深海新型M23家族蛋白酶pseudoalterin降解肽聚糖和弹性蛋白的分子机制、生态学功能及应用潜力评价
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摘要
深海有机氮的降解是海洋生态系统氮循环的重要环节。在海洋中,生物合成的高分子量有机氮(HMWON)以颗粒有机氮(PON)的形式,从海水表层沉降到深海沉积物中。PON在微生物的作用下降解为高分子量的溶解有机氮(DON),DON再经过微生物的分解、氨化、硝化和反硝化作用,最终完成整个氮循环。因此,深海沉积物中PON的降解在整个氮循环过程中具有非常关键的作用。深海沉积物中PON的主要成分是难以被降解的氨基化合物和生活在沉积物中的生物体,其中氨基化合物可能主要包括一些难溶于水的蛋白质如胶原蛋白和弹性蛋白等。弹性蛋白由于其特殊的亲疏水区交替的氨基酸序列和复杂的交联形式,稳定性非常高,很难被除弹性蛋白酶以外的其他蛋白酶降解。目前研究的弹性蛋白酶多来自哺乳动物,细菌弹性蛋白酶的报道较少,且多来自陆地致病菌。由于对海洋弹性蛋白酶缺乏研究,因此海洋中弹性蛋白是如何被降解的还不清楚。而深海沉积物中具有丰富的分泌弹性蛋白酶的微生物资源,且不同菌株对弹性蛋白的降解能力不同,表明它们可能是不同种类弹性蛋白酶。深海细菌肽聚糖中的肽段部分的存在使肽聚糖成为海洋中PON的组分之一,然而海洋降解肽聚糖的酶类研究很少。研究深海蛋白酶的作用机制对于阐明深海沉积物中氮的循环机制和开发新型蛋白酶具有重要的意义。
Pseudoalteromonas sp. CF6-2是我们从南中国海2441米海底沉积物中分离的
一株细菌,采样地点位于台湾西南九龙甲烷礁区。前期的研究表明,P. sp. CF6-2在弹性蛋白平板上可以产生很大的水解圈(水解圈/菌落直径=9.0),这说明P.sp.CF6-2是一株高产弹性蛋白酶的菌株。目前深入研究的弹性蛋白酶还没有来源于Pseudoalteromonas属细菌的,因此我们推测P. sp. CF6-2分泌的弹性蛋白酶可能是一个新型弹性蛋白酶。本论文纯化了菌株CF6-2分泌的弹性蛋白酶pseudoalterin,并对其酶学性质、基因序列、对弹性蛋白和细菌肽聚糖的降解机制、生态学功能和应用潜力进行了研究,取得了如下研究结果。
(1) Pseudoalterin的分离纯化、酶学性质研究以及基因克隆
通过阴离子交换层析,从P.sp.CF6-2发酵液中分离到一种电泳纯的蛋白酶,命名为pseudoalterin。通过研究它对不同底物的降解作用,发现该酶除了对弹性蛋白具有很高的酶活之外,对其他蛋白底物都几乎没有酶解活性,是一个特异性弹性蛋白酶。该酶在5℃时依然有10%的酶活,最适酶活温度为25℃,热稳定性很差,在35℃中保温20min,即会丧失70%的酶活,这表明pseudoalterin是典型的低温酶。该酶是一个碱性蛋白酶,pH9.5时酶活最高,pH低于8.0的缓冲体系中活性低于20%,而在pH10.5的体系中仍然保持了40%的活性,Ca2+和Sr2+显著增强pseudoalterin的酶活力量,Fe2+显著抑制pseudoalterin酶活,而Zn2+几乎完全抑制pseudoalterin(?)活性。该酶活力会被金属蛋白酶抑制剂抑制。根据以上结果推测它足一个依赖于Zn2+的金属蛋白酶。
通过使用PCR和Thermal asymmetric interlaced PCR(Tail PCR)等技术手段,克隆了pseudoalterin的全集因序列。基因全长1212bp,编码含有403个氨基酸残基的蛋白酶前体。基因序列已提交GenBankTM,序列号为HQ005379。序列分析农明,pseudoalterin为Zn金属蛋白酶,属于M23家族中的S亚家族,与该家族其他蛋白酶的序列最高相似度为39%,属于M23家族中一个新的蛋白酶。
对GenBankTM中21株深海沉积物和热泉细菌的基因组序列分析表明M23家族的蛋白酶在深海中广泛存在,因此可能在深海氮循环中发挥着重要的作用。然而它们具体的特性和作用却从未被报道。Pseudoalterin足第一个进行了性质研究的深海来源的M23家族蛋白酶。
(2)用生化方法和显微镜技术研究pseudoalterin对弹性蛋白的降解机制
M23家族蛋白酶都可以缓慢降解弹性蛋白。目前对M23家族蛋白酶降解弹性蛋白机制的研究仅限于它们在弹性蛋白或弹性蛋白类似小肽上的酶切位点的分析。而作为M23A亚家族的一个蛋白酶，pseudoalterin了以快速将不可溶弹性蛋白降解为可溶的肽段,所以研究它对弹性蛋白的降解机制对揭示M23家族蛋白酶的作用机制具有重要意义。通过光学显微镜观察pseudoalterin对牛弹性蛋白的酶解过程,发现pseudoalterin先将弹性蛋白纵向膨胀出丝状结构,推断pseudoalterin可以打开弹性蛋白内部的交联。用液相与质谱联用技术研究pseudoalterin对牛弹性蛋白的酶切位点,并通过合成小肽的酶解和锁链索(DES)的检测验证这些酶切位点,结果表明pseudoalterin可以优先降解弹性蛋白中交联区的序列。为了进一步研究pseudoalterin降解弹性蛋白的机制，我们用扫描电镜(SEM)观察了pseudoalterin对重组弹性蛋白和天然牛弹性蛋白的酶解过程。通过对酶解过程的观察,结合生化实验结果,我们提出了pseudoalterin降解弹性蛋白的机制模型：pseudoalterin优先降解弹性蛋白亲水的交联区,对疏水区作用较慢,因此在酶解过程中先使弹性蛋白分离成丝状,然后由丝状纤维进一步酶解为小球,最后酶解出弹性蛋白原单体并进一步降解为可溶的肽段。这是首次祥细阐明一个M23家族蛋白酶对弹性蛋白的降解机制。
目前报道的所有M23家族蛋白酶都仅显示出对疏水区甘氨酰键的酶解能力。因此我们揭示的pseudoalterin首先降解弹性蛋白亲水区再降解疏水区的弹性蛋白降解机制是一种崭新的M23家族蛋白酶降解弹性蛋白的机制。这也表明pseudoalterin是M23家族中一种新型的蛋白酶。我们对南海沉积物新型M23家族蛋白酶的弹性蛋白降解机制的分析为阐释深海沉积物中氮循环的研究提供了重要的证据,同时也为新型蛋白酶的开发利用提供了重要的理论基础。
(3) Pseudoalterin、myroilysin和pseudolysin三个不同家族的弹性蛋白酶对弹性蛋白降解机制的比较
陆地细菌分泌的弹性蛋白酶中研究比较多的是Pseudomonas aerugonosa分泌的M4家族pseudolysin,而深海来源的细菌弹性蛋白酶中对弹性蛋白酶解方式有所研究的是来源于Myroides profundi D25的M12家族蛋白酶myroilysin。对这两种弹性蛋白酶的作用机制虽然也有研究,但都没有通过SEM直接观察过酶解弹性蛋白的过程,对弹性蛋白降解机制也没有全面的研究。我们通过生化实验和电镜观察详细研究了pseudolysin和nyroilysin降解弹性蛋白的机制,并与pseudoalterin进行了比较。显微镜观察结果表明这两种弹性蛋白酶对弹性蛋白的降解模式与pseudoalterin完全不同。光镜下二者对弹性蛋白的降解方式略有不同,myroilysin倾向于优先将弹性蛋白横切成段状,pseudolysin对弹性蛋白则进行不规则切割。在SEM下,pseudolysin和myroilysin都首先在弹性蛋白表面形成孔洞,这跟pseudoalterin先将弹性蛋白拆分成丝状结构的降解方式不同。对这两种酶在牛弹性蛋白上的酶切位点分析发现它们的作用位点都集中在弹性蛋白的疏水区,而不是交联区,这可能正是造成它们酶解现象不同的原因。研究和比较pseudoalterin、myroilysin和pseudolysin三个不同家族弹性蛋白酶对弹性蛋白的降解机制,可以对弹性蛋白酶的作用机制有史深入的了解。
(4) Pseudoalterin裂菌活性的生态作用及其吸附和降解肽聚糖的机制
M23家族的蛋白酶都是可以酶解细菌细胞壁肽聚糖的酰胺酶或内肽酶,为了研究pseudoalterin勺裂菌活性在海洋中的生态作用,我们研究了它对海洋来源的革兰氏阳性细菌和部分革兰氏阴性细菌的肽聚糖的裂解活性。结果表明pseudoalterin对这些细菌都具有裂解活性,虽然不同种属之间肽聚糖结构不同学致了这种裂解活性具有一定差异。因此我们认为M23家族蛋白酶的裂菌活性决定了它们在深海有机质循环中发挥着重要的作用。
M23家族的蛋白酶对金黄色葡萄球菌的裂解活性最强。因此我们选择金黄色葡萄球菌的肽聚糖作为对象研究了pseudoalterin对肽聚糖的降解机制。我们实验结果表明,pseudoalterin不仅可以快速降解金葡萄肽聚糖,而且对肽聚糖以及其他几种不可溶多糖如几丁质、壳聚糖、纤维素等具有吸附作用。因此我们推测pseudoalterin在降解肽链部分之前可能先通过吸附结构域吸附到肽聚糖的糖链上
为了阐明pseudoalterin降解肽聚糖的机制,我们分析了pseudoalterin对肽聚糖的酶切位点。对pseudoalterin酶解后的肽聚糖产物进行液质联分析，结果表明pseudoalterin既是一个N-乙酰-L-丙氨酸酰胺酶,又是一个内肽酶。Pseudoalterin除了可以从多个位点切断五甘氨酸肽桥,还可以切开N-acylmuramoyl-Ala之间的酰胺键、肽桥与肽尾连接处的肽键Ala-Lys,以及肽尾的肽键Ala-Glu。
