海洋溶胶细菌Flammeovirga sp.MY04琼胶酶（系）的研究
|
摘要
海洋细菌Flammeovirga sp. MY04能直接液化琼脂糖，生成水溶性低聚糖和寡糖，具备用于固相发酵的潜力。因此，MY04是1株高效的琼胶酶资源菌，具有良好的应用价值和开发意义。
本文先后通过MY04胞外琼胶酶系的分析、基因文库的构建与琼胶酶基因的筛选，及基因组的测序与琼胶酶系的分析，进行了Flammeovirga sp. MY04琼胶酶资源的初步发掘，分析了菌株具有溶胶能力的遗传学基础。本文还系统地研究了琼胶酶AgaG4的酶学性质、新颖结构的功能与相关机制。结果如下：
1. MY04胞外琼胶酶系的基本特征分析
在无琼脂糖底物的培养条件下，获得了MY04的胞外粗酶，证明胞外琼胶酶系由至少4条琼脂糖降解酶组成，分子量大小约30kDa—70kDa，整体上具有耐中等高温(0℃—50℃)、耐碱(pH6—10)和耐盐(0M—0.9M)的特性。经寡糖产物的纯化、阳离子质谱和13C-谱分析，将胞外酶降解琼脂糖后的主产物鉴定为新琼四糖、新琼六糖，间接证明MY04的胞外琼胶酶系是β-琼胶酶系。
2. MY04基因组DNA文库的构建与琼胶酶的基因筛选、分析
以pCC1Fos为载体，在E. coli EPI300中构建了MY04的基因组DNA文库，平均插入片段约30kb—36kb，覆盖约15Gbp的MY04基因组DNA。对文库进行的活性筛选没有获得任何琼胶酶基因。最后，通过文库中随机克隆插入片段末端的测序与分析、文库的PCR筛选与阳性质粒pAI2的鸟枪法测序，获得了1个编码GH16家族β-琼胶酶的基因agaG4。
琼胶酶AgaG4由503个氨基酸残基组成，序列中含有在GH16家族β-琼胶酶中保守的8个糖基结合位点、2个催化位点和3个Ca2+结合位点。AgaG4具有模块化的组成特征，包括N-端信号肽、1个GH16模块、1个富含Gly-/Ser-残基的链接区、1个推定的碳水化合物结合模块和1个与膜定位相关的模块。这些功能模块的组成方式有两个新颖之处：一是GH16模块中含有不常见的由连续57个氨基酸(N246-G302)组成的肽段；二是C-端非催化结构域由可归类于CBM2或CBM5家族的几丁质结合域(ChtBD3)、por分泌系统膜整合结构域组成，这是琼胶酶一个新颖的模块化组织方式。系统发育分析表明，AgaG4与F. yaeyamensisYT的β-琼胶酶AgaYT、Microscilla sp. PRE1推定的β-琼胶酶MS116，构成GH16家族β-琼胶酶的一个亚家族分支。
3.琼胶酶AgaG4的异源表达、酶学鉴定，及其特殊模块结构的功能、机制分析
(1)琼胶酶AaG4的异源表达、重组酶制备与性质鉴定
用质粒pBAD/gIIIA在E. coli TOP10菌株中表达了基因agaG4，菌体中重组蛋白rAgaG4的表达量大于200mg/mL，含量接近30%。通过包涵体的纯化、变性溶解，及重组蛋白的Ni-亲和层析、复性，获得纯度大于97%的重组蛋白rAgaG4。复性后的rAgaG4具有专一的琼胶酶活性，酶活144U/mg，最适温度为50℃、最适pH为7.5，在0℃-50℃、pH6-9的范围内分别具有热稳定性和酸碱耐受性。重组酶rAgaG4在降解琼脂糖时表现为内切型降解模式。本章还建立了不依赖于13C-谱分析的琼寡糖、新琼寡糖的MS/MS鉴定方法，将两个寡糖终产物分别鉴定为新琼四糖、新琼六糖。这表明，尽管AgaG4与F. yaeyamensis YT的琼胶酶AgaYT具有98%的序列一致性，但它们的琼脂糖降解产物不同，因而是功能不同的琼胶酶。
本文的深入研究还通过寡糖总产物的荧光标记、HPLC分析，测定寡糖终产物中新琼四糖、新琼六糖的摩尔浓度之比是1.5:1，且总含量大于99%；测得寡糖总产率为72%—78%。重组酶rAgaG4的最小降解单元是新琼八糖，最小寡糖产物是新琼四糖。而且，在rAgaG4降解新琼十糖时，新琼四糖产生自非还原性末端。
综上，琼胶酶AgaG4是热稳定的、内切型GH16家族β-琼胶酶，在新琼四糖与新琼六糖的专一性制备中具有良好应用前景。相对单一的琼脂糖、寡糖降解方式提示，AgaG4具有严谨型的底物降解机制。
(2)琼胶酶AgaG4特殊模块结构的功能分析
用PCR技术对agaG4基因进行了截短改造，获得截除了AgaG4中C-端非催化结构域的编码序列、仅编码GH16模块的基因截短体agaG4-GH16，以及截除AgaG4的GH16模块中特殊肽段编码序列的基因截短体agaG4-T57。按照与基因agaG4同样的研究方法，分别获得纯度大于95%的重组蛋白截短体rAgaG4-GH16、rAgaG4-T57。
重组蛋白截短体rAgaG4-GH16与全长重组酶rAgaG4的酶学性质相似，差别在于起始复性浓度远低于rAgaG4、酶活大小是rAgaG4的35倍，这表明C-端非催化结构域对琼胶酶AgaG4的水溶性、活性大小具有显著影响。重组蛋白截短体rAgaG4-T57与重组酶rAgaG4的显著差异包括：最适温度为40℃；降解琼脂糖后的终产物包括新琼四糖、新琼六糖和新琼八糖，三者摩尔浓度之比为2.68:2.82:1；且rAgaG4-T57不能降解新琼八糖，其最小降解单元为新琼十糖。这些结果表明，GH16模块中的特殊肽段与琼胶酶AgaG4对新琼八糖的识别、降解作用密切相关。
(3) GH16模块中特殊肽段的关键位点及其功能、机制的分析
通过在线同源建模，获得了AgaG4的立体结构数据，分析后发现特殊肽段中的Y276位点位于催化腔的端口，侧链基团为含苯环的羟基，具有糖基结合潜力，判定为与新琼八糖降解相关的候选关键位点。
通过大引物错配PCR，扩增能表达微量水溶性AgaG4重组酶的pCTFG4质粒，获得一系列突变体。将空质粒pCold TF、重组质粒pCTFG4及其突变体，在E. coli BL21(DE3)中进行诱导表达，分别获得含有水溶性重组蛋白的粗酶。结合关键位点突变后氨基酸残基的结构特征，比较了粗酶液降解琼脂糖后的产物，发现：Y276位点突变为Gly-位点后使全酶失活；Y276突变为侧链基团含苯环的Phe-位点时，含重组酶突变体的粗酶降解琼脂糖后，终产物无变化，仅含有新琼四糖与新琼六糖，与rAgaG4的产物一致；Y276突变为侧链基团不含苯环结构的Asp-、Glu-、Asn-、Ser-或Thr-位点时，含重组酶突变体的粗酶降解琼脂糖后，终产物除含有新琼四糖与新琼六糖外，还含有新琼八糖，与rAgaG4-T57降解琼脂糖后的终产物一致。
上述结果证明，在琼胶酶AgaG4中，除前文预测的8个糖基结合位点之外，GH16模块中特殊肽段的Y276位点是1个新的糖基结合位点，与酶对琼脂糖、新琼八糖的降解作用密切相关。推测该位点侧链基团中的苯环在酶对新琼八糖的结合中具有重要作用。
4. MY04全基因组序列的测定与琼胶酶系的分析
本文首次获得火色杆菌属细菌的全基因组序列。MY04的染色体基因组大小约7.2M，共编码与已知琼胶酶序列一致性大于等于30%的15条β-琼胶酶序列，涵盖GH16、GH50和GH86等3个琼胶酶家族的成员，数量分别为4条、1条和10条。其中12条琼胶酶基因在染色体3.5M—5.1M处聚集。相似性分析表明，GH16家族成员与Zobellia galactanivorans Dsij、Microbulbifer thermotolerans的琼胶酶序列一致性最高，介于30%—42%，其它家族成员与Saccharophagusdegradans2-40的琼胶酶序列最高，介于30%—45%。对MY04中所有琼胶酶序列进行的系统发育分析表明，GH86家族成员分别聚集成与Saccharophagusdegradans2-40的琼胶酶GH86C、GH86E相关的2个类群。
这表明，Flammeovirga sp. MY04含有丰富的琼胶酶资源，琼胶酶系组成完整，具有一定的多样性组成特征。但是，在MY04基因组可注释的序列中，未发现胞外琼胶酶系中大小为30kDa-40kDa琼胶酶的编码基因，表明该菌株琼胶酶资源的全面发掘、研究仍然需要将基因组学与酶学、蛋白质组学等技术相结合。
综上，Flammeovirga sp. MY04基因组所编码的琼胶酶资源丰富，琼胶酶系组成完整，是菌株具有溶胶能力的重要遗传学基础。MY04的胞外琼胶酶系组成复杂，具有耐中等高温、耐碱性环境，和耐盐的稳定性特征，在新琼寡糖的制备中具有一定应用价值。琼胶酶AgaG4含有特殊模块结构、且进化地位特殊，具有热稳定性，在新琼四糖、新琼六糖的专一性生产中具有良好应用前景；GH16模块中的特殊肽段与新琼八糖的识别、降解相关；Y276是特殊肽段中关键的糖基结合位点，侧链基团中的苯环在酶对底物的识别、降解作用中具有重要作用。上述研究为深入、全面地开发Flammeovirga sp. MY04的琼胶酶资源奠定了坚实基础。
Agarose is a neutral complex polysaccharide composed of3,6-anhydro-L-galactopyranose-α-1,3-D-galactose units that are joined by β1-4bonds. Agarases areglycoside hydrolases (GHs) that catalyze the cleavage of α1-3linkages or β1-4linkages of agarose, and are grouped into α and β types respectively. The α-agarases(E.C.3.2.1.158) degrade agarose into agaro-oligosaccharides (AOs), withβ1,4-3,6-anhydro-L-galactopyranose as the reducing end, while β-agarases (E.C.3.2.1.81) depolymerise agarose into neoagaro-oligosaccharides (NAOs), withα-1,3-D-galactose as the reducing end. Nearly50agarases have been characterized,and most of them are β-agarases.
