摘要
随着社会经济的发展,人类面临着由资源的匮乏和能源的短缺所带来的诸多挑战。另一方面,木质纤维素作为地球上最为丰富的可再生资源,将会给人类社会的进步以及经济的发展带来新的资源基础,每年地球上的植物通过光合作用能够产生高达1011吨的纤维素。然而,其特殊的结构特征如水不溶性、高度结晶、相邻纤维素链间强烈的作用力(包括氢键、疏水作用)等造成了木质纤维素材料的生物转化效率低下。因此,实现纤维素的高效降解已经成为可再生能源研究的一个主要关注点。
1938年,Walker和Warren首次从土壤中分离得到了好氧的革兰氏阴性细菌Cytophaga hutchinsonii。它能够迅速的降解滤纸,具有高效的纤维素降解能力。初步研究表明,C. hutchinsonii具有独特的纤维素降解机制,可能不同于其它两种已得到广泛研究的纤维素降解策略,即真菌游离纤维素酶系及厌氧细菌纤维小体复合酶系。Cytophaga hutchinsonii同某些其他纤维素降解菌一样需要吸附到纤维素表面实现对纤维素的降解,因此,菌体细胞对纤维素底物的吸附可能是C. hutchinsonii对纤维素进行有效降解的第一步。因此研究Cytophaga hutchinsonii通过何种方式与纤维素进行吸附,阐明其吸附机制,将有利于深入了解其独特的纤维素降解机制,对纤维素这一丰富的可再生资源的利用具有重要的理论意义。
因此,本论文围绕Cytophaga hutchinsonii吸附突变株的筛选及其生理生化表征,从以下几个方面进行了研究：
1)突变株的诱变筛选。
采用传统的物理化学方法如紫外线诱变、EMS诱变以及自发突变和转座子随机插入突变等方法对C. hutchinsonii进行突变筛选,得到两株吸附突变株、两株滑动突变株及多株未知功能的突变株。所获得的吸附突变株其吸附能力出现稳定性的降低,对结晶纤维素吸附效率为野生型的20%-40%。突变株无论是在葡萄糖还是在纤维素为唯一碳源的培养基中,其生长能力均不如野生型,推测原因有可能是突变使细胞表面的某些蛋白功能发生缺失或变化,从而影响到糖的转运或者是菌体对纤维素的吸附而对菌体的生长造成影响。
有研究表明,C. hutchinsonii可能需要通过在纤维素表面上的滑动来实现对纤维素的有效降解,而滑动性的增加有可能会增加菌体对纤维素的降解能力。通过紫外线诱变我们得到两株在琼脂培养基上生长扩散发生明显变化的菌株,但其在纤维素底物上的生长特征没有出现明显提高,具体原因有待进一步研究。通过转座子随机插入诱变得到几株突变株,但对其吸附、运动以及生长特性进行研究,均没有发现与野生型明显的差异。经质粒拯救得到其基因失活的座位,保守序列进行分析发现CHU 0456含有一个FN3 Superfamily结构域,而此结构域预测可能是与底物吸附相关的,但对其进行吸附性质研究时却没出现吸附现象,CHU 0456在基因组中发挥着怎样的功能,这有待于后续试验的进一步研究。
2) Cytophaga hutchinsonii膜相关蛋白提取方法的研究
目前对膜蛋白的研究已经受到越来越多的关注。基因组序列分析表明,在Homo sapiens, Escherichia coli和Saccharomyces cerevisae中有30%的编码蛋白质是膜整合蛋白。由于膜蛋白所具有的一些特有的性质如高疏水性,低丰度等,使其成为较难研究的对象之一。初步研究表明,Cytophaga hutchinsonii对结晶纤维素的吸附降解极有可能与其膜表面的酶(蛋白)有关,因此我们对其膜蛋白的提取方法进行了初步探索,包括总膜蛋白的提取以及膜蛋白的分级分离。结果表明,Cytophaga hutchinsonii膜蛋白提取的最适去污剂为1% DDM。在4℃条件下处理3h可以释放最大量的膜蛋白,并通过实验表征了此两种方法的可行性。
3)纤维素吸附膜相关蛋白的鉴定
由于纤维素对吸附蛋白具有浓缩的作用,因此可以通过富集来鉴定与纤维素吸附相关的膜蛋白。通过将提取的葡萄糖培养外膜蛋白与纤维素混合进行吸附,随后SDS/PAGE电泳检测比较吸附前后富集的蛋白条带,发现有6条蛋白条带发生浓缩,将这些蛋白条带进行质谱鉴定,得到4个富集蛋白。其中3个是是功能未知的蛋白,另外1个是与滑动相关的蛋白。对此滑动蛋白进行的异源表达尝试没有成功,可能是由于膜蛋白结构的特殊性使其难以表达。
此外,为了比较吸附缺陷突变株与野生型膜相关蛋白的差异,通过SDS/PAGE电泳检测比较吸附缺陷突变株与野生型全膜蛋白与纤维素吸附之后相关差异膜蛋白。发现吸附缺陷突变株中有1条蛋白条带发生缺失,并在最上方出现一条明显的浓缩条带,将这些蛋白条带进行质谱鉴定,得到2个未知功能蛋白。对此缺失蛋白进行的异源表达尝试同样没有成功。
4)不同碳源培养条件下膜相关蛋白的双向电泳分析
C. hutchinsonii需要吸附到纤维素底物上才可以完成对纤维素的降解,因此在以纤维素为碳源培养过程中,某些膜蛋白可能会得到大量表达,因此比较以纤维素及葡萄糖为碳源培养C. hutchinsonii全膜及全膜吸附相关蛋白对于相关吸附蛋白的寻找具有重要作用。为了寻找以纤维素及葡萄糖为碳源培养C. hutchinsonii之间表达差异膜蛋白,采用双向电泳技术对纤维素、葡萄糖培养获得的总膜蛋白及吸附膜蛋白进行双向电泳检测差异蛋白点。纤维素、葡萄糖培养的总膜蛋白中,有14个差异点；吸附膜蛋白中,发现存在14个差异蛋白点,后续试验需要质谱鉴定这些差异蛋白,并对这些差异蛋白进行性质研究,以揭示可能的吸附机制。
5)不同碳源培养条件下膜相关蛋白的同位素相对标记与绝对定量技术(isobaric tags for relative and absolute quantitation, iTRAQ)定量分析