为了研究蛋白酶pseudoalterin吸附肽聚糖的机制,我们对蛋白酶pseudoalterin进行了结晶,通过X-射线衍射得到了辨率为1.5A的分子结构。对pseudoalterin的结构分析表明,一个pseudoalterin分子山1个α螺旋(α1)和9个β折叠片(β1-β9)组成,其中α螺旋非常短(104-108),位于分子外侧。5个较长的β折叠片β1(30-35)、β2(56-61)、β3(64-69)、β4(72-78)和β5(119-125)反平行排列并被loop包围在分子内部,形成一个较大的空腔,从空腔上半部分的Zn2+可以推测出这足该酶的催化腔。β6(134-137)、β7(140-143)、β8(157-160)和β9(164-165)这四个C端的β折叠片较短,反平行排列在分子右上方外侧,形成一个小沟。在靠近金属离子的一端,催化腔与这个小沟呈T字形排列。与M23家族已有的3个结构相比,pseudoalterin与它们都属于β型蛋白。但是pseudoalterin没有左下方的β domain,只有右上方的β domain,而且β折叠片更长,我们推测这个β domain的功能可能跟多糖的吸附附有关
为了研究蛋白酶pseudoalterin与肽聚糖糖链的相互作用方式,我们将壳四糖((NAG)4)通过Auto dock与pseudoalterin的β6-β9形成的小沟进行分子共模拟。结果表明,(NAG)4能够与pseudoalterin该区域相互作用,嵌于pseudoalterin的loop之间。利用pymol软件计算与(NAG)4的空间距离在3.2A内可能与底物形成氢键的氨基酸残基。结果表明位于吸附腔附近的氨基酸残基Asn134、Arg141、 Asp143、Tyr157、Glu159和Arg164与(NAG)4的距离都在此范围内。另外位于这一区域但与(NAG)4距离稍远的氨基酸His133、Asn154和Arg163也可能参与了这一过程。通过点突变构建了以上氨基酸的突变体并分析了这些突变体对肽聚糖的吸附能力,结果表明pseudoalterin C端20个氨基酸中的Tyr157,Glu159,Arg163和Arg164对pseudoalterin的吸附具有重要影响,从而确定了pseudoalterin右上方的β domain确实是一个多糖吸附结构域,阐明了pseudoalterin对肽聚糖的吸附机制。这是首次对一个M23家族蛋白酶吸附和降解肽聚糖的机制进行详细研究,这对进一步揭示该家族蛋白酶裂解细菌的机制具有重要意义。
(5) Pseudoalterin的应用潜力研究及其发酵条件的优化
多重抗药菌的出现导致对非抗生素类杀菌剂的强烈需求。溶菌酶由于降解肽聚糖保守结构成分,细菌对其不易产生抗药性,在医疗上具有比较好的应用潜力。我们的实验结果表明,pseudoalterin是一个可以通过酰胺酶和内肽酶活性快速降解细菌肽聚糖的溶菌酶,具有良好的应用前景。我们以多种陆地来源的革兰氏阳性菌和阴性菌的细胞壁为底物,研究蛋白酶pseudoalterin在特定浓度和作用时间下对各种细菌的裂解能力,特别是对多抗性金黄色葡萄球菌的裂解能力。并且将其裂解细菌的能力与商品化的溶菌酶mutanolysin和lysostaphin进行了比较。结果表明,pseudoalterin对taphylococcus aureus细胞壁的裂解能力比lysostaphin高出100多倍,而且对多种细菌细胞壁都具有裂解能力。Pseudoalterin作为单一酶,对一些细菌细胞壁的裂解能力甚至比mutanolysin这种复合酶效果还要好。Pseudoalterin对多抗菌的裂解能力与普通金葡萄一样,说明多抗菌对这种溶菌酶没有抗性。对金葡菌活细胞的杀灭实验也证明pseudoalterin确实可以通过降解肽聚糖杀灭细菌,因而在临床上具有潜在的应用价值。
Pseudoalterin是一个诱导酶,在发酵中需要加入弹性蛋白诱导其产生。但是弹性蛋白的昂贵价格限制了pseudoalterin的大规模发酵。本论文研究了不同诱导物对pseudoalterin产量的影响,实验发现心管能完全代替弹性蛋白诱导酶的产生,而不降低产量。利用单因子实验确定了三个对酶发酵影响显著的因子,分别为心管用量、培养温度和培养时间。用响应面法优化了这三个因子的组合,分别为心管粉1.2%，培养温度20.17℃,培养时间28.04h。在此条件下发酵液的酶活达到100.02±9.0U/mL。相比于优化前的46.9±3.9U/mL提高了一倍。因此,我们的研究结果不仅提高了pseudoalterin的产量,而且大幅度降低了发酵成本,这为pseudoalterin的进一步开发应用奠定了很好的基础。
Degradation of organic nitrogen is an important part of global nitrogen cycling. In the ocean, High-Molecular-Weight Organic Nitrogen (HMWON) produced by organisms in the seawater settles and accumulates in the sediment in the form of particulate organic nitrogen (PON). Most of PON will be degraded into dissolved organic nitrogen (DON) by microorganisms, and then participates into the nitrogen cycling through ammonium regeneration, nitrification and denitrification in the sediment. Thus, the recycling of PON in deep-sea sediment would be a non-negligible part of ocean nitrogen cycling. Recent studies indicate that a significant fraction of PON is composed of insoluble amides and organisms living in the sediment. Because of the insoluble properties, collagens and elastins are speculated to be important components of sedimental PON. Elastins are made up of tropoelastin monomers with alternating hydrophobic and hydrophilic domains which are cross-linked complicatedly. Because of the rigid structure, elastins are resistant to most proteases except for a limited number of elastases. Up to now, the reports about elastolytic proteases were mostly focused on elastases from animals. The studies of elastases from microorganisms are relatively fewer and most of bacterial elastases are pathogenic and terrestrial. Thus the elastolytic mechanism of the ocean is unclear. There are abundant elastase-produced bacteria in deep-sea sediment. Elastases from different strains show different elastolytic abilities which imply the diversity of elastases in deep-sea. The peptidoglycans of bacteria living in sediment are also important components of sedimental PON. However, the studies of enzymes from deep sea that could degrade peptidoglycans are limited. Research into the function and mechanism of marine proteases will provide important implications for deep-sea nitrogen cycling and novel proteases development.
Pseudoalteromonas sp. CF6-2was isolated from the deep-sea sediment at a water depth of2,441m in the Jiulong methane reef area off the southwest of the island of Taiwan during the South China Sea Open Cruise of R/V Shiyan3. Our previous study showed that P. sp. CF6-2could produce hydrolytic zone on elastin agar plates with the hydrolytic zone diameter/the colony diameter (H/C)=9.0, suggesting that P. sp. CF6-2is a good producer of elastase. Because no elastase from bacteria in Pseudoalteromonas genus has been reported, we speculated that strain CF6-2secrets a novel elastase. In this thesis, the elastase, named pseudoalterin. was purified from the fermentation broth of P. sp. CF6-2and its enzymatic characteristics, gene sequence, degradation mechanisms to elastin and peptidoglycan and application potential were studied. The results are as following:
(1) Purification, characteristics and gene cloning of pseudoalterin
A protein with a molecular mass of19kDa was purified from the fermentation broth by a DEAE-Sepharose Fast Flow chromatography. which was designated as pseudoalterin. The determination of the substrate specificity showed that pseudoalterin could hardly hydrolyze any protein substrate except elastin. This indicated that it is a strict elastase. Pseudoalterin retained10%activity at5℃and its optimum temperature was25℃. Its thermostability was very low. and70%activity would be lost after it was incubated at35℃for20min. These properties indicated that this protease was a typical cold-adapted enzyme. Pseudoalterin was an alkaline elastase with an optimum pH of9.5. Lower than20%activity could be detected under pH8.0while40%activity was retained at pH10.5. Ca2+and Sr2+markedly increased the enzyme activity of pseudoalterin, and Fe2+severely inhibited the enzyme activity. Zn2+abolished the elastolytic activity completely. Moreover, the activity of pseudoalterin could be inhibited by metalloprotease inhibitors. So pseudoalterin was a Zn2+dependent metallopeptidasc.
The full-length gene of pseudoalterin was cloned from P. sp. CF6-2by PCR and thermal asymmetric interlaced PCR (Tail PCR) techniques. The ORF contains1212bp, encoding a precursor of403amino acid residues. The nucleotide sequence was submitted to the GenBankTM with accession number HQ005379. Analysis of the amino acid sequence showed that pseudoalterin is a zinc metalloprotease. Among the characterized proteases, pseudoalterin shows the highest identity (39%) to staphylolysin (M23A subfamily) from Pseudomoiuis aeruginasa PAO1(accession numberAAG05260). The conserved residues in and around the active site of M23 proteases are also found in pseudoalterin. These results all point to pseudoalterin as a novel metalloprotease of the M23A subfamily.
Analysis of the genomes of21bacteria isolated from deep-sea sediments and hydrothermal vents, which are in GenBankTM database, indicates that most of these bacteria could secrete one or several M23family proteases. This finding suggests that the M23family proteases may be popular in deep sea sediments and may therefore play an important role in sedimentary PON degradation. However, the properties and functionality of marine M23proteases have not been reported. Pseudoalterin was the first characterized marine elastase in M23family.
(2) Elastolytic mechanism of pseudoalterin
M23proteases could slowly degrade elastions. However, researches of elastolytic mechanism of M23proteases were limited to the analysis of their cleavage sites on elastin or elastin-like peptides. As a M23A subfamily protease, pseudoalterin could quickly degrade insoluble elastin into soluble peptides. Studying its elastolytic mechanism is helpful for clarifying the degradation mechanism of M23elastases. Microscopic observations indicated that pseudoalterin swelled elastin and separated elastin into filaments, implying that this enzyme initially broke down cross-links between the filaments. Analysis of the cleavage sites of pseudoalterin on bovine elastin and synthetical peptides indicated that pseudoalterin preferentially hydrolyzed elastin cross-linking domains. To further study the elastolytic mechanism of pseudoalterin, scanning electron microscope (SEM) observation of recombinant tropoelastin spherules and bovine elastin degradation process was performed. Based on our biochemical results and SEM observation, the elastolytic mechanism of pseudoalterin was concluded:pseudoalterin successively released filaments, droplets, and spherules from elastic fibers by destroying the cross-links at each structural level. This is the first detailed research of a M23elastase about its elastolytic mechanism.
