【作者】 冉丽媛；

【导师】 张玉忠； 陈秀兰；

【作者基本信息】 山东大学 ， 微生物学， 2014， 博士

【摘要】 海洋覆盖了超过71%的地表面积,是地球上最重要的生态系统之一。海洋中蕴藏着数量巨大、种类繁多的微生物,是开发新能源的巨大宝库。全球海洋氮源中约有25%都埋藏在深海沉积物里,因此,由沉积物细菌胞外酶所介导的有机氮的降解和转化对全球生物化学循环产生着重大的影响。深海中的高分子量有机氮几乎全都是能抵抗化学水解和生物降解的氨基化合物,胶原蛋白在高等动物(包括海洋动物)体内含量非常丰富。由于它结构复杂而致密,很难被除胶原蛋白酶以外的其他蛋白酶降解,因此很可能是深海颗粒有机氮(PON)的重要组成成分。目前研究最多的胶原蛋白酶为哺乳动物的基质金属蛋白酶(MMPs),关于细菌胶原蛋白酶的研究多来自陆地致病菌。由于对海洋蛋白酶缺乏研究,海洋中的胶原蛋白是如何被降解的还不清楚。因此,研究深海胶原蛋白酶的作用机制对于最终揭示全球海洋氮循环机制具有重要意义。深海沉积物适冷菌Pseudoalteromonas sp. SM9913分泌的蛋白酶deseasinMCP-01属于丝氨酸蛋白酶S8家族,成熟的MCP-01包含催化结构域(CATD)、连接区域、P结构域和多囊性肾病(PKD)结构域四部分,是一个多结构域的subtilisin家族蛋白酶。MCP-01成熟酶和单独的CATD均可对胶原蛋白产生降解,C端的PKD结构域能吸附并膨胀胶原蛋白,但不破坏胶原蛋白三螺旋的结构。在前期工作的基础上,本论文着重从MCP-01催化结构域的晶体结构及其对胶原蛋白的结合和降解机制上进行了研究。Myroides profundi D25是本实验室从冲绳海槽1245米深的深海沉积物中分离、鉴定的一个新的产蛋白酶菌株。该菌能分泌一种M12家族的新型弹性蛋白酶myroilysin。该蛋白酶不具有胶原蛋白酶活,但却对胶原蛋白具有显著的膨胀作用,可与外源细菌胶原蛋白酶在胶原蛋白降解过程中产生协同。在本论文中,我们纯化了菌株D25分泌的一种胞外胶原蛋白酶myroicolsin,对其酶学性质、基因序列、结构域功能及胶原蛋白降解机制进行了研究；我们还探究了菌株D25分泌的弹性蛋白酶myroilysin和胶原蛋白酶myroicolsin在降解胶原蛋白中的协同作用,并对myroilysin的发酵产酶条件进行了优化。共取得了如下研究结果：(1)蛋白酶deseasin MCP-01催化结构域识别和降解胶原蛋白的分子机制研究为了分析MCP-01识别和降解胶原蛋白的机制,我们首先利用原子力显微镜观察了MCP-01全酶、催化结构域CATD和PKD结构域对胶原蛋白纤维的作用过程,并利用生化实验进行了证实。结果表明,MCP-01和CATD都能通过降解胶原纤维之间的蛋白聚糖和原纤维内部的端肽来破坏胶原纤维复杂而致密的结构,直至释放出胶原蛋白单体,并最终将单体降解为小肽和游离的氨基酸。为了从分子层面揭示蛋白酶MCP-01识别和降解胶原蛋白的机制,我们解析了CATD的晶体结构。Carlsberg是subtilisin家族的原型酶,我们发现CATD的拓扑结构与Carlsberg非常相似,但是Carlsberg却不能降解胶原蛋白。通过生化分析及突变实验验证,我们发现CATD中由三条loop (loop7,9,11)参与围成的较大的底物结合口袋是MCP-01识别和结合大分子量底物(如胶原蛋白)所必需的；同时,这三条loop上含有的大量的芳香族氨基酸和酸性氨基酸能够形成一个带负电的疏水环境,有利于CATD与表面带正电且具疏水性的胶原蛋白的结合。利用液质联用技术分析了CATD在胶原蛋白肽链上的酶切位点。我们发现CATD在胶原蛋白肽链上的酶切位点特异性不是非常严格,它的P1位通常是Pro,而P1’位常是Gly,这可能与胶原蛋白肽链独特的氨基酸组成有关；此外碱性氨基酸残基(Lys, Arg)也多次出现在P1位上,这与本实验室前期实验结果相符,同时也是CATD不同于其它S8家族蛋白酶的地方。结构分析和突变实验证明,位于S1底物结合口袋中的His211是影响MCP-01对P1位碱性氨基酸偏好性的关键位点。综上所述,我们的研究结果揭示了S8家族丝氨酸胶原蛋白酶MCP-01识别和降解胶原蛋白的分子机制。由于目前有很多环境微生物和致病微生物都能分泌S8家族胶原蛋白酶,因此我们的结果不仅有助于研究环境中有机氮的降解机制,也为开发以S8家族胶原蛋白酶为致病因子的疾病的治疗药物奠定一定的基础。此外,由于deseasins类蛋白酶在海洋沉积物中广泛存在,我们的研究将为阐明这类蛋白酶在深海PON降解过程中的作用机制奠定基础。(2)蛋白酶myroicolsin的分离纯化、基因克隆及其酶学性质和结构域功能的研究通过硫酸铵沉淀、阴离子交换层析及分子筛层析等步骤,从菌株D25的胞外发酵液中纯化得到电泳纯的蛋白酶myroicolsin。Myroicolsin对胶原蛋白的底物特异性非常广泛,可以降解多种天然胶原蛋白(Ⅰ型、Ⅱ型及Ⅳ型)及明胶,其中对鱼不可溶胶原蛋白的活性最为显著。以牛不可溶Ⅰ型胶原蛋白作为底物,其最适酶活温度为60℃,在0℃下仍保持了近10%的活力；最适pH为8.5,在pH7.0-9.5的缓冲体系中可保持60%以上的酶活；同时myroicolsin对NaCl具有较好的耐受性,在0.5M NaCl中活性最高,在4M NaCl中可保持50%以上的活性,这些性质充分体现了蛋白酶myroicolsin对深海低温、弱碱和高盐环境的适应,说明该蛋白酶很可能在深海PON的降解中发挥着作用。Ca2+能显著增强myroicolsin的酶活力,而抑制剂PMSF可强烈抑制myroicolsin的酶活,推测该蛋白酶属于丝氨酸蛋白酶家族。根据成熟酶的N端氨基酸序列及丝氨酸蛋白酶家族的保守区,我们利用PCR及Tail-PCR的方法克隆了蛋白酶myroicolsin的全基因序列共2040bp,该基因编码一个包含679个氨基酸残基的蛋白酶前体,该基因序列已提交GenBankTM,序列号为JF514144。序列比对表明myroicolsin属于蛋白酶S8家族subtilisin亚家族,该蛋白酶序列新颖,与该家族中已有报道的蛋白酶相似性均低于30%,很可能是该家族中的新成员。Myroicolsin是一个多结构域的蛋白酶,其前体形式主要包含信号肽、N端前导肽、催化结构域、连接区域、β折叠结构域及C端前导肽。根据成熟酶N端序列及分子量大小,我们推测信号肽、N端前导肽及C端前导肽在myroicolsin成熟过程中自然脱落,成熟后的myroicolsin仅包括催化结构域、连接区及β折叠结构域,共计507个氨基酸残基。缺失突变的实验结果表明：myroicolsin的C端前导肽与N端前导肽的切除有关,它影响着蛋白酶的成熟但与蛋白的折叠和分泌无关；具有柔性的连接区可能与蛋白的正确折叠息息相关；而p折叠结构域在蛋白的折叠、分泌和成熟过程中均未发现有明显作用。通过底物吸附实验发现,β折叠结构域在0℃及25℃对不可溶胶原蛋白均无明显的吸附作用,这与其他胶原蛋白酶多在C端含有一个胶原蛋白结合结构域来协助催化结构域对底物进行降解有所不同,预示着myroicolsin可能有不同于其他胶原蛋白酶的独特的胶原蛋白降解机制。