将以纤维素和葡萄糖为唯一碳源分别培养的C. hutchinsonii提取总膜蛋白,或以结晶纤维素吸附后所得蛋白进行iTRAQ检测差异分析。在葡萄糖及纤维素上培养的总膜蛋白中,共有182个差异表达的蛋白,其中有90个纤维素上培养的膜蛋白发生上调表达,多数这些差异膜蛋白在分子功能上是与吸附及催化相关的；在葡萄糖上培养的总膜蛋白及吸附蛋白,有89个蛋白在吸附蛋白中的量多于总膜蛋白中的,这89个蛋白可能是吸附之后浓缩了的蛋白,可能参与到纤维素的吸附；纤维素上培养的总膜蛋白及吸附蛋白,有84个蛋白在吸附蛋白中的量多于总膜蛋白中的,这84个蛋白可能是吸附之后浓缩了蛋白,可能参与到纤维素的吸附。以纤维素及葡萄糖为唯一碳源培养提取总膜蛋白经纤维素吸附同时浓缩的蛋白有40个,且浓缩都较为明显。
另外,通过超速离心方法提取C. hutchinsonii膜相关蛋白,经iTRAQ检测发现有17个可能是纤维素降解或吸附膜相关蛋白,其中9个与Gary Xie等对纤维素酶的预测相一致。
除此之外,我们检测到8种Gary Xie等没有预测到的膜蛋白可能参与纤维素降解或吸附相关膜蛋白,根据功能可以将这17个可能参与纤维素降解或吸附的膜蛋白分成三类即有2个膜相关蛋白仅参与纤维素的吸附,7个膜相关蛋白仅参与纤维素的降解,8个膜相关蛋白既参与纤维素的吸附有参与纤维素的降解,但这仍需后续试验进一步的验证。
Along with progress of society and the development of economy, the human beings have to be confront with many challenges, such as meager resources and shortage of energy, which is the undoubted barriers of society and economy. On the other hand, as the most abound renewable resource on the earth, cellulose would be brought new fundamental resources for society and economy. More than 1011 tons of cellulose was produced every year. However, because of its water-insolubility, high crystallization and strong force between cellulose chains(eg. hydrogen bond, hydrophobic interaction), biotransformation of cellulose is inefficient. Thus, it is one of the main concerns on the research of renewable resources to develop efficient methods of degradation of cellulose.
In 1938, Cytophaga hutchinsonii, the abundant aerobic gram-negative celluloytic soil bacterium, was first isolated from the soil by Walker and Warren. Cytophaga hutchinsonii could degrade quickly the filters and posses the efficient capability of cellulose degradation. One research suggests that a third strategy to degrade cellulose is employed which is different from that of most aerobic cellulolytic microorganisms are employed by secreting a set of individual cellulases and that of most anaerobic microorganisms are used by cellulosomes complex. Similar to other cellulolytic bacteria, it is needed for Cytophaga hutchinsonii to direct contact with cellulose for efficient digestion, thus the adherence between strains and cellulose might be the first step to disgest the cellulose efficiently. Consequently, to research the mechanism of adhesion to cellulose would be benefit for understanding its unique strategy of cellulose degradation, which is crucial for the utilization of cellulose.
In this thesis, we embarked on the isolation of adherence-defective mutants and their physiological and biochemical characterizations. The research was conducted as the following aspects. Firstly, isolation of mutants.