All the proteases in M23family with investigation only cleaved Gly-Xaa bonds in hydrophobic domains in elastin. Therefore, that pseudoalterin hydrolyzed cross-linking domains in elastin before hydrophobic domains represents a new-type elatolytic mechanism in M23family. It also revealed that pseudoalterin is a novel protease in this family. The insight into the novel elastase from the sediment and its mechanism provides important evidences for nitrogen cycling in deep-sea sediment. The research also provides theoretical basis for the development of novel proteases.
(3) Comparison of pseudoalterin, myroilysin and pseudolysin on their elastolytic mechanism
Of all the elastases from terrestrial bacteria, pseudolysin in M4family from pathogenic bacteria Pseiidomonas aerugonosa was detailed investigated. Myroilysin was the only marine elastase of which the elastin degradation pattern was studied. Myroilysin is an M12family protease from deep-sea sediment bacterium Myroides profundi D25. The detailed elastolytic mechanisms of pseudolysin and myroilysin are still unclear. We studied the elastolytic mechanisms of pseudolysin and myroilysin by biochemical experiments and SEM observation in detail, and compared them with that of pseudoalterin. Although the degradation patterns between myroilysin and pseudolysin were slightly different under light microscope, they both formed cavities at the surface of elastin fibres under SEM. This was completely different from pseudoalterin that separated elastin into filaments firstly. Analysis of the cleavage sites of myroilysin and pseudolysin showed that they foeused on elastin hydrophobic domains but not hydrophilic domains involved in cross-linking, which may explain the different elastolytic patterns among them.
Investigation and comparison of the elastolytic mechanism of pseudoalterin. myroilysin and pseudolysin. which are from three distinct families, would be helpful to further understand the elastolytic mechanisms of elastases.
(4) The ecological role of the pseudoalterin and its mechanism to absorb and degrade peptidoglycan
Enzymes in M23family are all amidases and/or endopeptidases in peptidoglycan degradation. So the peptidoglycans of various marine bacteria were used as substrates to test the antimierobial spectrum of pseudoalterin. It was showed that pseudoalterin lysed both Gram positive and Gram negative bacteria isolated from marine environments, although the activities were not equal because of different structures among genus. Therefore, the bacteriolytic activity of pseudoalterin may have significant ecological role in deep sea and play important roles in the cycling of sedimentary organic matter.
Among various kinds of peptidoglycans, the peptidoglycan of Staphylococcus aureus is the favorite substrate of M23proteases. Therefore, the peptidoglycan of S. aureus was chosen to determine the degradation mechanism of pseudoalterin. Our result showed that in addition to hydrolyze the peptidoglycan of S. aureus quickly, pseudoalterin could also adsorb to it and other insoluble polysaccharides, such as chitin, chitose and cellulose. We hypothesized that pseudoalterin could bind to the glycans and degrade the peptides in peptidoglycan degradation.
In order to study the peptidoglycan degradation mechanism of pseudoalterin, analysis of cleavage sites on peptidoglycan by pseudoalterin was performed. The result showed that pseudoalterin was both an amidase and an endopeptidase. Except for the glycyl bonds in peptide bridges, the amido bond N-acylmuramoyl-Ala and peptide bonds Ala-Lys and Ala-Glu in peptidoglycan could also be hydrolyzed by pseudoalterin.
To gain insights into the structural basis of its adsorption and degradation mechanisms to peptidoglycan, pseudoalterin was crystallized at a resolution of1.5A. Structure analysis showed that a molecule of pseudoalterin is composed of a short α-helix (α1) and9βsheets (β1-β9). The helix α1(104-108) is located in lateral region of the molecule. Five longer antiparallel β sheets:β1(30-35), β2(56-61), β3(64-69), β4(72-78) and β5(119-125), are surrounded by loops at the center, forming a big cleft. Zn2+is located at the upper part of this cleft, indicating that this cleft is the catalytic center. Four shorter antiparallel sheets β6(134-137), β7(140-143), β8(157-160) and β9(164-165) arrange at the top right corner of the pseudoalterin molecule and form a smaller groove near Zn2+, which may interact with substrates. Pseudoalterin is a β protein, just like the other three M23proteases with tertiary structures. However, the structure of pseudoalterin has some difference from those of the other three M23proteases. In addition to the catalytic cleft, pseudoalterin has only one β domain at its top right, but the other proteases have two, one at its top right and the other at the bottom left of the molecule. Moreover, compared to those of the other proteases, the β-sheets at the top right of pseudoalterin are longer and form a wider groove, which may be responsible for peptidoglycan binding.
A proposed model of the pseudoalterin-(NAG)4complex was obtained using docking algorithm. The mode indicates that (NAG)4can be easily bound in the groove formed by (36-P9sheets in pseudoalterin. The distances shorter than3.2A between (NAG)4and pseudoalterin amino acid residues were calculated with pymol software. Analysis showed that Asn134, Argl41, Asp143, Tyr157, Glu159and Arg164were likely to form hydrogen bonds with (NAG)4。 Three amino acid residues His133, Asnl54and Arg163located in this region were also taken in our consideration. Mutations of these residues were constructed to verify the key amino acids in the adsorption process. The binding assays revealed that the last20amino acids paly important roles in pseudoalterin adsorption and the mutation of Tyrl57. Glu159, Arg163and Arg164caused dramatically decreases in pseudoalterin adsorption. Therefore, the top-right β domain is confirmed to be responsible for substrate binding. This is the first comprehensive study of an M23protease about its peplidoglycan adsorption and degradation mechanisms, which play important roles in clarifying the bacteriolytic mechanism of M23proteases.
(5) The application potential of pseudoalterin and the optimization of its production
Non-antibiotic drugs are becoming an urgent need because of increasing of multiple resistant bacteria. Because lysozymes degrade conserved structures of bacterial peptidoglycan and hardly lead to bacterial resistance, they have great application potential in anti-bacterial therapy. Pseudoalterin may have the promising use in therapy as both an amidase and an endopeptidase to degrade peptidoglycan rapidly. Peptidoglycans of various terrestrial Gram positive and Gram negative bacteria (including methieillin-resistance Staphylococcus aureus, MRSA) were extracted to test the bacteriolytic activity of pseudoalterin. The bacteriolytic effect of pseudoalterin was compared with commercialized lysozyines:mutanolysin and lysostaphin. The results showed that pseudoalterin had lytic activity to most of the gram positive bacteria and the lytic activity to S. aureus was100fold higher than lysostaphin. As a single enzyme, pseudoalterin even had better bacteriolytic effect than complex enzyme mutanolysin to some bacteria. Pseudoalterin had the highest bacteriolytic activity to S. aureus and MRSA, which were both about75%, indicating that MRSA has no resistance to pseudoalterin. The ability of pseudoalterin on killing live cells of S. aureus suggested that pseudoalterin has potential as an anti-bacterial agent.