(3)蛋白酶myroicolsin对胶原蛋白降解机制的研究我们利用扫描电子显微镜和原子力显微镜观察发现nyroicolsin能破坏胶原纤维的结构,暴露出胶原原纤维并最终释放出胶原蛋白单体。同时结合一系列的生化实验证明了myroicolsin通过降解胶原纤维之间或胶原原纤维内部的蛋白聚糖和端肽交联而实现其对胶原蛋白的逐步降解。圆二色光谱扫描结果证明myroicolsin能破坏胶原蛋白的三螺旋结构,从中释放出a肽链,并将其降解为小肽和游离的氨基酸。通过液质联用技术分析,得到myroicolsin在胶原蛋白肽链上的酶切位点,我们发现myroicolsin对天然胶原和热变性胶原的酶切方式不同：对天然胶原蛋白,酶切位点的P1位大多数是Gly、Arg、Pro或Phe等,而P1’位主要是Gly；对热变性的胶原蛋白P1位则全被碱性氨基酸(Arg和Lys)占据,而P1’位仍然大多数都是Gly。根据以往报道,S8家族subtilisin-like蛋白酶通常偏好于降解P1位是疏水性氨基酸的肽段,而S1家族trypsin-like的丝氨酸蛋白酶通常降解P1位是碱性氨基酸的肽键,myroicolsin同时表现出subtilisin-like和trypsin-like的酶切位点特异性,同家族的细菌胶原蛋白酶MCP-01也具有这种特性,这可能是该家族胶原蛋白酶的共同之处。(4)S8家族细菌胶原蛋白酶降解胶原蛋白的机制模型蛋白酶MCP-01和myroicolsin都是由深海沉积物细菌分泌的S8家族胶原蛋白酶,两者对胶原蛋白的降解机制有一定的相似性：它们都能通过降解胶原纤维之间的蛋白聚糖而打散胶原蛋白致密的纤维状结构,暴露出大量的胶原原纤维；进而通过降解胶原原纤维内部的端肽,释放吡啶交联,加速了胶原纤维结构的瓦解,释放出大量的胶原蛋白单体；然后通过降解胶原蛋白单体内维系三螺旋稳定性的关键氨基酸周围的肽键,破坏其三螺旋结构,释放α肽链并最终将其降解为小肽和游离的氨基酸。基于蛋白酶MCP-01和myroicolsin对胶原蛋白的降解机制,提出了S8家族细菌胶原蛋白酶对胶原蛋白降解的机制模型,这让我们对深海细菌分泌的S8家族胶原蛋白酶的底物降解机制以及细菌胞外蛋白酶如何参与深海氮循环的过程有了新的认识和理解,也为新型蛋白酶资源的开发、利用奠定了重要的理论基础。(5)弹性蛋白酶myroilysin对蛋白酶myroicolsin降解胶原蛋白的协同作用研究及其中试发酵工艺本实验室前期工作发现菌株D25分泌的新型弹性蛋白酶myroilysin不具有胶原蛋白酶活性,但是却能显著的膨胀胶原蛋白,与外源胶原蛋白酶在胶原蛋白降解过程中能产生协同。本论文进一步研究了菌株D25自身分泌的弹性蛋白酶myroilysin和胶原蛋白酶myroicolsin在胶原蛋白降解中的协同作用,结果表明,myroilysin与myroicolsin确实在胶原蛋白降解过程中产生了协同,提高了myroicolsin对胶原蛋白的降解效率,这说明深海沉积物中细菌分泌的不同蛋白酶可能具有协同降解PON的生态学作用。由于胶原蛋白在生物医学领域具有广泛的用途,myroilysin膨胀却不降解胶原蛋白的特性预示着该酶可能是一种优良的胶原蛋白改性剂。因此我们采用单因子实验法分析了影响蛋白酶myroilysin产量的关键因素,得出在摇瓶中发酵的最佳培养条件：4%麸皮,2%豆粕,1%t米粉,0.4%Na2HPO4,0.03%KH2PO4,0.1%CaCl2,pH8.0,装液量100mL/500mL三角瓶,1%接种量,于15℃,200rpm培养72-84h。根据摇瓶优化的结果,进行5L发酵罐小试和200L发酵罐的中试培养,通过对发酵过程中关键参数的优化,确定了蛋白酶myroilysin的小试和中试发酵工艺,使产酶量达到1100U/mL以上,为工业发酵生产myroilysin和开发其在生物医学领域的应用潜力奠定了基础。更多还原

【Abstract】 The ocean, covering more than71%of the earth’s surface, is one of the most important ecosystems on the earth. There are various and abundant microorganisms in the ocean, which offer a great potential for the discovery of new resources. Since deep sea sediments contain at least25%of the global ocean nitrogen burial, degradation and transformation of organic nitrogen mediated by extracellular enzymes from deep sea sediment bacteria may have significant effect on the global biogeochemical cycles. High molecular weight organic nitrogen is mostly present as biological and chemical hydrolysis-resistant amides on the deep sea floor. Collagen is the most abundant fibrous protein in all higher organisms, including marine animals. Due to its tight and complicated structure, collagen is resistant to common proteases and can only be degraded by a limited number of collagenolytic proteases. Collagen is therefore an important component of deep sea sedimentary particulate organic nitrogen (PON). Of all the collagenolytic proteases, mammalian matrix metalloproteinases (MMPs) have been well investigated and reports on collagenolytic proteases secreted by microorganisms are relatively fewer. Most of bacterial collagenases so far reported are pathogenic and terrestrial, and only several ones are oceanic. Thus, the degradation mechanisms of the ocean collagen are still rather unclear. Research