To achieve C. hutchinsonii mutants, several mutant methods including UV mutagenesis, EMS mutagenesis, Spontaneous mutation and Transposon mutagenesis are employed. Two adherence-defective mutants, two gliding mutants and other unknown functional mutants of C. hutchinsonii were isolated.
One adherence-defective mutant was isolated by employing the EMS mutagenesis and Spontaneous mutation, respectively. the capability of adherence was decreased steadily to 20-40%. Furthermore, the growth of these two adherence-defective mutants on glucose and cellulose were determined by measuring cellular proteins by digesting with NaOH. Whether growth on glucose or cellulose, the growth of mutants preformed weaker than that of wild type C. hutchinsonii, which is possible that the expression of some proteins such as the proteins related to transportation of oligosaccharides or proteins related to adhesion are changed or deleted in mutants.
Based on the former researches, gliding along cellulose might be necessary for C. hutchinsonii to degrade the cellulose, which could enhance the ability of digestion of cellulose. Two mutants that whose spread capability on the agar medium were changed significantly, were isolated by employing UV mutagenesis. However, the growth of these two mutants kept in the same level with the wild type C. hutchinsonii.
The further researches are needed to understand this phenomenon.
Furthermore, several unknown functional mutants were also isolated through transposon mutagenesis. There are few change regardless of any aspects such as adhesion, mobility and growth. However, the inactivated genes were identified by plasmid rescue. The further researches of specific effects of these genes on C hutchinsonii are necessary.
Secondly, isolation of membrane proteins.
Nowadays, there is a trend that increasing researches focused on membrane proteins, including their structures, functions and generations. O Sequence analysis of genomes of Homo sapiens, Escherichia coli and Saccharomyces cerevisae suggests that about third of all genes of various organisms encode membrane proteins. However, due to their high hydrophobicity and low abundance, membrane proteins are demonstrated to be one of the most difficult researches.
The former researches suggest that the adherence of C. hutchinsonii to cellulose might be related to the surface proteins of cells. Thus, the primary extraction and separation procedures of membrane proteins were developed in this thesis. We found that 1%DDM might be the ideal detergent to release the maximum membrane proteins being treated 3 hours at 4℃.
Thirdly, identification of adherence membrane proteins through SDS/PAGE
Because of enrichment of adherence membrane proteins by binding to cellulose, they could be separated through the enrichment.. Six concentrated outer membrane proteins growth on glucose were detected by employing SDS/PAGE. Four proteins were identified by mass spectrum, of which three were hypothetical proteins, another One was related to mobility. The heterologous expression of this gliding motility-like protein was attempted yet failed, which was probably mainly due to the special structure of membrane proteins. Besides, the adherence membrane proteins of adherence-defective mutants and wild type C. hutchinsonii were also compared with similar method. Two more membrane proteins related to adherence were achieved.
Fourthly, determination of differential membrane proteins under different cultural conditions through Two Dimensional Electrophoresis (IEF/SDS-PAGE)
Cytophaga hutchinsonii requires direct contact with cellulose for efficient digestion, so some proteins might be over expressed on the cellulose. It is crucial for adherence membrane proteins through comparison of all membrane proteins and adherence membrane proteins when cells were cultivated on different carbon sources through Two Dimensional Electrophoresis (IEF/SDS-PAGE). In the comparison of differential proteins of all membrane, fourteen protein spots were detected, fourteen as well as in the comparison of adherence-related differential membrane proteins. The further researches about such as the identification of these proteins by MALDI-TOF MS and the characterizations of adherence membrane proteins are needed to explain the adherence mechanism.
Fifthly, determination and analysis of differential membrane proteins on different carbon sources through Isobaric Tags for Relative and Absolute Quantitation (iTRAQ)
We determined the differential membrane proteins by comparison of all membrane proteins and adherence membrane proteins respectively growth on glucose and cellulose through Isobaric Tags for Relative and Absolute Quantitation (iTRAQ). According to In the comparison of all differential membrane proteins on glucose and cellulose, amount about one hundred and eighty-two differential proteins were detected. Most of them were related to adherence and catalytic in molecular function. Among them, the expression level of ninety differential membrane proteins was up-regulated on cellulose.
Besides, We also detected several enriched membrane proteins when binding to cellulose, no matter C. hutchinsonii growing on glucose or cellulose. There are 89 and 84 enriched membrane proteins growing on glucose and cellulose, respectively, which might be related to cellulose adherence. No matter cultivated on glucose or cellulose, forty of all these detected membrane proteins were both enriched when binding to cellulose. Furthermore, seventeen membrane proteins perhaps related to cellulose degradation or adherence were identified. Nine of them were the same as the proteins predicted by Gary Xie, et al. in 2007. The other eight membrane proteins r were not reported before. Based on their predicted functions and Itraq results, three categories were classified:two of them are only related to cellulose adherence; seven of them are only related to cellulose degradation; eight of them are related to cellulose adherence and cellulose degradation. Future studies are needed.
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