Pseudoalterin was an induced elastase and elastin was indispensable compound in the fermentation medium for pseudoalterin production. However, purified elastin was too expensive to be used in large scale production of pseudoalterin. To reduce the cost for pseudoalterin production, various inducers were tested for their induction effects on pseudoalterin production. The result showed that bovine artery could be used as an inducer to replace elastin without loss in enzyme production. In order to find out the key factors significantly affecting pseudoalterin production of strain CF6-2, the relative significances of variables were investigated using one-factor experiment. The results showed that artery dosage, ferment temperature and ferment time were the greatest important variables for pseudoalterin production. Thus, artery dosage, ferment temperature and ferment time were selected for further optimization using central composite design (CCD). By solving the inverse matrix using Expert-Design software, the optimal values for pseudoalterin production were1.2%for artery,20.17℃for ferment temperature and28.04h for ferment time. Under the optimal conditions mentioned above, the maximum pseudoalterin activity in fermentation broth reached100.02±9.0U/mL. By optimization of the medium composition and the culture conditions using response surface methodology, pseudoalterin activity in fermentation broth was doubled. Our results improved the production of enzyme and greatly reduced the cost of fermentation, which provide a foundation for developing pseudoalterin as an anti-bacterial agent.
Pseudoalteromonas sp. CF6-2是我们从南中国海2441米海底沉积物中分离的
一株细菌,采样地点位于台湾西南九龙甲烷礁区。前期的研究表明,P. sp. CF6-2在弹性蛋白平板上可以产生很大的水解圈(水解圈/菌落直径=9.0),这说明P.sp.CF6-2是一株高产弹性蛋白酶的菌株。目前深入研究的弹性蛋白酶还没有来源于Pseudoalteromonas属细菌的,因此我们推测P. sp. CF6-2分泌的弹性蛋白酶可能是一个新型弹性蛋白酶。本论文纯化了菌株CF6-2分泌的弹性蛋白酶pseudoalterin,并对其酶学性质、基因序列、对弹性蛋白和细菌肽聚糖的降解机制、生态学功能和应用潜力进行了研究,取得了如下研究结果。
(1) Pseudoalterin的分离纯化、酶学性质研究以及基因克隆
通过阴离子交换层析,从P.sp.CF6-2发酵液中分离到一种电泳纯的蛋白酶,命名为pseudoalterin。通过研究它对不同底物的降解作用,发现该酶除了对弹性蛋白具有很高的酶活之外,对其他蛋白底物都几乎没有酶解活性,是一个特异性弹性蛋白酶。该酶在5℃时依然有10%的酶活,最适酶活温度为25℃,热稳定性很差,在35℃中保温20min,即会丧失70%的酶活,这表明pseudoalterin是典型的低温酶。该酶是一个碱性蛋白酶,pH9.5时酶活最高,pH低于8.0的缓冲体系中活性低于20%,而在pH10.5的体系中仍然保持了40%的活性,Ca2+和Sr2+显著增强pseudoalterin的酶活力量,Fe2+显著抑制pseudoalterin酶活,而Zn2+几乎完全抑制pseudoalterin(?)活性。该酶活力会被金属蛋白酶抑制剂抑制。根据以上结果推测它足一个依赖于Zn2+的金属蛋白酶。
通过使用PCR和Thermal asymmetric interlaced PCR(Tail PCR)等技术手段,克隆了pseudoalterin的全集因序列。基因全长1212bp,编码含有403个氨基酸残基的蛋白酶前体。基因序列已提交GenBankTM,序列号为HQ005379。序列分析农明,pseudoalterin为Zn金属蛋白酶,属于M23家族中的S亚家族,与该家族其他蛋白酶的序列最高相似度为39%,属于M23家族中一个新的蛋白酶。
对GenBankTM中21株深海沉积物和热泉细菌的基因组序列分析表明M23家族的蛋白酶在深海中广泛存在,因此可能在深海氮循环中发挥着重要的作用。然而它们具体的特性和作用却从未被报道。Pseudoalterin足第一个进行了性质研究的深海来源的M23家族蛋白酶。
(2)用生化方法和显微镜技术研究pseudoalterin对弹性蛋白的降解机制
M23家族蛋白酶都可以缓慢降解弹性蛋白。目前对M23家族蛋白酶降解弹性蛋白机制的研究仅限于它们在弹性蛋白或弹性蛋白类似小肽上的酶切位点的分析。而作为M23A亚家族的一个蛋白酶，pseudoalterin了以快速将不可溶弹性蛋白降解为可溶的肽段,所以研究它对弹性蛋白的降解机制对揭示M23家族蛋白酶的作用机制具有重要意义。通过光学显微镜观察pseudoalterin对牛弹性蛋白的酶解过程,发现pseudoalterin先将弹性蛋白纵向膨胀出丝状结构,推断pseudoalterin可以打开弹性蛋白内部的交联。用液相与质谱联用技术研究pseudoalterin对牛弹性蛋白的酶切位点,并通过合成小肽的酶解和锁链索(DES)的检测验证这些酶切位点,结果表明pseudoalterin可以优先降解弹性蛋白中交联区的序列。为了进一步研究pseudoalterin降解弹性蛋白的机制，我们用扫描电镜(SEM)观察了pseudoalterin对重组弹性蛋白和天然牛弹性蛋白的酶解过程。通过对酶解过程的观察,结合生化实验结果,我们提出了pseudoalterin降解弹性蛋白的机制模型：pseudoalterin优先降解弹性蛋白亲水的交联区,对疏水区作用较慢,因此在酶解过程中先使弹性蛋白分离成丝状,然后由丝状纤维进一步酶解为小球,最后酶解出弹性蛋白原单体并进一步降解为可溶的肽段。这是首次祥细阐明一个M23家族蛋白酶对弹性蛋白的降解机制。
目前报道的所有M23家族蛋白酶都仅显示出对疏水区甘氨酰键的酶解能力。因此我们揭示的pseudoalterin首先降解弹性蛋白亲水区再降解疏水区的弹性蛋白降解机制是一种崭新的M23家族蛋白酶降解弹性蛋白的机制。这也表明pseudoalterin是M23家族中一种新型的蛋白酶。我们对南海沉积物新型M23家族蛋白酶的弹性蛋白降解机制的分析为阐释深海沉积物中氮循环的研究提供了重要的证据,同时也为新型蛋白酶的开发利用提供了重要的理论基础。
(3) Pseudoalterin、myroilysin和pseudolysin三个不同家族的弹性蛋白酶对弹性蛋白降解机制的比较
陆地细菌分泌的弹性蛋白酶中研究比较多的是Pseudomonas aerugonosa分泌的M4家族pseudolysin,而深海来源的细菌弹性蛋白酶中对弹性蛋白酶解方式有所研究的是来源于Myroides profundi D25的M12家族蛋白酶myroilysin。对这两种弹性蛋白酶的作用机制虽然也有研究,但都没有通过SEM直接观察过酶解弹性蛋白的过程,对弹性蛋白降解机制也没有全面的研究。我们通过生化实验和电镜观察详细研究了pseudolysin和nyroilysin降解弹性蛋白的机制,并与pseudoalterin进行了比较。显微镜观察结果表明这两种弹性蛋白酶对弹性蛋白的降解模式与pseudoalterin完全不同。光镜下二者对弹性蛋白的降解方式略有不同,myroilysin倾向于优先将弹性蛋白横切成段状,pseudolysin对弹性蛋白则进行不规则切割。在SEM下,pseudolysin和myroilysin都首先在弹性蛋白表面形成孔洞,这跟pseudoalterin先将弹性蛋白拆分成丝状结构的降解方式不同。对这两种酶在牛弹性蛋白上的酶切位点分析发现它们的作用位点都集中在弹性蛋白的疏水区,而不是交联区,这可能正是造成它们酶解现象不同的原因。研究和比较pseudoalterin、myroilysin和pseudolysin三个不同家族弹性蛋白酶对弹性蛋白的降解机制,可以对弹性蛋白酶的作用机制有史深入的了解。
(4) Pseudoalterin裂菌活性的生态作用及其吸附和降解肽聚糖的机制
M23家族的蛋白酶都是可以酶解细菌细胞壁肽聚糖的酰胺酶或内肽酶,为了研究pseudoalterin勺裂菌活性在海洋中的生态作用,我们研究了它对海洋来源的革兰氏阳性细菌和部分革兰氏阴性细菌的肽聚糖的裂解活性。结果表明pseudoalterin对这些细菌都具有裂解活性,虽然不同种属之间肽聚糖结构不同学致了这种裂解活性具有一定差异。因此我们认为M23家族蛋白酶的裂菌活性决定了它们在深海有机质循环中发挥着重要的作用。
