方法49-54
        3.2.1 菌种与质粒49-50
        3.2.2 培养基与溶液50
        3.2.3 主要试剂和仪器50-51
        3.2.4 Klebsiella sp. SHN-1 的培养及基因组 DNA 的提取51
        3.2.5 普鲁兰酶编码基因的克隆51
        3.2.6 PCR 扩增产物的回收、纯化及浓缩51-52
        3.2.7 PCR 产物与 T-Vector 连接及测序52
        3.2.8 外源基因与质粒 pET-28a 的连接52-53
        3.2.9 感受态细胞的制备及 DNA 转化53
        3.2.10 阳性克隆的挑取及验证53-54
        3.2.11 工程菌株的诱导表达54
        3.2.12 工程菌株胞内外蛋白的 SDS-PAGE 分析54
        3.2.13 酶活及细胞浓度的测定54
        3.2.14 亚细胞组份的制备54
    3.3 结果与讨论54-60
        3.3.1 Klebsiella sp. SHN-1 基因组 DNA 的提取及普鲁兰酶编码基因的克隆54-57
        3.3.2 表达系统 E. coli BL21(DE3)/pET-28a-pul 的构建57-58
        3.3.3 工程菌的诱导表达58-59
        3.3.4 重组蛋白在亚细胞部分中的分布59-60
    3.4 本章小结60-61
第四章 工程菌普鲁兰酶分泌调控研究61-72
    4.1 前言61
    4.2 材料与方法61-63
        4.2.1 菌种与质粒61
        4.2.2 培养基与溶液61-62
        4.2.3 主要试剂和仪器62
        4.2.4 质粒 T-pul 的提取62
        4.2.5 成熟普鲁兰酶编码基因的克隆62
        4.2.6 无信号肽重组表达质粒的构建62
        4.2.7 重组普鲁兰酶生产条件的考察62-63
        4.2.8 重组普鲁兰酶的纯化63
        4.2.9 重组普鲁兰酶酶学性质的考察63
    4.3 结果与讨论63-71
        4.3.1 无信号肽编码序列重组表达质粒的构建63-65
        4.3.2 质粒 pET-28a-pul-sig-在大肠杆菌中的诱导表达及重组蛋白分布研究65-67
        4.3.3 工程菌 E. coli BL21 (DE3)/pET-28a-pul-sig-产酶条件的研究67-69
        4.3.4 重组普鲁兰酶纯化及酶学性质研究69-71
    4.4 本章小结71-72
第五章 基于自诱导与温度控制策略生产胞外普鲁兰酶72-90
    5.1 前言72
    5.2 材料与方法72-74
        5.2.1 菌株72
        5.2.2 主要试剂和仪器72
        5.2.3 储液和培养基72-73
        5.2.4 固体自诱导培养基可视化筛选稳定克隆子73
        5.2.5 培养方法与条件73
        5.2.6 细胞浓度与生物量的测定73
        5.2.7 生长曲线的绘制73
        5.2.8 普鲁兰酶酶活的测定73-74
        5.2.9 质粒稳定性的测定74
        5.2.10 发酵液中底物与产物的 HPLC 分析74
        5.2.11 不同细胞渗透物对胞外普鲁兰酶产量的影响74
        5.2.12 分批发酵产胞外普鲁兰酶74
        5.2.13 分批补料发酵生产胞外普鲁兰酶74
        5.2.14 胞外普鲁兰酶样品的制备及 SDS-PAGE74
    5.3 结果与讨论74-88
        5.3.1 可视化筛选高产胞外普鲁兰酶克隆子74-77
        5.3.2 种龄时机的选择77-78
        5.3.3 自诱导培养生产胞外普鲁兰酶78-81
        5.3.4 双温度调控策略高效生产胞外普鲁兰酶81-83
        5.3.5 细胞渗透物对胞外普鲁兰酶产量的影响83-86
        5.3.6 发酵罐水平发酵生产胞外普鲁兰酶86-88
    5.4 本章小结88-90
主要结论与展望90-93
    主要结论90-91
    展望91-93
论文主要创新点93-94
致谢94-95
参考文献95-108
附录:图(表)108-111
附录:作者在攻读博士学位期间发表的论文111


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