Agarases are useful in the preparation of algal protoplasts and the recovery of DNAfrom agarose gels. Recent studies have discovered some biological activities of NAOs,such as anti-oxidation, and prebiotic-and whitening-effects, which imply potentialapplications of agarases and oligosaccharides in food, pharmaceutical, and cosmeticindustries. However, few cheap and efficient agarases have been industrially produced.Thus, it is essential to find more bacteria with high agarase-yielding level for theexploration of more efficient agarases.
Flammeovirga is a bacterial genus belonging to the family Flammeovirgaceaewithin the α-Proteobacteria. Five species have been reported in this genus: F. aprica, F.arenaria, F. yaeyamensis, F. kamogawensis, and F. pacifica. All the type strains areagarolytic strains, while only one β-agarase, AgaYT from F. yaeyamensis strain YT,has been reported so far. Flammeovirga sp. MY04is a polysaccharide-degradingmarine bacterium with agarose liquefying ability, suggesting that agarases areabundant in Flammeovirga sp. MY04.
In this thesis, the agarase system of Flammeovirga sp. MY04has been exploredand studied, and the detailed results are,
1．The extracellular agarase system of Flammeovirga sp. MY04
The agarolytic marine isolate, MY04, is a member of the genus Flammeovirga. TheMY04strain is able to utilise multiple CPs as a sole carbon source and grows best onagarose, mannan, or xylan. This strain produces high concentrations of extracellularproteins (490±18.2mg/l liquid culture) that exhibit efficient and extensive degradation activities on various polysaccharides, especially agarose, for which theseproteins have an activity of310±9.6U/mg proteins. The extracellular agarase system(EAS), comprised in the crude extracellular enzymes, contains at least four agarosedepolymerases, with molecular masses of approximately30-70kDa. The EAS isstable at a wide range of pHs (6.0-11.0), temperatures (0-50℃), and sodium chlorideconcentrations (0-0.9M). Two major degradation products generated from agarose bythe EAS were identified to be neoagarotetraose and neoagarohexaose, suggesting thatthe major constituents of the MY04EAS are β-agarases.
2. Gene cloning and sequence analyses of the agarase AgaG4from the genomicDNA library
(1) The genomic library of Flammeovirga sp. MY04has been constructed using theEpifos fosmid library system. Five hundred nanograms of36-45kb blunt-repairedgenomic DNA has been ligated into the copy control vector pCC1FOS and transfectedinto E. coli EPI300-T1R. Totally, the genomic library of Flammeovirga sp. MY04contains more than500,000fosmids with insert sizes ranging from30kb to36kb,covering more than15.0Gbp of the genomic DNA.
(2) More than3,000clones have been surveyed for activity screening of agarasegenes, but none of them showed any clear halos around bacterial colonies. Individualfosmids have been isolated from120random clones of the genomic library, and weresequenced using the T7primer of the pCC1FOS vector. After the trimming of vectorsequences, total118credible sequence tags with reading length longer than600bpwere obtained and analyzed. Partial agarase gene (agaG4) was initially identified atthe T7end of the fosmid pG4from clone R075. Special primers were designed,synthesized, and applied in PCR screening of agaG4from the genomic library. As aresult, the full-length gene of AgaG4was found in the fosmid pAI2from clone R012.Shotgun sequencing of pAI2revealed a31655bp DNA insert with eighteen deducedORFs, including the agarse gene agaG4and a potential gene cluster that may beinvolved in the degrading of mannan and cellulose. The5’-flanking region of agaG4lacked universal promoter elements such as the-35,-10, and RBS motifs, meaningthat the promoter can hardly be driven by the transcription systems of E. coli cells.GC contents of the ORF and GC3s are much lower than those of E. coli genes. These molecular characteristics, however, might lead to the unsuccessful screening of agaG4from the DNA library.
(3) Several functional sites that were conservative among most GH16β-agaraseshave been found in the protein sequence of AgaG4. These conservative sites includedeight sugar-binding sites (N72, W74, W145, D151, F178, R180, I325, and V327), acatalytic motif of two sites (E154, and E159,), and three calcium binding sites (E22、N49、S365). AgaG4contains an N-termianl signal peptide, followed by a novel GH16module, a Ser-/Gly rich linker, a hypothetical chitin binding module (ChtBD3), and apor secretion system sorting domain. Although AgaG4shares high identity (98%)with the GH16β-agarase AgaYT from F. yaeyamensis YT, two novel modularproperties and a distinctive revolutionary position of both AgaG4and AgaYT havebeen discovered in this study. The catalytic modules of AgaG4and AgaYT arenon-typical GH16module, the inner peptide (N246-G302) of which share fewhomologous regions among the characterized GH16β-agarases except amongthemselves but with five different sites. The non-catalytic domains at the C-terminalconsists of a hypothetical chitin binding module (ChtBD3) and a por secretion systemsorting module, a novel modular organization type of agarase. Moreover, the agarasesAgaG4, together with the agarase AgaYT and the predicted agarase MS116fromMicroscilla sp. PRE1, was classified into a new β-agarase subclass of the GH16family.
3. Heteologous expression of AgaG4and characterization of the novelmodular structures
(1) The recombinant protein rAgaG4was overexpressed in E. coli using thepBAD/gIII expression system. Recombinant proteins were obtained from inclusionbodies using urea, purified by Ni-affinity chromatograph, and refolded throughdialysis. The agarase rAgaG4， with purity high than97%, showed optimalcharacteristics at50°C and pH7.5respectively, and was stable for a temperature rangeof0to50℃and a pH range of6-9. A novel non-13C-NMR spectroscopic methodusing2-amino benzamide labelling and MS/MS experiments was applied in thecharacterization of agarose-derived oligosaccharides by rAgaG4. The finaldegradation products of agarose by rAgaG4were confirmed as neoagarotetraose(NA4) and neoagarohexaose (NA6), with a mol ratio of1.53:1, and amounting to total99%proportion. Furthermore, rAgaG4did not digest NA4and NA6, while dividedneoagarooctaose (NA8) into NA4, and degraded neoagarodecaose (NA10) into NA4 and NA6. Moreover, NA4was produced from the non-reducing ends of NA10.Although AgaG4and AgaYT from F. yaeyamensis YT share98%identity of aminoacid sequences, they produce different final products in agarose degradation, thereforethey are different agarases. The biochemical characteristics of rAgaG4indicate that itis a thermostable endo-β-agarase of family GH16. The agarose-degrading propertiesshowed great promise of applying rAgaG4in the preparation of NA4and NA6specially.
(2) The gene agaG4was truncated using PCR method, resulting one gene fragment(agaG4-GH16) that encoded the GH16module and the other fragment (agaG4-T57)that encoded partial AgaG4with the deletion of the unusual peptide. The genes wereover-expressed in E. coli strain TOP10using the pBAD/gIII expression system.Recombinant proteins were obtained from inclusion bodies using similar approachesas that of rAgaG4.The truncated agarase rAgaG4-GH16showed similar enzymecharacteristics to those of rAgaG4, but a lower starting refolding concentration and ahigher enzymatic activity, suggesting that the non-catalytic domain at the C-terminalof AgaG4had much to do with the protein solubility and enzyme activity. The othertruncated protein rAgaG4-T57showed optimum temperature at40°C, and degradedagarose into NA4, NA6and neoagarooctaose (NA8), with a final mol ratio of2.68:2.82:1. Furthermore, rAgaG4T57was unable to hydrolyze NA8, but degradedNA10into NA4and NA6. The results indicated that the special peptide in the GH16module of AgaG4plyays an essential role in binding and degrading of NA8.
