海洋细菌Glaciecola mesophila KMM241产木聚糖酶的结构、功能和应用潜力研究
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摘要
海洋总面积占到地球面积70%以上,海水占到地球总水量的97%,因此海洋环境是地球生态环境的重要组成部分,海洋碳循环在地球生物圈碳循环中起到至关重要的作用。在海洋碳循环过程中,海洋微生物扮演着重要的角色,它们将海洋浮游植物和光合细菌通过光合作用储存的有机碳重新分解,以二氧化碳的形式重新释放到大气圈中。木聚糖是一种最主要的半纤维素,是地球上仅次于纤维素的第二大碳源物质,也是海洋环境中有机碳的重要组成部分。与纤维素相比,木聚糖结构更为复杂,木聚糖的完全降解需要多种酶类的协同作用。β-1,4-内切木聚糖酶(EC3.2.1.8)能够水解木聚糖主链的β-1,4-木糖苷键,在木聚糖的降解中起到关键作用,因此在整个海洋碳循环中也具有重要意义。海洋环境普遍具有高盐,高流动性的特点,而深海区域(深度超过2000米)更是具有高压,低温,黑暗,寡营养等极端特征。海洋中的微生物为了适应海洋环境,进化出了许多与之相适应的特点,并且在其基因组中往往蕴含着许多有价值的基因资源。因此海洋不但是物种多样性的宝库,也是基因的宝库。对这些基因资源的序列分析和功能鉴定对于研究海洋生境中的许多生化过程具有重要理论意义。
Glaciecola mesophila KMM241是一株分离自海洋无脊椎动物的海洋细菌。我们的前期研究表明,该菌株能够产生多种木聚糖酶,并且在前期研究中我们已经对该菌株产生的一个属于糖苷水解酶第十家族的木聚糖酶XynA进行了基因克隆和性质研究。在此基础上,本论文对该菌株所产的另一个木聚糖酶XynB进行了基因序列、分子结构、生化性质及其N端结构域的功能研究,并探讨了木聚糖酶XynA在提高面包品质中的应用潜力,取得了以下主要结果。
1.木聚糖酶XynB的基因克隆和序列分析
由于菌株G mesophila KMM241与另一株基因组序列已经公布的海洋细菌Pseudoalteromonas atlantica T6c的16S rDNA序列相似性为99%。根据菌株Pseudoalteromonas atlantica T6c基因组中木聚糖酶基因(Gene ID:4172855)的序列设计的一对特异性引物,以菌株G mesophila KMM241基因组DNA为模板,PCR扩增得到了木聚糖酶基因xynB的全序列。序列分析表明,xynB基因序列全长2739bp,编码的木聚糖酶XynB的前体含912个氨基酸残基。木聚糖酶XynB的前体由N端的信号肽序列(Metl-Ala43),一个功能未知的N端结构域(NTD, Glu44-Arg584)和一个C端的糖苷水解酶第八家族(GH8)催化结构域(CD,Ser585-Glu912)组成。通过序列比对发现只有三个酶与XynB氨基酸全序列具有同源性,分别是来自菌株P. atlanlica T6c (98%), Glaciecola chathamensis S18K6(80%)和Glaciecola polaris LMG21857(73%)的糖苷水解酶,但这三个酶都是从基因组序列中推定的糖苷水解酶,并没有被研究报道。序列比对结果表明,XynB的催化结构域与其它酶的最高相似性只有41%,在已经报道的第八家族木聚糖酶中,与之相似性最高的是来自环境DNA文库的Xyn8(39%, ABB71891).将XynB的催化结构域与已报道的其它4个第八家族木聚糖酶氨基酸序列进行比对分析,发现了三个在第八家族木聚糖酶中高度保守的氨基酸残基,分别为第586位的谷氨酸,第646位和第786位的天冬氨酸,这三个残基被认为在第八家族糖苷水解酶的催化反应中起到重要作用。序列分析表明,XynB (?)的NTD的氨基酸序列与数据库中已知功能的序列均没有明显的同源性,表明该NTD可能是一个结构新颖的结构域。因此,对XynB的序列分析表明,该酶是GH8家族中一个结构新颖的多结构域木聚糖酶。2.木聚糖酶XynB的异源表达和性质研究
XynB是一个多结构域木聚糖酶,通过异源表达得到有活性的酶对酶的催化性质研究非常重要。我们通过pET-22b原核表达系统,成功构建了表达载体pET-22b-xynB,并且在大肠杆菌BL21(DE3)中实现了可溶性表达。为了获得较高的表达量和表达活性,我们进行了产酶条件优化实验,确定了最佳表达条件为在0.5mM的IPTG诱导下15℃培养96h,在此条件下重组大肠杆菌的胞内酶活最高可达到4.2U／ml菌液。由于电泳分析表明在最适条件下培养48h,胞内酶蛋白的表达量基本已达到最大,并且此时目的蛋白占细胞总蛋白的比例较高,我们选择在最适条件下培养48h后从胞内分离纯化重组木聚糖酶XynB,这不但利于重组酶的纯化,而且缩短了培养时间。通过细胞超声破碎获得胞内可溶蛋白成分,再通过阴离子交换色谱层析和镍柱亲和层析两步纯化,得到了电泳纯的重组XynB, SDS-PAGE显示分子量约为100kDa。根据氨基酸序列计算表明,XynB去掉信号肽之后的分子量为97.1kDa。因此,重组表达的XynB是一个含有NTD和CD的多结构域木聚糖酶。
对重组XynB(?)的底物特异性研究结果表明,该酶是一个特异性的内切木聚糖酶,能高效水解山毛榉木聚糖和燕麦木聚糖,对羧甲基纤维素、淀粉、海藻多糖、几丁质以及甘露聚糖均没有活性。XynB的最适酶活pH为60-7.0,在酸性条件下酶活丧失较快,在弱碱性条件下仍能保持较高的酶活,是一个中性略偏碱性的木聚糖酶。该酶在中性偏碱性较广的pH范围(pH6.0-pH10.0)内比较稳定,在15℃放1h后还能保持80%以上的残余酶活XynB的最适酶活温度为35-40℃,在0℃和5℃时分别保持有最高酶活的7.5%和14.6%,该酶的热稳定性很差,在35℃保温1h后只保持不到40%的残余酶活,在45℃保温20min便丧失全部酶活。这些结果说明该酶具有一定的适冷特征。Co2+、SDS、EDTA、Zn2+、Ni2+、Mn2+和Li2+在5mM浓度下能够强烈抑制XynB酶活性,但在1mM浓度下只有Co2+的抑制作用明显。由于Fe3+和Cu2+两种重金属离子在1mM浓度下对酶活几乎没有抑制作用,这可能使该酶在某些工业应用中具有优势。NaCl对XynB的活性并没有促进作用,在低浓度NaCl存在时(0.5M及以下),XynB的酶活性受到轻微抑制作用(残余酶活在85%以上)；当NaCl浓度从1.0M增加到接近饱和的4.0M时,该酶的残余酶活仅从67%下降到约40%,表现出了较强的NaCl耐受性,这反映了该酶对海洋高盐环境的适应。XynB对山毛榉木聚糖和燕麦木聚糖的Km值分别为5.82mg/ml和11.86mg/ml, keat/Km值分别为104.64ml/mg. s和60.03ml/mg.s,表明XynB对山毛榉木聚糖具有更高的亲和力和催化效率。XynB不能水解木三糖和木四糖,能够高效水解木六糖生成木四糖、木三糖和木二糖,该酶水解木五糖的效率比水解木六糖时低很多,只能产生非常少量的木三糖和木二糖,另外所有的酶解反应中均没有木糖产生,这表明XynB是一个严格的内切木聚糖酶,需要至少5个糖基单元才能进行有效的水解。
木聚糖酶在制浆造纸、食品、饲料、纺织等行业都有广泛的应用,特别是制浆造纸工业是应用木聚糖酶最多的行业。XynB具有对热敏感的特征,在一些不能进行高温加热的食品加工领域中可能具有应用价值。另外,XynB具有耐盐的特征,在海产品和其他含盐产品的加工中也可能具有应用价值。
3.NTD的异源表达以及NTD对不可溶多糖的结合功能研究