M23家族的蛋白酶对金黄色葡萄球菌的裂解活性最强。因此我们选择金黄色葡萄球菌的肽聚糖作为对象研究了pseudoalterin对肽聚糖的降解机制。我们实验结果表明,pseudoalterin不仅可以快速降解金葡萄肽聚糖,而且对肽聚糖以及其他几种不可溶多糖如几丁质、壳聚糖、纤维素等具有吸附作用。因此我们推测pseudoalterin在降解肽链部分之前可能先通过吸附结构域吸附到肽聚糖的糖链上
为了阐明pseudoalterin降解肽聚糖的机制,我们分析了pseudoalterin对肽聚糖的酶切位点。对pseudoalterin酶解后的肽聚糖产物进行液质联分析，结果表明pseudoalterin既是一个N-乙酰-L-丙氨酸酰胺酶,又是一个内肽酶。Pseudoalterin除了可以从多个位点切断五甘氨酸肽桥,还可以切开N-acylmuramoyl-Ala之间的酰胺键、肽桥与肽尾连接处的肽键Ala-Lys,以及肽尾的肽键Ala-Glu。
为了研究蛋白酶pseudoalterin吸附肽聚糖的机制,我们对蛋白酶pseudoalterin进行了结晶,通过X-射线衍射得到了辨率为1.5A的分子结构。对pseudoalterin的结构分析表明,一个pseudoalterin分子山1个α螺旋(α1)和9个β折叠片(β1-β9)组成,其中α螺旋非常短(104-108),位于分子外侧。5个较长的β折叠片β1(30-35)、β2(56-61)、β3(64-69)、β4(72-78)和β5(119-125)反平行排列并被loop包围在分子内部,形成一个较大的空腔,从空腔上半部分的Zn2+可以推测出这足该酶的催化腔。β6(134-137)、β7(140-143)、β8(157-160)和β9(164-165)这四个C端的β折叠片较短,反平行排列在分子右上方外侧,形成一个小沟。在靠近金属离子的一端,催化腔与这个小沟呈T字形排列。与M23家族已有的3个结构相比,pseudoalterin与它们都属于β型蛋白。但是pseudoalterin没有左下方的β domain,只有右上方的β domain,而且β折叠片更长,我们推测这个β domain的功能可能跟多糖的吸附附有关
为了研究蛋白酶pseudoalterin与肽聚糖糖链的相互作用方式,我们将壳四糖((NAG)4)通过Auto dock与pseudoalterin的β6-β9形成的小沟进行分子共模拟。结果表明,(NAG)4能够与pseudoalterin该区域相互作用,嵌于pseudoalterin的loop之间。利用pymol软件计算与(NAG)4的空间距离在3.2A内可能与底物形成氢键的氨基酸残基。结果表明位于吸附腔附近的氨基酸残基Asn134、Arg141、 Asp143、Tyr157、Glu159和Arg164与(NAG)4的距离都在此范围内。另外位于这一区域但与(NAG)4距离稍远的氨基酸His133、Asn154和Arg163也可能参与了这一过程。通过点突变构建了以上氨基酸的突变体并分析了这些突变体对肽聚糖的吸附能力,结果表明pseudoalterin C端20个氨基酸中的Tyr157,Glu159,Arg163和Arg164对pseudoalterin的吸附具有重要影响,从而确定了pseudoalterin右上方的β domain确实是一个多糖吸附结构域,阐明了pseudoalterin对肽聚糖的吸附机制。这是首次对一个M23家族蛋白酶吸附和降解肽聚糖的机制进行详细研究,这对进一步揭示该家族蛋白酶裂解细菌的机制具有重要意义。
(5) Pseudoalterin的应用潜力研究及其发酵条件的优化
多重抗药菌的出现导致对非抗生素类杀菌剂的强烈需求。溶菌酶由于降解肽聚糖保守结构成分,细菌对其不易产生抗药性,在医疗上具有比较好的应用潜力。我们的实验结果表明,pseudoalterin是一个可以通过酰胺酶和内肽酶活性快速降解细菌肽聚糖的溶菌酶,具有良好的应用前景。我们以多种陆地来源的革兰氏阳性菌和阴性菌的细胞壁为底物,研究蛋白酶pseudoalterin在特定浓度和作用时间下对各种细菌的裂解能力,特别是对多抗性金黄色葡萄球菌的裂解能力。并且将其裂解细菌的能力与商品化的溶菌酶mutanolysin和lysostaphin进行了比较。结果表明,pseudoalterin对taphylococcus aureus细胞壁的裂解能力比lysostaphin高出100多倍,而且对多种细菌细胞壁都具有裂解能力。Pseudoalterin作为单一酶,对一些细菌细胞壁的裂解能力甚至比mutanolysin这种复合酶效果还要好。Pseudoalterin对多抗菌的裂解能力与普通金葡萄一样,说明多抗菌对这种溶菌酶没有抗性。对金葡菌活细胞的杀灭实验也证明pseudoalterin确实可以通过降解肽聚糖杀灭细菌,因而在临床上具有潜在的应用价值。
Pseudoalterin是一个诱导酶,在发酵中需要加入弹性蛋白诱导其产生。但是弹性蛋白的昂贵价格限制了pseudoalterin的大规模发酵。本论文研究了不同诱导物对pseudoalterin产量的影响,实验发现心管能完全代替弹性蛋白诱导酶的产生,而不降低产量。利用单因子实验确定了三个对酶发酵影响显著的因子,分别为心管用量、培养温度和培养时间。用响应面法优化了这三个因子的组合,分别为心管粉1.2%，培养温度20.17℃,培养时间28.04h。在此条件下发酵液的酶活达到100.02±9.0U/mL。相比于优化前的46.9±3.9U/mL提高了一倍。因此,我们的研究结果不仅提高了pseudoalterin的产量,而且大幅度降低了发酵成本,这为pseudoalterin的进一步开发应用奠定了很好的基础。
Degradation of organic nitrogen is an important part of global nitrogen cycling. In the ocean, High-Molecular-Weight Organic Nitrogen (HMWON) produced by organisms in the seawater settles and accumulates in the sediment in the form of particulate organic nitrogen (PON). Most of PON will be degraded into dissolved organic nitrogen (DON) by microorganisms, and then participates into the nitrogen cycling through ammonium regeneration, nitrification and denitrification in the sediment. Thus, the recycling of PON in deep-sea sediment would be a non-negligible part of ocean nitrogen cycling. Recent studies indicate that a significant fraction of PON is composed of insoluble amides and organisms living in the sediment. Because of the insoluble properties, collagens and elastins are speculated to be important components of sedimental PON. Elastins are made up of tropoelastin monomers with alternating hydrophobic and hydrophilic domains which are cross-linked complicatedly. Because of the rigid structure, elastins are resistant to most proteases except for a limited number of elastases. Up to now, the reports about elastolytic proteases were mostly focused on elastases from animals. The studies of elastases from microorganisms are relatively fewer and most of bacterial elastases are pathogenic and terrestrial. Thus the elastolytic mechanism of the ocean is unclear. There are abundant elastase-produced bacteria in deep-sea sediment. Elastases from different strains show different elastolytic abilities which imply the diversity of elastases in deep-sea. The peptidoglycans of bacteria living in sediment are also important components of sedimental PON. However, the studies of enzymes from deep sea that could degrade peptidoglycans are limited. Research into the function and mechanism of marine proteases will provide important implications for deep-sea nitrogen cycling and novel proteases development.