(3) The three-dimentional structural data was obtained by homologous constructiononline (http://swissmodel.expasy.org/), and observed using the soft Swiss PDBViewer3.7. The Y276residue, lying at the the entrance of the catalytic cleft andconsisting of a phenolic hydroxyl group, was determined as a candidate sugar-bindingsite in the special peptide of AgaG4. The plasmid pCTFG4, which can produce a fewsoluble fusion agarase of AgaG4in E. coli BL21(DE3), was mutated at the Y276siteusing PCR strategies, and formed a series of plasmid mutants. The plasmid pCTFG4,its mutants, and the negative plasmid pCold TF were individually transformed into thehost cells, and the recombinant fusion proteins were expressed by incubation usingIPTG. After the cells were centrifugated and sonicated, individual soluble fractionscontaing targeting proteins was prepared by futher centrifugation and reacted with the agarose solution retained at45℃as the crude enzyme。The degradation products wereanalyzed and compared usin TLC detection.
As a result, when Y276(Tye276) was mutated into the Phe256site, the finaldegradation products of agarose by the crude enzyme were NA4and NA6, similar tothat by rAgaG4. While the Y276site was mutated into the sites of Asp276, Glu276,Asn276, Ser276or Thr276, the final degradation products of agarsoe changed intoNA4, NA6, and NA8, similar to that by rAgaG4-T57. Interestingly, when Y276wasmutated into the Gly276site, the degradation products of agarose by wereundetectable. The results indicated that the Y276site of the spectial peptide in theGH16module is a NA8-binding site of AgaG4and plays an important role in thedegradation of agarose. It suggested that the benzene ring is essential in the binding ofNA8.
4. The agarase system encoded by the genome sequence MY04
The genome of Flammeovirga sp. MY04is a circular chromosome of7,244,701bp. A total of5536protein-encoding genes were predicted. Fifteen agarasesharing identities high than30%with reported agarases have been found. The fourGH16agarases of MY04shared identities ranging from30%to42%with agarasesfrom Zobellia galactanivorans Dsij or Microbulbifer thermotolerans. Only oneagarase belong to the family of GH50, and the other ten agarases belong to the GH86family. The GH50and GH86agarases of MY04shared identities ranging from30%to45%with agarases from Saccharophagus degradans2-40. Moreover, the GH86agarases were clustered into two subclass, with relation to the GH86C and GH86E ofSaccharophagus degradans2-40respectively. However, three extracellular agarasesranging form30kDa to40kDa in the EAS of MY04were not found in the predictedgenes. It meant that biochemical and proteomic methods, more than genomic strategy,were neededed in the exploration of all agarases of Flammeovirga sp. MY04.
Altogether, the agarases of Flammeovirga sp. MY04have been searched inthe extracellular agarase system, the genomic DNA library, and the chromosomegenome sequence. Moreover, the agarase AgaG4has been heterologouslyexpressed, biochemically characterized in detail. Furthermore, enzymatic functions and possible catalytic mechanism of the novel module structrures inAgaG4were analyzed and discovered.
本文先后通过MY04胞外琼胶酶系的分析、基因文库的构建与琼胶酶基因的筛选，及基因组的测序与琼胶酶系的分析，进行了Flammeovirga sp. MY04琼胶酶资源的初步发掘，分析了菌株具有溶胶能力的遗传学基础。本文还系统地研究了琼胶酶AgaG4的酶学性质、新颖结构的功能与相关机制。结果如下：
1. MY04胞外琼胶酶系的基本特征分析
在无琼脂糖底物的培养条件下，获得了MY04的胞外粗酶，证明胞外琼胶酶系由至少4条琼脂糖降解酶组成，分子量大小约30kDa—70kDa，整体上具有耐中等高温(0℃—50℃)、耐碱(pH6—10)和耐盐(0M—0.9M)的特性。经寡糖产物的纯化、阳离子质谱和13C-谱分析，将胞外酶降解琼脂糖后的主产物鉴定为新琼四糖、新琼六糖，间接证明MY04的胞外琼胶酶系是β-琼胶酶系。
2. MY04基因组DNA文库的构建与琼胶酶的基因筛选、分析
以pCC1Fos为载体，在E. coli EPI300中构建了MY04的基因组DNA文库，平均插入片段约30kb—36kb，覆盖约15Gbp的MY04基因组DNA。对文库进行的活性筛选没有获得任何琼胶酶基因。最后，通过文库中随机克隆插入片段末端的测序与分析、文库的PCR筛选与阳性质粒pAI2的鸟枪法测序，获得了1个编码GH16家族β-琼胶酶的基因agaG4。
琼胶酶AgaG4由503个氨基酸残基组成，序列中含有在GH16家族β-琼胶酶中保守的8个糖基结合位点、2个催化位点和3个Ca2+结合位点。AgaG4具有模块化的组成特征，包括N-端信号肽、1个GH16模块、1个富含Gly-/Ser-残基的链接区、1个推定的碳水化合物结合模块和1个与膜定位相关的模块。这些功能模块的组成方式有两个新颖之处：一是GH16模块中含有不常见的由连续57个氨基酸(N246-G302)组成的肽段；二是C-端非催化结构域由可归类于CBM2或CBM5家族的几丁质结合域(ChtBD3)、por分泌系统膜整合结构域组成，这是琼胶酶一个新颖的模块化组织方式。系统发育分析表明，AgaG4与F. yaeyamensisYT的β-琼胶酶AgaYT、Microscilla sp. PRE1推定的β-琼胶酶MS116，构成GH16家族β-琼胶酶的一个亚家族分支。
3.琼胶酶AgaG4的异源表达、酶学鉴定，及其特殊模块结构的功能、机制分析
(1)琼胶酶AaG4的异源表达、重组酶制备与性质鉴定
用质粒pBAD/gIIIA在E. coli TOP10菌株中表达了基因agaG4，菌体中重组蛋白rAgaG4的表达量大于200mg/mL，含量接近30%。通过包涵体的纯化、变性溶解，及重组蛋白的Ni-亲和层析、复性，获得纯度大于97%的重组蛋白rAgaG4。复性后的rAgaG4具有专一的琼胶酶活性，酶活144U/mg，最适温度为50℃、最适pH为7.5，在0℃-50℃、pH6-9的范围内分别具有热稳定性和酸碱耐受性。重组酶rAgaG4在降解琼脂糖时表现为内切型降解模式。本章还建立了不依赖于13C-谱分析的琼寡糖、新琼寡糖的MS/MS鉴定方法，将两个寡糖终产物分别鉴定为新琼四糖、新琼六糖。这表明，尽管AgaG4与F. yaeyamensis YT的琼胶酶AgaYT具有98%的序列一致性，但它们的琼脂糖降解产物不同，因而是功能不同的琼胶酶。
本文的深入研究还通过寡糖总产物的荧光标记、HPLC分析，测定寡糖终产物中新琼四糖、新琼六糖的摩尔浓度之比是1.5:1，且总含量大于99%；测得寡糖总产率为72%—78%。重组酶rAgaG4的最小降解单元是新琼八糖，最小寡糖产物是新琼四糖。而且，在rAgaG4降解新琼十糖时，新琼四糖产生自非还原性末端。
综上，琼胶酶AgaG4是热稳定的、内切型GH16家族β-琼胶酶，在新琼四糖与新琼六糖的专一性制备中具有良好应用前景。相对单一的琼脂糖、寡糖降解方式提示，AgaG4具有严谨型的底物降解机制。
(2)琼胶酶AgaG4特殊模块结构的功能分析
用PCR技术对agaG4基因进行了截短改造，获得截除了AgaG4中C-端非催化结构域的编码序列、仅编码GH16模块的基因截短体agaG4-GH16，以及截除AgaG4的GH16模块中特殊肽段编码序列的基因截短体agaG4-T57。按照与基因agaG4同样的研究方法，分别获得纯度大于95%的重组蛋白截短体rAgaG4-GH16、rAgaG4-T57。
重组蛋白截短体rAgaG4-GH16与全长重组酶rAgaG4的酶学性质相似，差别在于起始复性浓度远低于rAgaG4、酶活大小是rAgaG4的35倍，这表明C-端非催化结构域对琼胶酶AgaG4的水溶性、活性大小具有显著影响。重组蛋白截短体rAgaG4-T57与重组酶rAgaG4的显著差异包括：最适温度为40℃；降解琼脂糖后的终产物包括新琼四糖、新琼六糖和新琼八糖，三者摩尔浓度之比为2.68:2.82:1；且rAgaG4-T57不能降解新琼八糖，其最小降解单元为新琼十糖。这些结果表明，GH16模块中的特殊肽段与琼胶酶AgaG4对新琼八糖的识别、降解作用密切相关。
(3) GH16模块中特殊肽段的关键位点及其功能、机制的分析
通过在线同源建模，获得了AgaG4的立体结构数据，分析后发现特殊肽段中的Y276位点位于催化腔的端口，侧链基团为含苯环的羟基，具有糖基结合潜力，判定为与新琼八糖降解相关的候选关键位点。
通过大引物错配PCR，扩增能表达微量水溶性AgaG4重组酶的pCTFG4质粒，获得一系列突变体。将空质粒pCold TF、重组质粒pCTFG4及其突变体，在E. coli BL21(DE3)中进行诱导表达，分别获得含有水溶性重组蛋白的粗酶。结合关键位点突变后氨基酸残基的结构特征，比较了粗酶液降解琼脂糖后的产物，发现：Y276位点突变为Gly-位点后使全酶失活；Y276突变为侧链基团含苯环的Phe-位点时，含重组酶突变体的粗酶降解琼脂糖后，终产物无变化，仅含有新琼四糖与新琼六糖，与rAgaG4的产物一致；Y276突变为侧链基团不含苯环结构的Asp-、Glu-、Asn-、Ser-或Thr-位点时，含重组酶突变体的粗酶降解琼脂糖后，终产物除含有新琼四糖与新琼六糖外，还含有新琼八糖，与rAgaG4-T57降解琼脂糖后的终产物一致。