为了研究NTD在XynB催化木聚糖降解中的作用,我们对NTD进行了单独异源表达。通过pET-22b原核表达系统,成功构建了NTD的表达质粒,并且在大肠杆菌BL21(DE3)中实现了NTD的可溶性表达。为了获得有活性的目的蛋白,对表达条件进行了优化,最终选择的表达条件为：在15℃条件下用0.5mM的IPTG诱导表达20h。然后通过超声波细胞破碎获得胞内可溶性蛋白,利用镍柱亲和层析法纯化得到电泳纯的重组NTD。为了验证NTD在XynB催化木聚糖降解中是否具有结合底物的功能,我们首先利用SDS-PAGE检测了NTD是否能结合不可溶木聚糖。实验结果表明,NTD对不可溶木聚糖具有明显的吸附结合能力。此外,NTD对也具有微晶纤维素很强的吸附结合能力,这些结果表明,NTD中可能含有多糖结合结构域(CBM)。由于NTD的氨基酸序列与已报道的CBM没有同源性,所以NTD中含有的CBM可能是新型的CBM。
4.NTD的蛋白结晶
由于NTD的氨基酸序列与目前数据库中已知功能的蛋白序列都没有明显的同源性,因此难以通过同源比对推测NTD中CBM的结构。为了进一步揭示NTD的结构与功能,我们决定通过结晶来解析NTD的结构,然后分析其中可能含有的新型CBM的结构。由于蛋的结晶需要大量高纯度的蛋白,我们首先对NTD进行了大批量高纯度制备。经过镍柱亲和层析,Q柱(强阴离子交换柱)层析和凝胶过滤层析3步纯化,得到高纯度的重组NTD蛋白样品。将样品浓缩到8mg/ml浓度,经过坐滴气相扩散法初筛,确定出了有利于NTD晶体生长的条件,获得一些雪花状的小晶体。由于这些晶体太小,分辨率太低,我们又利用悬滴气相扩散法进行了复筛,经过结晶条件的优化,得到了晶体生长的最佳条件为pH9.0的0.1M Tris-HCl缓冲体系,添加30%(w／v)的PEG6000。在此条件下目前已长出棒状晶体,该晶体经SDS-PAGE分析已证实为蛋白结晶。这些晶体体还在进一步生长之中,经过进一步的生长之后,即可通过X射线衍射分析其分辨率。这些研究结果为进一步得到NTD晶体结构进而阐明其多糖结合作用机制以及NTD与CD的协同作用机制奠定了基础。
5.木聚糖酶Xyn A提高面包品质的应用潜力研究
我们的前期研究表明,((?)laciecola mesophila KMM241分泌的木聚糖酶XynA是一个GH10家族的适冷木聚糖酶。本论文研究了木聚糖酶XynA提高面包品质的潜力,并与GH10家族的中温木聚糖酶EX1进行了比较。最适添加量的分析表明,EX1的最适添加量为270U/kg,而Xyn A(?)最适添加量为0.9U/kg。添加Xyn A和EX1都会使面粉的弱化度降低,在最适添加量下可使弱化度降低50%,但是在降低面团形成时间方面,Xyn A的作用较EX1更显著。在面包体积的影响来看,两种木聚糖酶的作用都很显著。这两种木聚糖酶对面包的抗老化作用方面的表现是相似的,相比较来说,EX1在降低面包初始硬度方面作用明显,而Xyn A在减慢面包老化速率方面作用显著。这些结果表明,GH10家族的这两种酶在改善面包和面团的质量方面各有优势,这也是它们具有作为添加剂用于焙烤工业的潜在优势。这是首次发现GH10家族的适冷木聚糖酶具有改善面包品质的潜力。
The area of oceans account for more than70percent of the earth's surface and sea water accounts for97percent of total water on earth. Marine environment is an important part of the global ecological environment and marine carbon cycle plays a critical role in the total carbon cycle of biosphere Marine microorganisms decompose the organic carbon stored through photosynthesis of marine phytoplankton and photosynthetic bacteria into carbon dioxide, and thus play an important role in the marine carbon cycle. Xylan is a main component of hemicellulose and also the second abundant carbon source on the earth, thus represents an important kind of marine organic carbon. Compared to cellulose, the structure of xylan is more complex and needs the synergy of many xylanolytic enzymes to be complete degraded. β-1,4-endo-xylanase (EC3.2.1.8) can cleave the β-1,4-xylosidic bonds of the main chain of xylan and plays a key role in the xylan breakdown. Therefore, it has significance in the marine carbon cycle. Marine environment is generally characterized by high salinity and high fluidity, and the deep sea (more than2000m deep) is specifically characterized by high pressure, low temperature, low transmission of light and oligotrophy. Marine organisms have evolved to adapt the extreme environment and preserve many gene resources which may have great values. Therefore, oceans are reservoir not only for species but also for genes. Sequence analysis and function identification of these gene resources are significant for the study of biochemical processes in the marine environment.
Glaciecola mesophila KMM241is a cold-adapted marine bacterium isolated from an invertebrate. Our previous study showed that G. mesophila KMM241could produce more than one kind of xylanases and possesses. We have cloned and characterized a xylanase XynA from this strain and studied the potential of XynA in improving the quality of wheat bread. In this study, the xylanase XynB from G. mesophila KMM241was studied, including gene cloning and sequence analysis, heterogenous expression and biochemical propery analyses In addition, the function of the N-terminal domain of XynB was studied and elucidated The main research results are listed as below:
1. Gene cloning and sequence analysis of gene xynB
The16S rRNA gene sequence of G. mesophila KMM241had more than99%identity to that of Pseudoalteromonas atlantica T6c The whole genome of strain T6c was sequenced and released in GenBank, which revealed that there is a gene encoding a hypothetical endo-1,4-[3-xylanase in the genome Based on the5' end and3' end sequence of this gene, a pair of specific primers for PCR were designed, and the total sequence of xylanase gene xynH was amplified through PCR using the genomic DNA of strain G. mesophila KMM241as template. Sequence analysis showed that gene xynB is2739bp in length, encoding a protein of912amino acids, which is the precursor of xylanase XynB. XynB precursor is composed of a signal peptide (Metl-Ala43), a N-terminal domain (NTD, Glu44-Arg584) with unknown function and a GH family8catalytic domain(CD, Ser585-Glu912) Sequence alignment showed that only three enzymes have hotnology with the whole amino acid sequence of XynB. They are all hypothetical glycoside hydrolases from strains P. atlantica T6c (98%), Gaciecola chathamensis S18K6(80%) and Glaciecola polaris LMG21857(73%), respectively, none of which has been studied. The amino acid sequence of the catalytic domain of XynB showed highest similarity of41%to a hypothetical xylanase. Among characterized xylanases, XynB had highest identity (39%) to the xylanase Xyn8from an uncultured bacterium (ABB71891) The multiple sequence alignment revealed that XynB contained glutamate and aspartate residues Glu586, Asp646and Asp786, which are considered to be crucial for catalytic activity of GH family8. The amino acid sequence of the NTD of XynB showed no homology to any functional sequence in the data bases, suggesting that the NTD may be a domain with new structure. These analyses of XynB sequence indicated that XynB may be a novel multidomain xylanase of GH8.
2. Heterologous expression and characterization of Xyn B
Obtaining the active enzyme through heterologous expression is very important for the study of the enzymatic properties of XynB. An expression plasmid pET-22b-xynB was constructed and successfully expressed in E. coli BL21(DE3). The best condition for XynB expression was determined through optimization experiments. Strain E. coli BL21(DE3) was induced by0.5mM IPTG and cultured at15℃for96hours. At this condition, a highest intracellular enzyme activity of4.2U/ml was obtained. For the purification of recombinant XynB, the E. coli strain was cultured for48hours when the proportion of target protein was relatively high. The recombinant XynB was purified from intracellular proteins through sonication, DEAE-Sepharose ion exchange chromatography and Ni-affinity chromatography. SDS-PAGE analysis showed that the Mr of XynB is about100kDa, which is in accordance with the calculated Mr of XynB (97.1kDa) without the predicted signal peptide. Therefore, the recombinant XynB is a multidomain xylanase containing the NTD and the CD.