into the function and mechanism of marine collagenases will provide important implications for global deep sea nitrogen cycling.The protease deseasin MCP-01belonging to the S8family of serine proteases is secreted by the deep sea cold-adapted bacterium Pseudoalteromonas sp. SM9913. Mature MCP-01is a multidomain protein composed of a catalytic domain (CATD), a linker, a P-proprotein domain and a ploycystic kidney disease (PKD) domain. The CATD alone can degrade type Ⅰ collagen with relatively lower efficiency than intact MCP-01. The PKD domain at its C-terminus is responsible for binding and swelling of collagen, but it cannot unwind the collagen triple helix. In this dissertation, structural analysis and biochemical experiments were performed to study the recognition and degradation mechanism of MCP-01on collagen.Myroides profundi D25is a protease-secreting bacterium isolated from the deep sea sediment of the southern Okinawa Trough at a water depth of1245m. The D25strain produces a novel elastinolytic protease of M12family, myroilysin, which cannot hydrolysis collagen but has strong collagen swelling ability and cooperates with collagenase in collagen hydrolysis. Here, a novel collagenolytic protease secreted by strain D25, designated myroicolsin, was purified from fermentation medium of strain D25and its enzymatic characteristics, gene sequence, function of domains and degradation mechanisms to collagen were studied. Moreover, the synergistic action between elastase myroilysin and collagenase myroicolsin and the pilot-scaled fermentation teechnology of myroilysin were further studied. The results are as following:(1) Structural and mechanistic insights into collagen recognition and degradation by deseasin MCP-01.In order to study the collagen recognition and degradation mechanism of MCP-01, the time-dependent progressive disintegration of collagen fiber by MCP-01, the CATD and PKD domain was observed by atomic force microscopy. Biochemical analysis was confirmed that MCP-01and the CATD, in the collagen fibers degradation process, progressively release single fibrils from collagen fibers and collagen monomers or monomer fragments from fibrils mainly by hydrolyzing the proteoglycans that interdigitate with fibrils and the telopeptides within or between fibrils. Collagen monomers are subsequently released and further degraded. To further study how the CATD binds and degrades collagen molecules, the crystal structure of the CATD (Serl-Phe333) was determined. Subtilisin Carlsberg is the prototype of the S8family, which shows a similar topology to CATD. There have been no reports that subtilisin Carlsberg is capable of degrading collagen. Structural analysis and mutational assays indicated that an enlarged substrate-binding pocket, mainly composed of loops7,9and11, was necessary for the CATD in collagen recognition. The acidic and aromatic residues on these loops form a negatively charged, hydrophobic environment for the binding of collagen which has a positively charged, hydrophobic