上述结果证明，在琼胶酶AgaG4中，除前文预测的8个糖基结合位点之外，GH16模块中特殊肽段的Y276位点是1个新的糖基结合位点，与酶对琼脂糖、新琼八糖的降解作用密切相关。推测该位点侧链基团中的苯环在酶对新琼八糖的结合中具有重要作用。
4. MY04全基因组序列的测定与琼胶酶系的分析
本文首次获得火色杆菌属细菌的全基因组序列。MY04的染色体基因组大小约7.2M，共编码与已知琼胶酶序列一致性大于等于30%的15条β-琼胶酶序列，涵盖GH16、GH50和GH86等3个琼胶酶家族的成员，数量分别为4条、1条和10条。其中12条琼胶酶基因在染色体3.5M—5.1M处聚集。相似性分析表明，GH16家族成员与Zobellia galactanivorans Dsij、Microbulbifer thermotolerans的琼胶酶序列一致性最高，介于30%—42%，其它家族成员与Saccharophagusdegradans2-40的琼胶酶序列最高，介于30%—45%。对MY04中所有琼胶酶序列进行的系统发育分析表明，GH86家族成员分别聚集成与Saccharophagusdegradans2-40的琼胶酶GH86C、GH86E相关的2个类群。
这表明，Flammeovirga sp. MY04含有丰富的琼胶酶资源，琼胶酶系组成完整，具有一定的多样性组成特征。但是，在MY04基因组可注释的序列中，未发现胞外琼胶酶系中大小为30kDa-40kDa琼胶酶的编码基因，表明该菌株琼胶酶资源的全面发掘、研究仍然需要将基因组学与酶学、蛋白质组学等技术相结合。
综上，Flammeovirga sp. MY04基因组所编码的琼胶酶资源丰富，琼胶酶系组成完整，是菌株具有溶胶能力的重要遗传学基础。MY04的胞外琼胶酶系组成复杂，具有耐中等高温、耐碱性环境，和耐盐的稳定性特征，在新琼寡糖的制备中具有一定应用价值。琼胶酶AgaG4含有特殊模块结构、且进化地位特殊，具有热稳定性，在新琼四糖、新琼六糖的专一性生产中具有良好应用前景；GH16模块中的特殊肽段与新琼八糖的识别、降解相关；Y276是特殊肽段中关键的糖基结合位点，侧链基团中的苯环在酶对底物的识别、降解作用中具有重要作用。上述研究为深入、全面地开发Flammeovirga sp. MY04的琼胶酶资源奠定了坚实基础。
Agarose is a neutral complex polysaccharide composed of3,6-anhydro-L-galactopyranose-α-1,3-D-galactose units that are joined by β1-4bonds. Agarases areglycoside hydrolases (GHs) that catalyze the cleavage of α1-3linkages or β1-4linkages of agarose, and are grouped into α and β types respectively. The α-agarases(E.C.3.2.1.158) degrade agarose into agaro-oligosaccharides (AOs), withβ1,4-3,6-anhydro-L-galactopyranose as the reducing end, while β-agarases (E.C.3.2.1.81) depolymerise agarose into neoagaro-oligosaccharides (NAOs), withα-1,3-D-galactose as the reducing end. Nearly50agarases have been characterized,and most of them are β-agarases.
Agarases are useful in the preparation of algal protoplasts and the recovery of DNAfrom agarose gels. Recent studies have discovered some biological activities of NAOs,such as anti-oxidation, and prebiotic-and whitening-effects, which imply potentialapplications of agarases and oligosaccharides in food, pharmaceutical, and cosmeticindustries. However, few cheap and efficient agarases have been industrially produced.Thus, it is essential to find more bacteria with high agarase-yielding level for theexploration of more efficient agarases.
Flammeovirga is a bacterial genus belonging to the family Flammeovirgaceaewithin the α-Proteobacteria. Five species have been reported in this genus: F. aprica, F.arenaria, F. yaeyamensis, F. kamogawensis, and F. pacifica. All the type strains areagarolytic strains, while only one β-agarase, AgaYT from F. yaeyamensis strain YT,has been reported so far. Flammeovirga sp. MY04is a polysaccharide-degradingmarine bacterium with agarose liquefying ability, suggesting that agarases areabundant in Flammeovirga sp. MY04.
In this thesis, the agarase system of Flammeovirga sp. MY04has been exploredand studied, and the detailed results are,
1．The extracellular agarase system of Flammeovirga sp. MY04
The agarolytic marine isolate, MY04, is a member of the genus Flammeovirga. TheMY04strain is able to utilise multiple CPs as a sole carbon source and grows best onagarose, mannan, or xylan. This strain produces high concentrations of extracellularproteins (490±18.2mg/l liquid culture) that exhibit efficient and extensive degradation activities on various polysaccharides, especially agarose, for which theseproteins have an activity of310±9.6U/mg proteins. The extracellular agarase system(EAS), comprised in the crude extracellular enzymes, contains at least four agarosedepolymerases, with molecular masses of approximately30-70kDa. The EAS isstable at a wide range of pHs (6.0-11.0), temperatures (0-50℃), and sodium chlorideconcentrations (0-0.9M). Two major degradation products generated from agarose bythe EAS were identified to be neoagarotetraose and neoagarohexaose, suggesting thatthe major constituents of the MY04EAS are β-agarases.
2. Gene cloning and sequence analyses of the agarase AgaG4from the genomicDNA library
(1) The genomic library of Flammeovirga sp. MY04has been constructed using theEpifos fosmid library system. Five hundred nanograms of36-45kb blunt-repairedgenomic DNA has been ligated into the copy control vector pCC1FOS and transfectedinto E. coli EPI300-T1R. Totally, the genomic library of Flammeovirga sp. MY04contains more than500,000fosmids with insert sizes ranging from30kb to36kb,covering more than15.0Gbp of the genomic DNA.
(2) More than3,000clones have been surveyed for activity screening of agarasegenes, but none of them showed any clear halos around bacterial colonies. Individualfosmids have been isolated from120random clones of the genomic library, and weresequenced using the T7primer of the pCC1FOS vector. After the trimming of vectorsequences, total118credible sequence tags with reading length longer than600bpwere obtained and analyzed. Partial agarase gene (agaG4) was initially identified atthe T7end of the fosmid pG4from clone R075. Special primers were designed,synthesized, and applied in PCR screening of agaG4from the genomic library. As aresult, the full-length gene of AgaG4was found in the fosmid pAI2from clone R012.Shotgun sequencing of pAI2revealed a31655bp DNA insert with eighteen deducedORFs, including the agarse gene agaG4and a potential gene cluster that may beinvolved in the degrading of mannan and cellulose. The5’-flanking region of agaG4lacked universal promoter elements such as the-35,-10, and RBS motifs, meaningthat the promoter can hardly be driven by the transcription systems of E. coli cells.GC contents of the ORF and GC3s are much lower than those of E. coli genes. These molecular characteristics, however, might lead to the unsuccessful screening of agaG4from the DNA library.