Study on the specificity of substrate of recombinant XynB showed that it is a specific endo-xylanase which could efficiently hydrolyze beechwood xylan and oat spelt xylan, but had no enzymatic activity on CMC, starch, laminarin, chitosan and mannan. The optimum pH for XynB is6.0-7.0. XynB is relatively more active in alkaline condition than in acid condition, which lost its enzymatic activity quickly in acid condition and retained most of its activity in alkalescent conditions. XynB was relatively stable in a wide pH range (pH6.0-10.0), retaining more than80%activity after incubation at pH6.0-10.0,15℃for1h. The optimal temperature for XynB was35℃. It retained7.5%and14.6%activity at0℃and5℃, respectively. XynB exhibited very low thermostability, retaining less than40%activity after60-min incubation at35℃and losing all the activity after20-min incubation at45℃. These results indicated that XynB had cold-adaptaion properties. Co2+(1mM and5mM) had the strongest inhibitory effect on the activity, while Zn2+, Ni2+, Mn2+and Li2+at a concentration of5mM had significantly inhibitory effects. XynB may have advantages in some industrial applications as two heavy metal ions Fe3+and Cu2+at a concentration of1mM had no evident inhibitory effect on its activity NaCl had no stimulative effect on the activity of recombinant XynB, on the contrary, NaCl in a low concentration (below0.5M) had slight inhibitory effect on its enzymatic activity (more than85%residual activity) However, XynB showed salt-tolerant ability, retaining approximately40%activity in4.0M NaCl, reflecting the adaptation of XynB to marine saline environment. XynB had lower Km and higher kcat/Km to beech wood xylan (5.82mg/ml,104.64ml/mg.s) than to oat spelt xylan (11.86mg/ml,6003ml/mg.s), indicating that XynB had stronger alffinity and higher catalytic efficiency to beech wood xylan than to oat spelt xylan XynB could not hydrolyze xylotetraose and xylotriose, and could hydrolyze xylohexaose and xylopentaose. Xylohexaose was completely hydrolyzed, producing xylobiose, xylotriose and xylotetraose The hydrolysis efficiency to xylopentaose was relatively low, producing a small amount of xylotriose and xylobiose. No xylose was detected in all hydrolysis process. These results showed that XynB is a strict endo-β-1,4-xylanase exhibiting an action pattern with a demand of at least five sugar moieties for effective cleavage.
Xylanases have potential applications in a wide range of industrial processes, such as food processing, animal feeds, paper and pulp, textiles and bioremediation, etc. As a strict endo-xylanase, XynB may have potential in some of these industrial processes. Especially, XynB is thermolabile, which may have advantage in the food industries where a high temperature for enzyme inactivation is not allowed. As a salt-tolerant enzyme, xynB may have potential in the biotechnological processes where the catalysis environment is highly salty, such as in the processing of sea food and saline food
3. Heterologous expression of the NTD and its binding ability to insoluble polysaccharides
In order to study the function of the NTD in xylan degradation by XynB, the NTD was expressed in E. coli. The expression vector for NTD was constructed through pET prokaryotic expression system. The NTD was successfully expressed in E. coli BL21(DE3). The expression conditions were optimized to obtain soluble NTD. E. coli BL21(DE3) was induced by0.5mM IPTG and cultured at15℃for20hours. The recombinant NTD was purified from intracellular proteins through sonication and Ni-affinity chromatography. To test whether the NTD has a function of binding the substrate during xylan degradation by XynB, the binding ability of the recombinant NTD to insoluble oat spelt xylan was studied. The result showed that the recombinant NTD exhibited strong binding ability to insoluble oat spelt xylan. In addition, the recombinant NTD also exhibited strong binding ability to avicel, but little binding ability to chitosan and chitin. These results suggested that the NTD may contain a carbohydrate binding module (CBM) As the amino acid sequence of the NTD showed no homology to other CBMs, the NTD may contain a new type of CBM.
4. Crystallization of the reconibinant NTD
Because the NTD has no homology to any protein sequence with known function, it is hard to predict the structure of the NTD. In order to further study the structure and function of the NTD, we decided to analyze the structure of the NTD by crystallization. Since a large quantity of protein with high purity is needed for crystallization, we first prepared the recombinant NTD of high purity through Ni-affinity chromatography, Q Sepharose Fast Flow chromatography and gel filtration chromatography. The purified protein of NTD was concentrated to8mg/ml and applied to crystal growing apparatus. Proper conditions for NTD crystallization were determined through preliminary screening and snowflake crystals were obtained, which is small and with low resolution. Therefore, proper conditions for NTD crystallization were further optimized. After optimization, rodlike crystals are now growing on the condition of0.1M Tris-HCl, pH9.0,30%(w/v) PEG6000, which has been testified to be protein crystals by SDS-PAGE These crystals are still growing. When their size is big enough, their resolution can be determined by X ray diffection. These results provide a good foundation for the study of the stucture and the polysaccharide-binding mechanism of the NTD.