surface.Analysis of the cleavage pattern of MCP-01on triple-helical type I collagen based on the cleavage sites was determined in our previous and present studies indicated that it displayed a non-strict preference for peptide bonds with Pro at the P1site and/or Gly at the PI’site in collagen. Moreover, MCP-01showed a different specificity from other S8proteases, tending to cleave the peptide bonds with basic residues (Lys and Arg) at the P1site in collagen. Structural analysis and mutation assays indicated that His211in the S1pocket is a key residue for the preference of MCP-01for basic residues at the P1site. In summary, our study gives structural and mechanistic insights into collagen degradation of an S8collagenolytic protease MCP-01. Because the S8collagenolytic proteases are secreted by both environmental and pathogenic microorganisms, our results are helpful in studying the environmental organic nitrogen degradation mechanism and in developing therapeutics for diseases with S8collagenolytic proteases as pathogenic factors. In addition, because deseasins (MCP-01-like proteases) are most likely widespread in marine sediment, this study sheds light on the degradation mechanism of deseasins for marine sedimentary PON.(2) Purification, gene cloning, characteristics and domain functions of collagenolytic protease myroicolsin.A protein was purified from the fermentation broth of strain D25through ammonium sulfate precipitation, DEAE-Sepharose Fast Flow chromatography and gel filtration chromatography, which was designated as myroicolsin. Substrate specificity analysis showed that myroicolsin had broad specificity for various collagens, especially fish-insoluble collagen. With insoluble bovine type I collagen fiber as the substrate, the optimal temperature of myroicolsin was60℃, and10%of the highest activity was retained at0℃. Myroicolsin displayed the greatest collagenolytic activity at pH8.5, more than60%activity was retained between pH7.0and9.5. Myroicolsin showed the highest activity in0.5M NaCl, and more than50%of the highest activity was retained in4M NaCl, showing its high salt tolerance. These results indicated that myroicolsin has evolved over time to accommodate itself to low temperature, slightly alkaline and high salt concentration of marine environment, fully participating in PON hydrolysis process in the deep-sea ecosystem. In addition,4mM Ca2+markedly increased the activity of myroicolsin. The activity of myroicolsin was almost completely abolished by the serine protease inhibitor PMSF, suggesting that myroicolsin is probably a serine proteases family.Based on the N-terminal amino acid sequence of myroicolsin and the conserved sequence in the catalytic domains of serine proteases, the complete gene of myroicolsin was cloned by a combination of PCR and thermal asymmetric interlaced PCR. The open reading frame of the whole gene is2040bp and was deduced to encode a protease precursor of679amino acid residues. The nucleotide sequence was submitted to the GenBankTM with accession