(3) Several functional sites that were conservative among most GH16β-agaraseshave been found in the protein sequence of AgaG4. These conservative sites includedeight sugar-binding sites (N72, W74, W145, D151, F178, R180, I325, and V327), acatalytic motif of two sites (E154, and E159,), and three calcium binding sites (E22、N49、S365). AgaG4contains an N-termianl signal peptide, followed by a novel GH16module, a Ser-/Gly rich linker, a hypothetical chitin binding module (ChtBD3), and apor secretion system sorting domain. Although AgaG4shares high identity (98%)with the GH16β-agarase AgaYT from F. yaeyamensis YT, two novel modularproperties and a distinctive revolutionary position of both AgaG4and AgaYT havebeen discovered in this study. The catalytic modules of AgaG4and AgaYT arenon-typical GH16module, the inner peptide (N246-G302) of which share fewhomologous regions among the characterized GH16β-agarases except amongthemselves but with five different sites. The non-catalytic domains at the C-terminalconsists of a hypothetical chitin binding module (ChtBD3) and a por secretion systemsorting module, a novel modular organization type of agarase. Moreover, the agarasesAgaG4, together with the agarase AgaYT and the predicted agarase MS116fromMicroscilla sp. PRE1, was classified into a new β-agarase subclass of the GH16family.
3. Heteologous expression of AgaG4and characterization of the novelmodular structures
(1) The recombinant protein rAgaG4was overexpressed in E. coli using thepBAD/gIII expression system. Recombinant proteins were obtained from inclusionbodies using urea, purified by Ni-affinity chromatograph, and refolded throughdialysis. The agarase rAgaG4， with purity high than97%, showed optimalcharacteristics at50°C and pH7.5respectively, and was stable for a temperature rangeof0to50℃and a pH range of6-9. A novel non-13C-NMR spectroscopic methodusing2-amino benzamide labelling and MS/MS experiments was applied in thecharacterization of agarose-derived oligosaccharides by rAgaG4. The finaldegradation products of agarose by rAgaG4were confirmed as neoagarotetraose(NA4) and neoagarohexaose (NA6), with a mol ratio of1.53:1, and amounting to total99%proportion. Furthermore, rAgaG4did not digest NA4and NA6, while dividedneoagarooctaose (NA8) into NA4, and degraded neoagarodecaose (NA10) into NA4 and NA6. Moreover, NA4was produced from the non-reducing ends of NA10.Although AgaG4and AgaYT from F. yaeyamensis YT share98%identity of aminoacid sequences, they produce different final products in agarose degradation, thereforethey are different agarases. The biochemical characteristics of rAgaG4indicate that itis a thermostable endo-β-agarase of family GH16. The agarose-degrading propertiesshowed great promise of applying rAgaG4in the preparation of NA4and NA6specially.
(2) The gene agaG4was truncated using PCR method, resulting one gene fragment(agaG4-GH16) that encoded the GH16module and the other fragment (agaG4-T57)that encoded partial AgaG4with the deletion of the unusual peptide. The genes wereover-expressed in E. coli strain TOP10using the pBAD/gIII expression system.Recombinant proteins were obtained from inclusion bodies using similar approachesas that of rAgaG4.The truncated agarase rAgaG4-GH16showed similar enzymecharacteristics to those of rAgaG4, but a lower starting refolding concentration and ahigher enzymatic activity, suggesting that the non-catalytic domain at the C-terminalof AgaG4had much to do with the protein solubility and enzyme activity. The othertruncated protein rAgaG4-T57showed optimum temperature at40°C, and degradedagarose into NA4, NA6and neoagarooctaose (NA8), with a final mol ratio of2.68:2.82:1. Furthermore, rAgaG4T57was unable to hydrolyze NA8, but degradedNA10into NA4and NA6. The results indicated that the special peptide in the GH16module of AgaG4plyays an essential role in binding and degrading of NA8.
(3) The three-dimentional structural data was obtained by homologous constructiononline (http://swissmodel.expasy.org/), and observed using the soft Swiss PDBViewer3.7. The Y276residue, lying at the the entrance of the catalytic cleft andconsisting of a phenolic hydroxyl group, was determined as a candidate sugar-bindingsite in the special peptide of AgaG4. The plasmid pCTFG4, which can produce a fewsoluble fusion agarase of AgaG4in E. coli BL21(DE3), was mutated at the Y276siteusing PCR strategies, and formed a series of plasmid mutants. The plasmid pCTFG4,its mutants, and the negative plasmid pCold TF were individually transformed into thehost cells, and the recombinant fusion proteins were expressed by incubation usingIPTG. After the cells were centrifugated and sonicated, individual soluble fractionscontaing targeting proteins was prepared by futher centrifugation and reacted with the agarose solution retained at45℃as the crude enzyme。The degradation products wereanalyzed and compared usin TLC detection.
As a result, when Y276(Tye276) was mutated into the Phe256site, the finaldegradation products of agarose by the crude enzyme were NA4and NA6, similar tothat by rAgaG4. While the Y276site was mutated into the sites of Asp276, Glu276,Asn276, Ser276or Thr276, the final degradation products of agarsoe changed intoNA4, NA6, and NA8, similar to that by rAgaG4-T57. Interestingly, when Y276wasmutated into the Gly276site, the degradation products of agarose by wereundetectable. The results indicated that the Y276site of the spectial peptide in theGH16module is a NA8-binding site of AgaG4and plays an important role in thedegradation of agarose. It suggested that the benzene ring is essential in the binding ofNA8.
4. The agarase system encoded by the genome sequence MY04
The genome of Flammeovirga sp. MY04is a circular chromosome of7,244,701bp. A total of5536protein-encoding genes were predicted. Fifteen agarasesharing identities high than30%with reported agarases have been found. The fourGH16agarases of MY04shared identities ranging from30%to42%with agarasesfrom Zobellia galactanivorans Dsij or Microbulbifer thermotolerans. Only oneagarase belong to the family of GH50, and the other ten agarases belong to the GH86family. The GH50and GH86agarases of MY04shared identities ranging from30%to45%with agarases from Saccharophagus degradans2-40. Moreover, the GH86agarases were clustered into two subclass, with relation to the GH86C and GH86E ofSaccharophagus degradans2-40respectively. However, three extracellular agarasesranging form30kDa to40kDa in the EAS of MY04were not found in the predictedgenes. It meant that biochemical and proteomic methods, more than genomic strategy,were neededed in the exploration of all agarases of Flammeovirga sp. MY04.
Altogether, the agarases of Flammeovirga sp. MY04have been searched inthe extracellular agarase system, the genomic DNA library, and the chromosomegenome sequence. Moreover, the agarase AgaG4has been heterologouslyexpressed, biochemically characterized in detail. Furthermore, enzymatic functions and possible catalytic mechanism of the novel module structrures inAgaG4were analyzed and discovered.
引文
[1] Araki, C.H. Acetylation of agar like substance of Gelidium amansii. J. Chem. Soc.1937,58,1338–1350.
[2]王静雪,江晓路,胡晓珂.细菌降解琼胶的研究进展.中国水产科学.2001,8(3):94–96.
[3]杜宗军,王祥红,李筠.琼胶酶研究进展.微生物学通报.2003,30(1):64–67.
[4]马翠萍,石超.琼胶酶研究进展.微生物学通报.2008,35(1):107–111.
[5]胡晓珂,江晓路,管华诗.海藻多糖降解酶的性质和作用机理.微生物学报.2001,41(6):762–766.
[6] Cantarel, B.L., Coutinho, P.M., Rancurel, C., Bernard, T., Lombard V., Henrissat, B. TheCarbohydrate-Active Enzymes database (CAZy): an expert resource for Glycogenomics.Nucleic Acids Res.2009,37(suppl1): D233-D238.
[7] Potin, P.; Richard, C.; Rochas, C.; Kloareg, B. Purification and characterization of theα-agarase from Alteromonas agarlyticus (Cataldi) comb. nov., strain GJ1B. Eur J Biochem.1993,214:599–607.
[8] Kirimura, K.; Masuda, N.; Iwasaki, Y.; Nakagawa, H.; Kobayashi, R.; Usami, S. Purificationand characterization of a novel β-agarase from an alkalophilic bacterium, Alteromonas sp.E-1. J Biosci Bioeng.1999,87,436–441.
[9] Rochas, C., Potin, P., and Kloareg, B., NMR spectroscopic investigation of agarose oligomersproduced by an α-agarase. Carbohyd Res.1994,253:69–77.
[10] Morrice, L.M., Mclean, M.W., Williamson, F.B., and Long W.F., Beta-agarases I and II fromPseudomonas atlanticaa: Purification and some properties. Eur J Biochem.1983,135:553–558.
[11] Morrice, L.M., Maitland, W.M., Long, W.F., and Williamson, F.B. Beta-agarases I and II fromPseudomonas atlanticaa: Substrate specificities. Eur J Biochem,1983,137:149–154.
[12] Bibb, M.J., Jones, G.H., Joseph, R., Buttner, M.J., Ward, J.M. The agarase gene (dag A) ofStreptomyces coelicolor A3(2): Affinity purification and characterization of the cloned geneproduct. J Gen Microbiol.1987,133:2089–2096.