5Improvement of the baking quality by the psychrophilic xylanase XynA
Although a lot of xylanases have improvement on breadmaking and are even widely used in baking industry, only one GH10xylanase has been reported to be effective in baking. In this study, two GH10xylanases, cold-adapted Xyn A and mesophilic EX1, were compared for their effectiveness in breadmaking. the optimum dosage of the two enzymes to improve the quality of wheat flour dough and bread was270U/kg flour for EX1and0.9U/kg flour for Xyn A, showing that the dosage of cold-adapted xylanase was much smaller. With the optimum dosage, Xyn A and HX1had the same dough-softening ability,50%reduce in BU However, Xyn A was more effective in reducing DDT than EX1. Xyn A and EX1showed similar effect on improvement of bread volume (around30%increase). Although they exhibited similar anti-staling effect on bread based on decrease in bread hardness, Xyn A showed more affect on reducing firming rate, and EX1more on reducing initial crumb hardness. These results showed that the two GH10xylanases have respective advantages in improving the quality of dough and bread, and suggested their potential as additives in breadmaking. It is found for the first time that cold-adapted GH10xylanase has potential in improving the baking quality of bread.
Glaciecola mesophila KMM241是一株分离自海洋无脊椎动物的海洋细菌。我们的前期研究表明,该菌株能够产生多种木聚糖酶,并且在前期研究中我们已经对该菌株产生的一个属于糖苷水解酶第十家族的木聚糖酶XynA进行了基因克隆和性质研究。在此基础上,本论文对该菌株所产的另一个木聚糖酶XynB进行了基因序列、分子结构、生化性质及其N端结构域的功能研究,并探讨了木聚糖酶XynA在提高面包品质中的应用潜力,取得了以下主要结果。
1.木聚糖酶XynB的基因克隆和序列分析
由于菌株G mesophila KMM241与另一株基因组序列已经公布的海洋细菌Pseudoalteromonas atlantica T6c的16S rDNA序列相似性为99%。根据菌株Pseudoalteromonas atlantica T6c基因组中木聚糖酶基因(Gene ID:4172855)的序列设计的一对特异性引物,以菌株G mesophila KMM241基因组DNA为模板,PCR扩增得到了木聚糖酶基因xynB的全序列。序列分析表明,xynB基因序列全长2739bp,编码的木聚糖酶XynB的前体含912个氨基酸残基。木聚糖酶XynB的前体由N端的信号肽序列(Metl-Ala43),一个功能未知的N端结构域(NTD, Glu44-Arg584)和一个C端的糖苷水解酶第八家族(GH8)催化结构域(CD,Ser585-Glu912)组成。通过序列比对发现只有三个酶与XynB氨基酸全序列具有同源性,分别是来自菌株P. atlanlica T6c (98%), Glaciecola chathamensis S18K6(80%)和Glaciecola polaris LMG21857(73%)的糖苷水解酶,但这三个酶都是从基因组序列中推定的糖苷水解酶,并没有被研究报道。序列比对结果表明,XynB的催化结构域与其它酶的最高相似性只有41%,在已经报道的第八家族木聚糖酶中,与之相似性最高的是来自环境DNA文库的Xyn8(39%, ABB71891).将XynB的催化结构域与已报道的其它4个第八家族木聚糖酶氨基酸序列进行比对分析,发现了三个在第八家族木聚糖酶中高度保守的氨基酸残基,分别为第586位的谷氨酸,第646位和第786位的天冬氨酸,这三个残基被认为在第八家族糖苷水解酶的催化反应中起到重要作用。序列分析表明,XynB (?)的NTD的氨基酸序列与数据库中已知功能的序列均没有明显的同源性,表明该NTD可能是一个结构新颖的结构域。因此,对XynB的序列分析表明,该酶是GH8家族中一个结构新颖的多结构域木聚糖酶。2.木聚糖酶XynB的异源表达和性质研究
XynB是一个多结构域木聚糖酶,通过异源表达得到有活性的酶对酶的催化性质研究非常重要。我们通过pET-22b原核表达系统,成功构建了表达载体pET-22b-xynB,并且在大肠杆菌BL21(DE3)中实现了可溶性表达。为了获得较高的表达量和表达活性,我们进行了产酶条件优化实验,确定了最佳表达条件为在0.5mM的IPTG诱导下15℃培养96h,在此条件下重组大肠杆菌的胞内酶活最高可达到4.2U／ml菌液。由于电泳分析表明在最适条件下培养48h,胞内酶蛋白的表达量基本已达到最大,并且此时目的蛋白占细胞总蛋白的比例较高,我们选择在最适条件下培养48h后从胞内分离纯化重组木聚糖酶XynB,这不但利于重组酶的纯化,而且缩短了培养时间。通过细胞超声破碎获得胞内可溶蛋白成分,再通过阴离子交换色谱层析和镍柱亲和层析两步纯化,得到了电泳纯的重组XynB, SDS-PAGE显示分子量约为100kDa。根据氨基酸序列计算表明,XynB去掉信号肽之后的分子量为97.1kDa。因此,重组表达的XynB是一个含有NTD和CD的多结构域木聚糖酶。
对重组XynB(?)的底物特异性研究结果表明,该酶是一个特异性的内切木聚糖酶,能高效水解山毛榉木聚糖和燕麦木聚糖,对羧甲基纤维素、淀粉、海藻多糖、几丁质以及甘露聚糖均没有活性。XynB的最适酶活pH为60-7.0,在酸性条件下酶活丧失较快,在弱碱性条件下仍能保持较高的酶活,是一个中性略偏碱性的木聚糖酶。该酶在中性偏碱性较广的pH范围(pH6.0-pH10.0)内比较稳定,在15℃放1h后还能保持80%以上的残余酶活XynB的最适酶活温度为35-40℃,在0℃和5℃时分别保持有最高酶活的7.5%和14.6%,该酶的热稳定性很差,在35℃保温1h后只保持不到40%的残余酶活,在45℃保温20min便丧失全部酶活。这些结果说明该酶具有一定的适冷特征。Co2+、SDS、EDTA、Zn2+、Ni2+、Mn2+和Li2+在5mM浓度下能够强烈抑制XynB酶活性,但在1mM浓度下只有Co2+的抑制作用明显。由于Fe3+和Cu2+两种重金属离子在1mM浓度下对酶活几乎没有抑制作用,这可能使该酶在某些工业应用中具有优势。NaCl对XynB的活性并没有促进作用,在低浓度NaCl存在时(0.5M及以下),XynB的酶活性受到轻微抑制作用(残余酶活在85%以上)；当NaCl浓度从1.0M增加到接近饱和的4.0M时,该酶的残余酶活仅从67%下降到约40%,表现出了较强的NaCl耐受性,这反映了该酶对海洋高盐环境的适应。XynB对山毛榉木聚糖和燕麦木聚糖的Km值分别为5.82mg/ml和11.86mg/ml, keat/Km值分别为104.64ml/mg. s和60.03ml/mg.s,表明XynB对山毛榉木聚糖具有更高的亲和力和催化效率。XynB不能水解木三糖和木四糖,能够高效水解木六糖生成木四糖、木三糖和木二糖,该酶水解木五糖的效率比水解木六糖时低很多,只能产生非常少量的木三糖和木二糖,另外所有的酶解反应中均没有木糖产生,这表明XynB是一个严格的内切木聚糖酶,需要至少5个糖基单元才能进行有效的水解。