Pseudoalteromonas sp. CF6-2was isolated from the deep-sea sediment at a water depth of2,441m in the Jiulong methane reef area off the southwest of the island of Taiwan during the South China Sea Open Cruise of R/V Shiyan3. Our previous study showed that P. sp. CF6-2could produce hydrolytic zone on elastin agar plates with the hydrolytic zone diameter/the colony diameter (H/C)=9.0, suggesting that P. sp. CF6-2is a good producer of elastase. Because no elastase from bacteria in Pseudoalteromonas genus has been reported, we speculated that strain CF6-2secrets a novel elastase. In this thesis, the elastase, named pseudoalterin. was purified from the fermentation broth of P. sp. CF6-2and its enzymatic characteristics, gene sequence, degradation mechanisms to elastin and peptidoglycan and application potential were studied. The results are as following:
(1) Purification, characteristics and gene cloning of pseudoalterin
A protein with a molecular mass of19kDa was purified from the fermentation broth by a DEAE-Sepharose Fast Flow chromatography. which was designated as pseudoalterin. The determination of the substrate specificity showed that pseudoalterin could hardly hydrolyze any protein substrate except elastin. This indicated that it is a strict elastase. Pseudoalterin retained10%activity at5℃and its optimum temperature was25℃. Its thermostability was very low. and70%activity would be lost after it was incubated at35℃for20min. These properties indicated that this protease was a typical cold-adapted enzyme. Pseudoalterin was an alkaline elastase with an optimum pH of9.5. Lower than20%activity could be detected under pH8.0while40%activity was retained at pH10.5. Ca2+and Sr2+markedly increased the enzyme activity of pseudoalterin, and Fe2+severely inhibited the enzyme activity. Zn2+abolished the elastolytic activity completely. Moreover, the activity of pseudoalterin could be inhibited by metalloprotease inhibitors. So pseudoalterin was a Zn2+dependent metallopeptidasc.
The full-length gene of pseudoalterin was cloned from P. sp. CF6-2by PCR and thermal asymmetric interlaced PCR (Tail PCR) techniques. The ORF contains1212bp, encoding a precursor of403amino acid residues. The nucleotide sequence was submitted to the GenBankTM with accession number HQ005379. Analysis of the amino acid sequence showed that pseudoalterin is a zinc metalloprotease. Among the characterized proteases, pseudoalterin shows the highest identity (39%) to staphylolysin (M23A subfamily) from Pseudomoiuis aeruginasa PAO1(accession numberAAG05260). The conserved residues in and around the active site of M23 proteases are also found in pseudoalterin. These results all point to pseudoalterin as a novel metalloprotease of the M23A subfamily.
Analysis of the genomes of21bacteria isolated from deep-sea sediments and hydrothermal vents, which are in GenBankTM database, indicates that most of these bacteria could secrete one or several M23family proteases. This finding suggests that the M23family proteases may be popular in deep sea sediments and may therefore play an important role in sedimentary PON degradation. However, the properties and functionality of marine M23proteases have not been reported. Pseudoalterin was the first characterized marine elastase in M23family.
(2) Elastolytic mechanism of pseudoalterin
M23proteases could slowly degrade elastions. However, researches of elastolytic mechanism of M23proteases were limited to the analysis of their cleavage sites on elastin or elastin-like peptides. As a M23A subfamily protease, pseudoalterin could quickly degrade insoluble elastin into soluble peptides. Studying its elastolytic mechanism is helpful for clarifying the degradation mechanism of M23elastases. Microscopic observations indicated that pseudoalterin swelled elastin and separated elastin into filaments, implying that this enzyme initially broke down cross-links between the filaments. Analysis of the cleavage sites of pseudoalterin on bovine elastin and synthetical peptides indicated that pseudoalterin preferentially hydrolyzed elastin cross-linking domains. To further study the elastolytic mechanism of pseudoalterin, scanning electron microscope (SEM) observation of recombinant tropoelastin spherules and bovine elastin degradation process was performed. Based on our biochemical results and SEM observation, the elastolytic mechanism of pseudoalterin was concluded:pseudoalterin successively released filaments, droplets, and spherules from elastic fibers by destroying the cross-links at each structural level. This is the first detailed research of a M23elastase about its elastolytic mechanism.
All the proteases in M23family with investigation only cleaved Gly-Xaa bonds in hydrophobic domains in elastin. Therefore, that pseudoalterin hydrolyzed cross-linking domains in elastin before hydrophobic domains represents a new-type elatolytic mechanism in M23family. It also revealed that pseudoalterin is a novel protease in this family. The insight into the novel elastase from the sediment and its mechanism provides important evidences for nitrogen cycling in deep-sea sediment. The research also provides theoretical basis for the development of novel proteases.
(3) Comparison of pseudoalterin, myroilysin and pseudolysin on their elastolytic mechanism
Of all the elastases from terrestrial bacteria, pseudolysin in M4family from pathogenic bacteria Pseiidomonas aerugonosa was detailed investigated. Myroilysin was the only marine elastase of which the elastin degradation pattern was studied. Myroilysin is an M12family protease from deep-sea sediment bacterium Myroides profundi D25. The detailed elastolytic mechanisms of pseudolysin and myroilysin are still unclear. We studied the elastolytic mechanisms of pseudolysin and myroilysin by biochemical experiments and SEM observation in detail, and compared them with that of pseudoalterin. Although the degradation patterns between myroilysin and pseudolysin were slightly different under light microscope, they both formed cavities at the surface of elastin fibres under SEM. This was completely different from pseudoalterin that separated elastin into filaments firstly. Analysis of the cleavage sites of myroilysin and pseudolysin showed that they foeused on elastin hydrophobic domains but not hydrophilic domains involved in cross-linking, which may explain the different elastolytic patterns among them.