[13] Hosoda, A., Sakai, M., Kanazawa S. Isolation and characterization of agar-degradingPaenibacillus spp. Associated with the rhizosphere of spinach. Biosci Biotechnol Biochem.2003,67(5):1048–1055.
[14]韩文君,古静燕,李天东,罗英,阮期平.一株产琼胶酶土壤细菌Paenibacillus sp.MY03的筛选与分子鉴定.绵阳师范学院学报.2007,26(2):86–90.
[15] Fu, X.T., Kim, S.M. Agarase: Review of major sources, categories, purification method,enzyme characteristics and applications. Mar Drugs.2010,8:200–218.
[16] Voget, S., Leggewie, C., Uesbeck, A., Raasch, C., Jaeger, K.-E., Streit, W.R. Prospecting fornovel biocatalysts in soil metagenome. Appl Environ Microbiol,2003,69(10):6235–6242.
[17] Mello, L.V., Chen, X., Rigden, D.J. Mining metagenomic data for novel domains: BACON, anew carbohydrate-binding module. FEBS Lett.2010,584(11):2421–2426.
[18] Sim, S.J., Baik, S.K., Park, S.C., Choe, H.N., Seong, C.N., Shin, T.S., Woo, H.C., Cho, J.Y.,Kim, D. Characterization of alginate lyase gene using a mentagenomic library constructedfrom the gut microflora of abalone. J Ind Microbial Biotechnol.2011, Doi:10.1007/s10295-011-1054-0.
[19] Li, J.G., Zhang, K.G., Han, W.J. Cloning and biochemical characterization of a novel lipolyticgene from activated sludge metagenome, and its gene product. Microbial Cell Factories.2010,9:83.
[20] Glogauer, A., Martini, V.P., Faoro, H., Couto, G.H., Muller-Santos, M., Monteiro, R.A.,Mitchell, D.A., Souza, E.M., Pedrosa, F.O., Krieger, N. Identification and characterization ofa new true lipase isolated through metagenomic approach. Microbial Cell Factories.2011,10:54.
[22] Ohta, Y., Hatada, Y., Miyazaki, M., Nogi, Y., Ito, S., Horikoshi, K. Purification andcharacterization of a novel α-agarase from a Thalassomonas sp. Curr Microbiol.2005,50:212–216.
[23] Lahaye, M., Yaphe, W., Phan, Viet, M.T., and Rochas, C.,1989.13C-NMR spectroscopicinvestigation of metylated and charged agarose oligosaccharides and polysaccharides.Carbohydrate Research,90:249-265. Li, J.B., Han, F., Lu, X.Z., Fu, X.Y., Ma, C.P., Chu, Y.,and Yu, W.G.,2007. A simple method of preparing diverse neoagaro-oligosaccharides withβ-agarase. Carbohyd Res.342:1030–1033.
[24] Mei, J.F., Shao, J.T., Wang, Qi., Wang, H., Yi, Y., Ying, G.Q. Separation and quantification ofneoagaro-oligosaccharides. J Food Sci Technol.2011, Doi:10.1007/sl3197-011-0448-3.
[25] Michel, G., Barbeyron, T., Kloareg, B., Czjzek. The famly6carbohydrate-binding moduleshave coevolved with their appended catalytic modules toward similar substrate specificity.Glycobiol.2009,19(6):615–623.
[26] Flament, D., Barbeyron, T., Jam, M., Potin, P., Czjzek, M., Kloareg, B., Michel, G.Alpha-agarases define a new family of glycoside hydrolases, distinct from beta-agarasesfamilies. Appl Environ Microbiol.2007,73(14):4691–4694.
[27] Coutinho, P. M., and B. Henrissat. Carbohydrate-active enzymes: an integrated databaseapproach, p.3–12. In H. J. Gilbert, G. Davies, B. Henrissat, and B. Svensson (ed.), Recentadvances in carbohydrate bioengineering.1999. The Royal Society of Chemistry, Cambridge,United Kingdom.
[28] Sugano, Y., Matsumoto, T., Kodama, H., Noma, M. Cloning and sequencing of agaA, aunique agarase0107gene from a marine bacterium, Vibrio sp. strain JT0107. Appl EnvironMicrobiol.1993,59:3750–3756.
[29] Ohta, Y., Hatada, Y., Nogi, Y., Li, Z.J., Ito, S., Horikoshi, K. Cloning, expression, andcharacterization of a glycoside hydrolase family86β-agarase from a deep-seaMicrobulbifer-like isolate. Appl Microbiol Biotechnol.2004,66:266–275.
[30] Lu, X.Z., Chu, Y., Wu, Q.Q., Gu, Y.C., Han, F., Yu, W.G. Cloning, expression andcharacterization of a new agarase-encoding gene from marine Pseudoalteromonas sp..Biotechnol Lett.2009,31:1565–1570.
[31] Kim, H.T., Lee, S.Y., Lee, D.H., Kim, H.S., Bang, W.G., Kim, G.C. Overexpression andmolecular characterization of Aga50D from Saccharophagus degradans2-40: anexo-typeβ-agarase producing neoagarobiose. Appl Microbiol Biotechnol.2010,86:227–234.
[32] Jam, M., Flament, D., Allouch, J., Potin, P., Thion, L., Kloareg, B., Czjzek, M., Helbert, W.,Michel, G., Barbeyron, T. The endo-β-agarase AgaA and AgaB from the marine bacteriumZobellia galactanivorans: two paralogue enzymes with different molecular organizations andcatalytic behaviours. Biochem J.2005,385:703–713.
[33] Ma, C.P., Lu, X.Z., Shi, C., Li, J.B., Gu, Y.C., Ma, Y.M., Chu, Y., Han, F., Gong, Q.H., andYu, W.G. Molecular cloning and characterization of a novel beta-agarase, AgaB, frommarine Pseudoalteromonas sp. CY24. J Biol Chem.2007,282(6):3747–3754.
[34] Allouch, J., Jam, M., Helbert, W., Barbeyron, T., Kloareg, B., Henrissat, B., Czjzek, M. Thethree-deimensional structures of two β-agarases. J Biol Chem.2003,278(47):47171–47180.
[35] Shi, C., Lu, X.Z., Ma, C.P., Ma, Y.M., Fu, X.Y., Yu, W.G. Enhancing the thermostability of anovel β-agarase AgaB through directed evolution. Appl Biochem Biotechnol.2008,151:51–59.
[36] Jang, M.K., Lee, S.W., Lee, D.G., Kim, N.Y., Yu, K.H., Jang, H.J., Kim, S.K., Kim, A., Lee,S.H. Enhancement of the thermostability of a recombinant β-agarase, AgaB, from Zobelliagalactanivorans by random mutagenesis. Biotechnol Lett.2010,32:943–949.
[37] Lee, S.W., Lee, D.G., Jang, M.K., Jeon, M.J., Jang, H.J., Lee, S.H. Improvement in thecatalytic activity ofβ-agarase AgaA from Zobellia galactanivorans by site-directedmutagenesis. J Microbiol Biotechnol.2011,21(11):1116–1122.
[38] Henshaw, J., Horne-Bitschy, A., Lammerts van Bueren, A., Money, V.A., Bolam, D.N.,Czjzek, M., Ekborg, N.A., Weiner, R.M., Hutcheson, S.W., Davies, G.J., Boraston, A.B.,Gilbert, H.J. Family6carbohydrate binding modules in β-agarases display exquisiteselectivity for the non-reducing termini of agarose chains. J Biol Chem.2006,282(25):17099–17107.
[39] Allouch, J., Helbert, W., Henrissat, B., Czjzek, M. Parallel substrate binding sites in aβ-agarase suggest a novel mode of action on double-helical agarose. Structure,2004,12:622–632.
[39] Ekborg, N.A., Taylor, L.E., Longmire, A.G., Henrissat, B., Weiner, R.M., Hutcheson, S.W.Genomic and proteomic analyses of the agarolytic system expressed by Saccharophagusdegradans2-40. Appl Environ Microbiol.2006,72(5):3396–3405.
[40] Shin, M.H., Lee, D.Y., Wohlgemuth, G., Choi, I.G., Fiehn, O., Kim, K.H. Global metaboliteprofiling of agarose degradation by Saccharophagus degradans2-40. New Biotechnol.2010,27(2):156–168.
[41] Yun, E.J., Shin, M.H., Yoon, J.J., Kim, Y.J., Choi, I.G., Kim, K.H. Production of3,6-anhydro-L-galactose from agaose by agarolytic enzymes of Saccharophagus degradans2-40. Process Biochem.2011,46:88–93.
[42] Cole KD, kerman B. Enhanced capacity for electrophoretic capture of plasmid DNA byagarase treatment of agarose gels. Biomacromolecules.2000,1:771–81.