木聚糖酶在制浆造纸、食品、饲料、纺织等行业都有广泛的应用,特别是制浆造纸工业是应用木聚糖酶最多的行业。XynB具有对热敏感的特征,在一些不能进行高温加热的食品加工领域中可能具有应用价值。另外,XynB具有耐盐的特征,在海产品和其他含盐产品的加工中也可能具有应用价值。
3.NTD的异源表达以及NTD对不可溶多糖的结合功能研究
为了研究NTD在XynB催化木聚糖降解中的作用,我们对NTD进行了单独异源表达。通过pET-22b原核表达系统,成功构建了NTD的表达质粒,并且在大肠杆菌BL21(DE3)中实现了NTD的可溶性表达。为了获得有活性的目的蛋白,对表达条件进行了优化,最终选择的表达条件为：在15℃条件下用0.5mM的IPTG诱导表达20h。然后通过超声波细胞破碎获得胞内可溶性蛋白,利用镍柱亲和层析法纯化得到电泳纯的重组NTD。为了验证NTD在XynB催化木聚糖降解中是否具有结合底物的功能,我们首先利用SDS-PAGE检测了NTD是否能结合不可溶木聚糖。实验结果表明,NTD对不可溶木聚糖具有明显的吸附结合能力。此外,NTD对也具有微晶纤维素很强的吸附结合能力,这些结果表明,NTD中可能含有多糖结合结构域(CBM)。由于NTD的氨基酸序列与已报道的CBM没有同源性,所以NTD中含有的CBM可能是新型的CBM。
4.NTD的蛋白结晶
由于NTD的氨基酸序列与目前数据库中已知功能的蛋白序列都没有明显的同源性,因此难以通过同源比对推测NTD中CBM的结构。为了进一步揭示NTD的结构与功能,我们决定通过结晶来解析NTD的结构,然后分析其中可能含有的新型CBM的结构。由于蛋的结晶需要大量高纯度的蛋白,我们首先对NTD进行了大批量高纯度制备。经过镍柱亲和层析,Q柱(强阴离子交换柱)层析和凝胶过滤层析3步纯化,得到高纯度的重组NTD蛋白样品。将样品浓缩到8mg/ml浓度,经过坐滴气相扩散法初筛,确定出了有利于NTD晶体生长的条件,获得一些雪花状的小晶体。由于这些晶体太小,分辨率太低,我们又利用悬滴气相扩散法进行了复筛,经过结晶条件的优化,得到了晶体生长的最佳条件为pH9.0的0.1M Tris-HCl缓冲体系,添加30%(w／v)的PEG6000。在此条件下目前已长出棒状晶体,该晶体经SDS-PAGE分析已证实为蛋白结晶。这些晶体体还在进一步生长之中,经过进一步的生长之后,即可通过X射线衍射分析其分辨率。这些研究结果为进一步得到NTD晶体结构进而阐明其多糖结合作用机制以及NTD与CD的协同作用机制奠定了基础。
5.木聚糖酶Xyn A提高面包品质的应用潜力研究
我们的前期研究表明,((?)laciecola mesophila KMM241分泌的木聚糖酶XynA是一个GH10家族的适冷木聚糖酶。本论文研究了木聚糖酶XynA提高面包品质的潜力,并与GH10家族的中温木聚糖酶EX1进行了比较。最适添加量的分析表明,EX1的最适添加量为270U/kg,而Xyn A(?)最适添加量为0.9U/kg。添加Xyn A和EX1都会使面粉的弱化度降低,在最适添加量下可使弱化度降低50%,但是在降低面团形成时间方面,Xyn A的作用较EX1更显著。在面包体积的影响来看,两种木聚糖酶的作用都很显著。这两种木聚糖酶对面包的抗老化作用方面的表现是相似的,相比较来说,EX1在降低面包初始硬度方面作用明显,而Xyn A在减慢面包老化速率方面作用显著。这些结果表明,GH10家族的这两种酶在改善面包和面团的质量方面各有优势,这也是它们具有作为添加剂用于焙烤工业的潜在优势。这是首次发现GH10家族的适冷木聚糖酶具有改善面包品质的潜力。
The area of oceans account for more than70percent of the earth's surface and sea water accounts for97percent of total water on earth. Marine environment is an important part of the global ecological environment and marine carbon cycle plays a critical role in the total carbon cycle of biosphere Marine microorganisms decompose the organic carbon stored through photosynthesis of marine phytoplankton and photosynthetic bacteria into carbon dioxide, and thus play an important role in the marine carbon cycle. Xylan is a main component of hemicellulose and also the second abundant carbon source on the earth, thus represents an important kind of marine organic carbon. Compared to cellulose, the structure of xylan is more complex and needs the synergy of many xylanolytic enzymes to be complete degraded. β-1,4-endo-xylanase (EC3.2.1.8) can cleave the β-1,4-xylosidic bonds of the main chain of xylan and plays a key role in the xylan breakdown. Therefore, it has significance in the marine carbon cycle. Marine environment is generally characterized by high salinity and high fluidity, and the deep sea (more than2000m deep) is specifically characterized by high pressure, low temperature, low transmission of light and oligotrophy. Marine organisms have evolved to adapt the extreme environment and preserve many gene resources which may have great values. Therefore, oceans are reservoir not only for species but also for genes. Sequence analysis and function identification of these gene resources are significant for the study of biochemical processes in the marine environment.