Investigation and comparison of the elastolytic mechanism of pseudoalterin. myroilysin and pseudolysin. which are from three distinct families, would be helpful to further understand the elastolytic mechanisms of elastases.
(4) The ecological role of the pseudoalterin and its mechanism to absorb and degrade peptidoglycan
Enzymes in M23family are all amidases and/or endopeptidases in peptidoglycan degradation. So the peptidoglycans of various marine bacteria were used as substrates to test the antimierobial spectrum of pseudoalterin. It was showed that pseudoalterin lysed both Gram positive and Gram negative bacteria isolated from marine environments, although the activities were not equal because of different structures among genus. Therefore, the bacteriolytic activity of pseudoalterin may have significant ecological role in deep sea and play important roles in the cycling of sedimentary organic matter.
Among various kinds of peptidoglycans, the peptidoglycan of Staphylococcus aureus is the favorite substrate of M23proteases. Therefore, the peptidoglycan of S. aureus was chosen to determine the degradation mechanism of pseudoalterin. Our result showed that in addition to hydrolyze the peptidoglycan of S. aureus quickly, pseudoalterin could also adsorb to it and other insoluble polysaccharides, such as chitin, chitose and cellulose. We hypothesized that pseudoalterin could bind to the glycans and degrade the peptides in peptidoglycan degradation.
In order to study the peptidoglycan degradation mechanism of pseudoalterin, analysis of cleavage sites on peptidoglycan by pseudoalterin was performed. The result showed that pseudoalterin was both an amidase and an endopeptidase. Except for the glycyl bonds in peptide bridges, the amido bond N-acylmuramoyl-Ala and peptide bonds Ala-Lys and Ala-Glu in peptidoglycan could also be hydrolyzed by pseudoalterin.
To gain insights into the structural basis of its adsorption and degradation mechanisms to peptidoglycan, pseudoalterin was crystallized at a resolution of1.5A. Structure analysis showed that a molecule of pseudoalterin is composed of a short α-helix (α1) and9βsheets (β1-β9). The helix α1(104-108) is located in lateral region of the molecule. Five longer antiparallel β sheets:β1(30-35), β2(56-61), β3(64-69), β4(72-78) and β5(119-125), are surrounded by loops at the center, forming a big cleft. Zn2+is located at the upper part of this cleft, indicating that this cleft is the catalytic center. Four shorter antiparallel sheets β6(134-137), β7(140-143), β8(157-160) and β9(164-165) arrange at the top right corner of the pseudoalterin molecule and form a smaller groove near Zn2+, which may interact with substrates. Pseudoalterin is a β protein, just like the other three M23proteases with tertiary structures. However, the structure of pseudoalterin has some difference from those of the other three M23proteases. In addition to the catalytic cleft, pseudoalterin has only one β domain at its top right, but the other proteases have two, one at its top right and the other at the bottom left of the molecule. Moreover, compared to those of the other proteases, the β-sheets at the top right of pseudoalterin are longer and form a wider groove, which may be responsible for peptidoglycan binding.
A proposed model of the pseudoalterin-(NAG)4complex was obtained using docking algorithm. The mode indicates that (NAG)4can be easily bound in the groove formed by (36-P9sheets in pseudoalterin. The distances shorter than3.2A between (NAG)4and pseudoalterin amino acid residues were calculated with pymol software. Analysis showed that Asn134, Argl41, Asp143, Tyr157, Glu159and Arg164were likely to form hydrogen bonds with (NAG)4。 Three amino acid residues His133, Asnl54and Arg163located in this region were also taken in our consideration. Mutations of these residues were constructed to verify the key amino acids in the adsorption process. The binding assays revealed that the last20amino acids paly important roles in pseudoalterin adsorption and the mutation of Tyrl57. Glu159, Arg163and Arg164caused dramatically decreases in pseudoalterin adsorption. Therefore, the top-right β domain is confirmed to be responsible for substrate binding. This is the first comprehensive study of an M23protease about its peplidoglycan adsorption and degradation mechanisms, which play important roles in clarifying the bacteriolytic mechanism of M23proteases.
(5) The application potential of pseudoalterin and the optimization of its production
Non-antibiotic drugs are becoming an urgent need because of increasing of multiple resistant bacteria. Because lysozymes degrade conserved structures of bacterial peptidoglycan and hardly lead to bacterial resistance, they have great application potential in anti-bacterial therapy. Pseudoalterin may have the promising use in therapy as both an amidase and an endopeptidase to degrade peptidoglycan rapidly. Peptidoglycans of various terrestrial Gram positive and Gram negative bacteria (including methieillin-resistance Staphylococcus aureus, MRSA) were extracted to test the bacteriolytic activity of pseudoalterin. The bacteriolytic effect of pseudoalterin was compared with commercialized lysozyines:mutanolysin and lysostaphin. The results showed that pseudoalterin had lytic activity to most of the gram positive bacteria and the lytic activity to S. aureus was100fold higher than lysostaphin. As a single enzyme, pseudoalterin even had better bacteriolytic effect than complex enzyme mutanolysin to some bacteria. Pseudoalterin had the highest bacteriolytic activity to S. aureus and MRSA, which were both about75%, indicating that MRSA has no resistance to pseudoalterin. The ability of pseudoalterin on killing live cells of S. aureus suggested that pseudoalterin has potential as an anti-bacterial agent.
Pseudoalterin was an induced elastase and elastin was indispensable compound in the fermentation medium for pseudoalterin production. However, purified elastin was too expensive to be used in large scale production of pseudoalterin. To reduce the cost for pseudoalterin production, various inducers were tested for their induction effects on pseudoalterin production. The result showed that bovine artery could be used as an inducer to replace elastin without loss in enzyme production. In order to find out the key factors significantly affecting pseudoalterin production of strain CF6-2, the relative significances of variables were investigated using one-factor experiment. The results showed that artery dosage, ferment temperature and ferment time were the greatest important variables for pseudoalterin production. Thus, artery dosage, ferment temperature and ferment time were selected for further optimization using central composite design (CCD). By solving the inverse matrix using Expert-Design software, the optimal values for pseudoalterin production were1.2%for artery,20.17℃for ferment temperature and28.04h for ferment time. Under the optimal conditions mentioned above, the maximum pseudoalterin activity in fermentation broth reached100.02±9.0U/mL. By optimization of the medium composition and the culture conditions using response surface methodology, pseudoalterin activity in fermentation broth was doubled. Our results improved the production of enzyme and greatly reduced the cost of fermentation, which provide a foundation for developing pseudoalterin as an anti-bacterial agent.
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