number JF514144. Sequence analysis showed that myroicolsin is a novel protease of the S8family with low identity (<30%) to characterized peptidases. The precursor of myroicolsin contains a signal sequence, an N-propeptide, a catalytic domain, a linker, a β-jelly roll domain, and a C-pro-secre-tail. Based on the molecular mass of the purified myroicolsin and its N-terminal sequence, it could be deduced that the signal sequence, the N-propeptide, and the C-pro-secre-tail were cleaved spontaneously during enzyme maturation. The mature form of myroicolsin consists of507residues, including the catalytic domain, the linker, and the β-jelly roll domain. Truncated mutation assay showed that the C-pro-secre-tail is essential for the cleavage of the N-propeptide but not for the secretion and folding of the protein, the linker may be required for protein folding and the β-jelly roll domain is not required for folding and maturation. Our collagen binding assay showed that the β-jelly roll domain showed little binding ability to insoluble collagen fiber at0℃or25℃. These results suggested that myroicolsin has no C-terminal collagen-binding domain, unlike other characterized collagenolytic proteases. Therefore, it seems that myroicolsin may have a special collagen degradation mechanism.(3) Study of collagenolytic mechanism of myroicolsin.Our scanning electron microscopy and atomic force microscopy observations showed that myroicolsin released fibrils from collagen fiber and that the fibrils were further degraded into collagen monomers. Biochemical assays indicated that this stepwise degradation of collagen fiber was achieved through the hydrolysis of the proteoglycans and telopeptides in collagen fibers and fibrils. Circular dichroism spectra revealed that myroicolsin could destroy the triple helix structure of the collagen monomer, suggesting that the polypeptide chains in monomers could be released and further hydrolyzed into peptides and amino acids. The cleavage pattern of myroicolsin on collagen polypeptide chains were analyzed by high performance liquid chromatography and mass spectrometry. Myroicolsin showed different cleavage patterns against native collagen and denatured collagen. In native collagen, the P1position is often occupied by Gly, Arg, Pro, or Phe, and the P1’ position is almost always occupied by Gly. In denatured collagen, the P1position is always a basic residue (Lys or Arg), and the P1’ position is still Gly. Among serine proteases, subtilisin-like proteases are usually nonspecific peptidases with a preference to cleave after hydrophobic residues, while trypsin-like proteases always cleave the peptide bonds with a basic residue at the P1site. Therefore, it seems that myroicolsin exhibits a mixed type of PI specificity, both trypsin-like and subtilisin-like. The bacterial collagenase MCP-01of S8family showed the same P1specificity as myroicolsin, which may be the common characteristic of the S8family collagenases.