Glaciecola mesophila KMM241is a cold-adapted marine bacterium isolated from an invertebrate. Our previous study showed that G. mesophila KMM241could produce more than one kind of xylanases and possesses. We have cloned and characterized a xylanase XynA from this strain and studied the potential of XynA in improving the quality of wheat bread. In this study, the xylanase XynB from G. mesophila KMM241was studied, including gene cloning and sequence analysis, heterogenous expression and biochemical propery analyses In addition, the function of the N-terminal domain of XynB was studied and elucidated The main research results are listed as below:
1. Gene cloning and sequence analysis of gene xynB
The16S rRNA gene sequence of G. mesophila KMM241had more than99%identity to that of Pseudoalteromonas atlantica T6c The whole genome of strain T6c was sequenced and released in GenBank, which revealed that there is a gene encoding a hypothetical endo-1,4-[3-xylanase in the genome Based on the5' end and3' end sequence of this gene, a pair of specific primers for PCR were designed, and the total sequence of xylanase gene xynH was amplified through PCR using the genomic DNA of strain G. mesophila KMM241as template. Sequence analysis showed that gene xynB is2739bp in length, encoding a protein of912amino acids, which is the precursor of xylanase XynB. XynB precursor is composed of a signal peptide (Metl-Ala43), a N-terminal domain (NTD, Glu44-Arg584) with unknown function and a GH family8catalytic domain(CD, Ser585-Glu912) Sequence alignment showed that only three enzymes have hotnology with the whole amino acid sequence of XynB. They are all hypothetical glycoside hydrolases from strains P. atlantica T6c (98%), Gaciecola chathamensis S18K6(80%) and Glaciecola polaris LMG21857(73%), respectively, none of which has been studied. The amino acid sequence of the catalytic domain of XynB showed highest similarity of41%to a hypothetical xylanase. Among characterized xylanases, XynB had highest identity (39%) to the xylanase Xyn8from an uncultured bacterium (ABB71891) The multiple sequence alignment revealed that XynB contained glutamate and aspartate residues Glu586, Asp646and Asp786, which are considered to be crucial for catalytic activity of GH family8. The amino acid sequence of the NTD of XynB showed no homology to any functional sequence in the data bases, suggesting that the NTD may be a domain with new structure. These analyses of XynB sequence indicated that XynB may be a novel multidomain xylanase of GH8.
2. Heterologous expression and characterization of Xyn B
Obtaining the active enzyme through heterologous expression is very important for the study of the enzymatic properties of XynB. An expression plasmid pET-22b-xynB was constructed and successfully expressed in E. coli BL21(DE3). The best condition for XynB expression was determined through optimization experiments. Strain E. coli BL21(DE3) was induced by0.5mM IPTG and cultured at15℃for96hours. At this condition, a highest intracellular enzyme activity of4.2U/ml was obtained. For the purification of recombinant XynB, the E. coli strain was cultured for48hours when the proportion of target protein was relatively high. The recombinant XynB was purified from intracellular proteins through sonication, DEAE-Sepharose ion exchange chromatography and Ni-affinity chromatography. SDS-PAGE analysis showed that the Mr of XynB is about100kDa, which is in accordance with the calculated Mr of XynB (97.1kDa) without the predicted signal peptide. Therefore, the recombinant XynB is a multidomain xylanase containing the NTD and the CD.