[43] Yeong, H.Y., Khalid, N., Phang, S.M. Protoplast isolation and regeneration from Gracilariachangii (Gracilariales, Rhodophyta). J Appl Phycol.2008,20:641–51.
[44] Gupta, V., Kumar, M., Kumari, P., Reddy, C.R.K., Jha, B. Optimization of protoplast yieldsfrom the red algae Gracilaria dura (C. Agardh) J. Agardh and G. verrucosa (Huds.)Papenfuss. J Appl Phycol.2010, doi:10.1007/s10811-010-9579-6.
[45] Kujundzic, E., Greenberg, A.R., Fong, R., Moore, B., Kujundzic, D., Hernandez, M.Biofouling potential of industrial fermentation broth components during microfiltration. JMemb Sci.2010,349:44–55.
[46] Kim, H.T., Lee, S., Kim, K.H., Choi, I.G. The complete enzymatic saccharification of agaroseand its application to simultaneous saccharification and fementation of agaorse for ethanolproduction. Bioresource Technol.2011, doi:10.1016/j.biortech.2011.11.120.
[47] Wu, S.C., Wang, F.J., Chen, J.R., Pan, C.L. Study on wines produced from monostromanitidum hydrolysate solution. J Mar Sci Technol.2011,19(4):430–443.
[48] Wu, S.C., Wen, T.N., Pan, C.L. Algal-oligosaccharide-lysates prepared by two bacterialagarases stepwise hydrolyzed and their anti-oxidative properties. Fisher Sci.2005,71:1149–1159.
[49] Wang, J.X., Jiang, X.L., Mou, H.J., Guan, H.S. Anti-oxidation of agar oligosaccharidesproduced by agarase from a marine bacterium. J Appl Phycol.2004,16:333–340.
[50] Hu, B., Gong, Q.H., Wang, Y., Ma, Y.M., Li, J.B., Yu, W.G. Prebiotic effects ofneoagaro-oligosaccharides prepared by enzymatic hydrolysis of agarose. Anaerobe.2006,12:260–266.
[51] Oh, C.H., Nikapitiya, C.L., Lee, Y.D., Whang, I., Kim, S.J., Kang, D.H, Lee, J.H. Cloning,purification and biochemical characterization of beta agarase from the marine bacteriumPseudoalteromonas sp. AG4. J Ind Microbiol Biotechnol.2010,37:483–494.
[1] Zhong, Z.P., Toukdarian, A., Helinski, D., Knauf, V., Sykes, S., Wilkinson, J.E., O’Bryne, C.,Shea, T., DeLoughery, C., Caspi R. Sequence analysis of a101-kilobase plasmid required foragar degradation by a Microscilla isolate. Appl Environ Microbiol.2001,67(12):5771–5779.
[2]分子克隆实验指南(上、下册)/(美) J.萨姆布鲁克(Sambrook, J.)等著;黄培堂等译.–3版.–北京:科学出版社,2002.
[3] Thompson, J.D., Gibson,T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G. The ClustalXwindows interface: flexible strategies for multiple sequence alignment aided by qualityanalysis tools. Nucl Acids Res.1997,25:4876–4882.
[4] Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S. MEGA5: Molecularevolutionary genetics analysis using maximum likelihood, evolutionary distance, andmaximum parsimony methods. Mol Biol Evol.2011,28(10):2731–2739.
[5] Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities ofprotein utilizing the principle of protein-dye binding. Anal Biochem.1976,72:248–254.
[6] Miller, G.L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. AnalChem.1959,31:426–428.
[7]精编蛋白质科学实验指南/(美) JE.科林根(Coligan, J.E.)等著;李慎等译.–北京:科学出版社,2007.
[8] Zhang, M., Jiang, Z., Yang, S., Hua, C., Li, L. Cloning and expression of a Paecilomycesthermophila xylanase gene in E. coli and characterization of the recombinant xylanase.Bioresour Technol.2010,101:688–695.
[9] Lahaye, M., Yaphe, W., Phan, V.M.T., Rochas, C.13C-NMR spectroscopic investigation ofmetylated and charged agarose oligosaccharides and polysaccharides. Carbohydr Res.1989,90:249–265.
[10] Nakagawa, Y., Hamana, K., Sakane, T., Yamasato, K. Reclassification of Cytophaga aprica(Lewin1969) Reichenbach1989in Flammeovirga gen. nov. as Flammeovirga aprica comb.nov. and of Cytophaga diffluens (ex Stanier1940; emend. Lewin1969) Reichenbach1989inPersicobacter gen. nov. as Persicobacter diffluens comb. nov. Int J Syst Evol Microbiol.1997,47:220–223.
[11] Takahashi, M., Suzuki, K.C., Nakagawa, Y. Emendation of the genus Flammeovirga andFlammeovirga aprica with the proposal of Flammeovirga arenaria nom. rev., comb. nov. andFlammeovirga yaeyamensis sp. nov. Int J Syst Evol Microbiol.2006,56:2095–2100.
[12] Hosoya, S., Yokota, A. Flammeovirga kamogawensis sp. nov., isolated from coastal seawaterin Japan. Int J Syst Evol Microbiol.2007,57:1327–1330.
[13] Xu, H., Fu, Y.Y., Yang, N., Ding, Z.X., Lai, Q.L., Zeng, R.Y. Flammeovirga pacifica sp. nov.,isolate from deep sea sediment of west Pacific Ocea. J Syst Evol Microbiol.2011, doi:10.1099/ijs.0.030676-0
[14] Yang, J.L., Chen, L.C., Shih, Y.Y., Hsieh, C.Y., Chen, C.Y., Chen, W.M., Chen, C.C. Cloningand characterization of β-agarase AgaYT from Flammeovirga yaeyamensis strain YT. JBiosci Bioeng.2011,112:225–232.
[1]分子克隆实验指南(上、下册)/(美) J.萨姆布鲁克(Sambrook, J.)等著;黄培堂等译.–3版.–北京:科学出版社,2002.
[1]分子克隆实验指南(上、下册)/(美) J.萨姆布鲁克(Sambrook, J.)等著;黄培堂等译.–3版.–北京:科学出版社,2002.
[2] Hall, T.A. BioEdit: a user-friendly biological sequence alignment editor and analysis programfor Windows95/98/NT. Nucl Acids Symp Ser.1999,41:95–98.
[3] Zhong, Z.P., Toukdarian, A., Helinski, D., Knauf, V., Sykes, S., Wilkinson, J.E., O’Bryne, C.,Shea, T., DeLoughery, C., Caspi R. Sequence analysis of a101-kilobase plasmid required foragar degradation by a Microscilla isolate. Appl Environ Microbiol.2001,67(12):5771–5779.
[4] Allouch, J., Jam, M., Helbert, W., Barbeyron, T., Kloareg, B., Henrissat, B., Czjzek, M. Thethree-deimensional structures of two β-agarases. J Biol Chem.2003,278(47):47171–47180.
[5] Yang, J.L., Chen, L.C., Shih, Y.Y., Hsieh, C.Y., Chen, C.Y., Chen, W.M., Chen, C.C. Cloningand characterization of β-agarase AgaYT from Flammeovirga yaeyamensis strain YT. JBiosci Bioeng.2011,112:225–232.
[6] Voget, S., Leggewie, C., Uesbeck, A., Raasch, C., Jaeger, K.-E., Streit, W.R. Prospecting fornovel biocatalysts in soil metagenome. Appl Environ Microbiol,2003,69(10):6235–6242.
[7] Mello, L.V., Chen, X., Rigden, D.J. Mining metagenomic data for novel domains: BACON, anew carbohydrate-binding module. FEBS Lett.2010,584(11):2421–2426.
[8] Sim, S.J., Baik, S.K., Park, S.C., Choe, H.N., Seong, C.N., Shin, T.S., Woo, H.C., Cho, J.Y.,Kim, D. Characterization of alginate lyase gene using a mentagenomic library constructedfrom the gut microflora of abalone. J Ind Microbial Biotechnol.2011, Doi:10.1007/s10295-011-1054-0.
[9] Michel, G., Barbeyron, T., Kloareg, B., Czjzek. The famly6carbohydrate-binding moduleshave coevolved with their appended catalytic modules toward similar substrate specificity.Glycobiol.2009,19(6):615–623.
[10] Lu, X.Z., Chu, Y., Wu, Q.Q., Gu, Y.C., Han, F., Yu, W.G. Cloning, expression andcharacterization of a new agarase-encoding gene from marine Pseudoalteromonas sp..Biotechnol Lett.2009,31:1565–1570.
[1] Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities ofprotein utilizing the principle of protein-dye binding. Anal Biochem.1976,72:248–254.
[2] Miller, G.L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. AnalChem.1959,31:426–428.
[3] Zhang, M., Jiang, Z., Yang, S., Hua, C., Li, L. Cloning and expression of a Paecilomycesthermophila xylanase gene in E. coli and characterization of the recombinant xylanase.Bioresour Technol.2010,101:688–695.