(4) A model for collagen degradation by S8bacterial collagenases.Collagenolytic proteases, MCP-01and myroicolsin, from deep sea sediment bacteria are both the members of the S8family. Our results showed that they have some similarities in collagen degradation. They both first break the interfibrillar proteoglycan bridges, leading to the disassembly of the tight structure of collagen fibers and the exposure of collagen fibrils. Then telopeptides within collagen fibrils and microfibrils are hydrolyzed by them, which accelerates the unfolding of the collagen structure. Thus, these collagenases gain access to collagen monomers. Finally, the fibrillar structure of collagen is completely destroyed, and collagen monomers are degraded into peptides and free amino acids. Based on the collagenolytic mechanism of MCP-01and myroicolsin, a mechanism model for collagen fiber degradation by the S8collagenases is proposed. The results provide important evidences for the substrate degradation mechanism of the S8collagenases, and give insight into nitrogen cycling in deep-sea sediment and also provide theoretical basis for the discovery and development of novel proteases.(4) Synergistic action of myroilysin and myroicolsin in collagenolytic process and the fermentation technology of myroilysin at pilot scale.Our previous research revealed that myroilysin, secreted by strain D25, displayed little collagenolytic activity but strong collagen swelling ability. Myroilysin could play a synergistic role with other terrigenous collagenase in collagen hydrolysis through swelling collagen. In this study, we found that strain D25could secrete a collagenolytic protease myroicolsin, and myroilysin also played a synergistic role with myroicolsin in collagen hydrolysis, which could increase the collagenolytic activity of myroicolsin. The results indicated that different proteases secreted by deep sea bacteria may have ecological effect to synergistically hydrolyed the PON in the deep sea sediment. In recent years, collagen has been considered as one of the most useful biomaterials in the biomedicine field. The strong collagen swelling ability of myroilysin showed that it could be an excellent collagen modifier, which has great application potential in the production of collagen medicine materials. In order to find out the key factors significantly affecting myroilysin production of strain D25, the relative significance of variables were investigated using single factor experiments. Finally, we determined the optimal conditions for myroilysin production in shake flask culture:4%bran,2%bean pulp,1%corn powder,0.4%Na2HPO4,0.03%KH2PO4,0.1%CaCl2, pH8.0; loading volume100mL/500mL;1%inoculum density,15℃,200rpm for72-84h. We further optimized the culture condition of myroilysin in fermenters of5L and200L, and the fermentation technology of myroilysin at polit scale was developed. Under the optimal conditions, the maximum myroilysin activity in fermentation broth reached1100U/mL, which provides a foundation for developing the application potential of myroilysin in the biomedicine field.更多还原