Study on the specificity of substrate of recombinant XynB showed that it is a specific endo-xylanase which could efficiently hydrolyze beechwood xylan and oat spelt xylan, but had no enzymatic activity on CMC, starch, laminarin, chitosan and mannan. The optimum pH for XynB is6.0-7.0. XynB is relatively more active in alkaline condition than in acid condition, which lost its enzymatic activity quickly in acid condition and retained most of its activity in alkalescent conditions. XynB was relatively stable in a wide pH range (pH6.0-10.0), retaining more than80%activity after incubation at pH6.0-10.0,15℃for1h. The optimal temperature for XynB was35℃. It retained7.5%and14.6%activity at0℃and5℃, respectively. XynB exhibited very low thermostability, retaining less than40%activity after60-min incubation at35℃and losing all the activity after20-min incubation at45℃. These results indicated that XynB had cold-adaptaion properties. Co2+(1mM and5mM) had the strongest inhibitory effect on the activity, while Zn2+, Ni2+, Mn2+and Li2+at a concentration of5mM had significantly inhibitory effects. XynB may have advantages in some industrial applications as two heavy metal ions Fe3+and Cu2+at a concentration of1mM had no evident inhibitory effect on its activity NaCl had no stimulative effect on the activity of recombinant XynB, on the contrary, NaCl in a low concentration (below0.5M) had slight inhibitory effect on its enzymatic activity (more than85%residual activity) However, XynB showed salt-tolerant ability, retaining approximately40%activity in4.0M NaCl, reflecting the adaptation of XynB to marine saline environment. XynB had lower Km and higher kcat/Km to beech wood xylan (5.82mg/ml,104.64ml/mg.s) than to oat spelt xylan (11.86mg/ml,6003ml/mg.s), indicating that XynB had stronger alffinity and higher catalytic efficiency to beech wood xylan than to oat spelt xylan XynB could not hydrolyze xylotetraose and xylotriose, and could hydrolyze xylohexaose and xylopentaose. Xylohexaose was completely hydrolyzed, producing xylobiose, xylotriose and xylotetraose The hydrolysis efficiency to xylopentaose was relatively low, producing a small amount of xylotriose and xylobiose. No xylose was detected in all hydrolysis process. These results showed that XynB is a strict endo-β-1,4-xylanase exhibiting an action pattern with a demand of at least five sugar moieties for effective cleavage.
Xylanases have potential applications in a wide range of industrial processes, such as food processing, animal feeds, paper and pulp, textiles and bioremediation, etc. As a strict endo-xylanase, XynB may have potential in some of these industrial processes. Especially, XynB is thermolabile, which may have advantage in the food industries where a high temperature for enzyme inactivation is not allowed. As a salt-tolerant enzyme, xynB may have potential in the biotechnological processes where the catalysis environment is highly salty, such as in the processing of sea food and saline food
3. Heterologous expression of the NTD and its binding ability to insoluble polysaccharides
In order to study the function of the NTD in xylan degradation by XynB, the NTD was expressed in E. coli. The expression vector for NTD was constructed through pET prokaryotic expression system. The NTD was successfully expressed in E. coli BL21(DE3). The expression conditions were optimized to obtain soluble NTD. E. coli BL21(DE3) was induced by0.5mM IPTG and cultured at15℃for20hours. The recombinant NTD was purified from intracellular proteins through sonication and Ni-affinity chromatography. To test whether the NTD has a function of binding the substrate during xylan degradation by XynB, the binding ability of the recombinant NTD to insoluble oat spelt xylan was studied. The result showed that the recombinant NTD exhibited strong binding ability to insoluble oat spelt xylan. In addition, the recombinant NTD also exhibited strong binding ability to avicel, but little binding ability to chitosan and chitin. These results suggested that the NTD may contain a carbohydrate binding module (CBM) As the amino acid sequence of the NTD showed no homology to other CBMs, the NTD may contain a new type of CBM.
4. Crystallization of the reconibinant NTD
Because the NTD has no homology to any protein sequence with known function, it is hard to predict the structure of the NTD. In order to further study the structure and function of the NTD, we decided to analyze the structure of the NTD by crystallization. Since a large quantity of protein with high purity is needed for crystallization, we first prepared the recombinant NTD of high purity through Ni-affinity chromatography, Q Sepharose Fast Flow chromatography and gel filtration chromatography. The purified protein of NTD was concentrated to8mg/ml and applied to crystal growing apparatus. Proper conditions for NTD crystallization were determined through preliminary screening and snowflake crystals were obtained, which is small and with low resolution. Therefore, proper conditions for NTD crystallization were further optimized. After optimization, rodlike crystals are now growing on the condition of0.1M Tris-HCl, pH9.0,30%(w/v) PEG6000, which has been testified to be protein crystals by SDS-PAGE These crystals are still growing. When their size is big enough, their resolution can be determined by X ray diffection. These results provide a good foundation for the study of the stucture and the polysaccharide-binding mechanism of the NTD.
5Improvement of the baking quality by the psychrophilic xylanase XynA
Although a lot of xylanases have improvement on breadmaking and are even widely used in baking industry, only one GH10xylanase has been reported to be effective in baking. In this study, two GH10xylanases, cold-adapted Xyn A and mesophilic EX1, were compared for their effectiveness in breadmaking. the optimum dosage of the two enzymes to improve the quality of wheat flour dough and bread was270U/kg flour for EX1and0.9U/kg flour for Xyn A, showing that the dosage of cold-adapted xylanase was much smaller. With the optimum dosage, Xyn A and HX1had the same dough-softening ability,50%reduce in BU However, Xyn A was more effective in reducing DDT than EX1. Xyn A and EX1showed similar effect on improvement of bread volume (around30%increase). Although they exhibited similar anti-staling effect on bread based on decrease in bread hardness, Xyn A showed more affect on reducing firming rate, and EX1more on reducing initial crumb hardness. These results showed that the two GH10xylanases have respective advantages in improving the quality of dough and bread, and suggested their potential as additives in breadmaking. It is found for the first time that cold-adapted GH10xylanase has potential in improving the baking quality of bread.
引文
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