[4] Bigge, J.C., Patel, T.P., Bruce, J.A., Goulding, P.N., Charles, S.M., Parekh, R.B. Nonselectiveand efficient fluorescent labeling of glycans using2-amino benzamide and anthranilic acid.Anal Biochem.1995,230:229–238.
[5] Yang, J.L., Chen, L.C., Shih, Y.Y., Hsieh, C.Y., Chen, C.Y., Chen, W.M., Chen, C.C. Cloningand characterization of β-agarase AgaYT from Flammeovirga yaeyamensis strain YT. JBiosci Bioeng.2011,112:225–232.
[6] Zhong, Z.P., Toukdarian, A., Helinski, D., Knauf, V., Sykes, S., Wilkinson, J.E., O’Bryne, C.,Shea, T., DeLoughery, C., Caspi R. Sequence analysis of a101-kilobase plasmid required foragar degradation by a Microscilla isolate. Appl Environ Microbiol.2001,67(12):5771–5779.
[7] Dong, J.H., Tamaru, Y., Araki, T. A unique β-agarase, AgaA, from a marine bacterium, Vibriosp. strain PO-303. Appl Microbiol Biotechnol.2007,74:1248–1255.
[8] Lu, X.Z., Chu, Y., Wu, Q.Q., Gu, Y.C., Han, F., Yu, W.G. Cloning, expression andcharacterization of a new agarase-encoding gene from marine Pseudoalteromonas sp..Biotechnol Lett.2009,31:1565–1570.
[9] Long, M.X., Yu, Z.N., Xu, X. A novel β-agarase with high pH stability from marineAgarivorans sp. LQ48. Mar Biotechnol.2010,12:62–69.
[10] Zhang, W.W., Sun, L. Cloning, characterization, and molecular application of a beta-agarasegene from Vibrio sp. strain V134. Appl Environ Microbiol.2007,2825–2831.
[11] Wu, S.C., Wen, T.N., Pan, C.L. Algal-oligosaccharide-lysates prepared by two bacterialagarases stepwise hydrolyzed and their anti-oxidative properties. Fisher Sci.2005,71:1149–1159.
[12] Wang, J.X., Jiang, X.L., Mou, H.J., Guan, H.S. Anti-oxidation of agar oligosaccharidesproduced by agarase from a marine bacterium. J Appl Phycol.2004,16:333–340.
[13] Hu, B., Gong, Q.H., Wang, Y., Ma, Y.M., Li, J.B., Yu, W.G. Prebiotic effects ofneoagaro-oligosaccharides prepared by enzymatic hydrolysis of agarose. Anaerobe.2006,12:260–266.
[14] Oh, C.H., Nikapitiya, C.L., Lee, Y.D., Whang, I., Kim, S.J., Kang, D.H, Lee, J.H. Cloning,purification and biochemical characterization of beta agarase from the marine bacteriumPseudoalteromonas sp. AG4. J Ind Microbiol Biotechnol.2010,37:483–494.
[1] Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities ofprotein utilizing the principle of protein-dye binding. Anal Biochem.1976,72:248–254.
[2] Miller, G.L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. AnalChem.1959,31:426–428.
[3] Zhang, M., Jiang, Z., Yang, S., Hua, C., Li, L. Cloning and expression of a Paecilomycesthermophila xylanase gene in E. coli and characterization of the recombinant xylanase.Bioresour Technol.2010,101:688–695.
[4] Bigge, J.C., Patel, T.P., Bruce, J.A., Goulding, P.N., Charles, S.M., Parekh, R.B. Nonselectiveand efficient fluorescent labeling of glycans using2-amino benzamide and anthranilic acid.Anal Biochem.1995,230:229–238.
[1] Guex, N., Peitsch, M.C. SWISS-MODEL and the Swiss-PdbViewer: An environment forcomparative protein modeling. Electrophoresis.1997,18:2714–2723.
[2] Guex, N., Peitsch, M.C. Swiss-PdbViewer: A fast and easy-to-use PDB Viewer for Macintoshand PC. Protein Data Bank Quaterly Newsletter.1996,77:7.
[3] Zhang, M., Jiang, Z., Yang, S., Hua, C., Li, L. Cloning and expression of a Paecilomycesthermophila xylanase gene in E. coli and characterization of the recombinant xylanase.Bioresour Technol.2010,101:688–695.
[1] Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S. MEGA5: Molecularevolutionary genetics analysis using maximum likelihood, evolutionary distance, andmaximum parsimony methods. Mol Biol Evol.2011,28(10):2731–2739.
[2] Allouch, J., Jam, M., Helbert, W., Barbeyron, T., Kloareg, B., Henrissat, B., Czjzek, M. Thethree-deimensional structures of two β-agarases. J Biol Chem.2003,278(47):47171–47180.
[3] Ekborg, N.A., Taylor, L.E., Longmire, A.G., Henrissat, B., Weiner, R.M., Hutcheson, S.W.Genomic and proteomic analyses of the agarolytic system expressed by Saccharophagusdegradans2-40. Appl Environ Microbiol.2006,72(5):3396–3405.
[4] Pagani, I., Chertkov, O., Lpidus, A., Lucas, S., Rio, T.G.D., Tice, H., Copeland, A., Cheng,J.F., Nolan, M., Saunders, E., Pitluck, S., Held, B., Goodwin, L., Liolios, K., Ovchinikova,G., Lvanova, N., Mavromatis, K., Pati, A., Chen, A., Palaniappan, K., Land, M., Hauser, L.,Jeffries, C.D., Detter, J.C., Han, C., Tapia, R., Ngatchou-Djao, O.D., Rohde, M., Goker, M.,Spring, S., Sikorski, J., Woyke, T., Bristow, J., Eisen, J.A., Markowitz, V., Hugenholtz, P.,Klenk, H.P., Kyrpides, N.C. Complete genome sequence of Marivirga tractuosa type strian(H-43)T. Stand Genomic Sci.2011,42(2):154–1622.
[5] Nakagawa, Y., Hamana, K., Sakane, T., Yamasato, K. Reclassification of Cytophaga aprica(Lewin1969) Reichenbach1989in Flammeovirga gen. nov. as Flammeovirga aprica comb.nov. and of Cytophaga diffluens (ex Stanier1940; emend. Lewin1969) Reichenbach1989inPersicobacter gen. nov. as Persicobacter diffluens comb. nov. Int J Syst Evol Microbiol.1997,47:220–223.
[6] Takahashi, M., Suzuki, K.C., Nakagawa, Y. Emendation of the genus Flammeovirga andFlammeovirga aprica with the proposal of Flammeovirga arenaria nom. rev., comb. nov. andFlammeovirga yaeyamensis sp. nov. Int J Syst Evol Microbiol.2006,56:2095–2100.
[7] Hosoya, S., Yokota, A. Flammeovirga kamogawensis sp. nov., isolated from coastal seawaterin Japan. Int J Syst Evol Microbiol.2007,57:1327–1330.
[8] Xu, H., Fu, Y.Y., Yang, N., Ding, Z.X., Lai, Q.L., Zeng, R.Y. Flammeovirga pacifica sp. nov.,isolate from deep sea sediment of west Pacific Ocea. J Syst Evol Microbiol.2011, doi:10.1099/ijs.0.030676-0
[9] Yang, J.L., Chen, L.C., Shih, Y.Y., Hsieh, C.Y., Chen, C.Y., Chen, W.M., Chen, C.C. Cloningand characterization of β-agarase AgaYT from Flammeovirga yaeyamensis strain YT. JBiosci Bioeng.2011,112:225–232.
[2]王静雪,江晓路,胡晓珂.细菌降解琼胶的研究进展.中国水产科学.2001,8(3):94–96.
[3]杜宗军,王祥红,李筠.琼胶酶研究进展.微生物学通报.2003,30(1):64–67.
[4]马翠萍,石超.琼胶酶研究进展.微生物学通报.2008,35(1):107–111.
[5]胡晓珂,江晓路,管华诗.海藻多糖降解酶的性质和作用机理.微生物学报.2001,41(6):762–766.
[6] Cantarel, B.L., Coutinho, P.M., Rancurel, C., Bernard, T., Lombard V., Henrissat, B. TheCarbohydrate-Active Enzymes database (CAZy): an expert resource for Glycogenomics.Nucleic Acids Res.2009,37(suppl1): D233-D238.
[7] Potin, P.; Richard, C.; Rochas, C.; Kloareg, B. Purification and characterization of theα-agarase from Alteromonas agarlyticus (Cataldi) comb. nov., strain GJ1B. Eur J Biochem.1993,214:599–607.
[8] Kirimura, K.; Masuda, N.; Iwasaki, Y.; Nakagawa, H.; Kobayashi, R.; Usami, S. Purificationand characterization of a novel β-agarase from an alkalophilic bacterium, Alteromonas sp.E-1. J Biosci Bioeng.1999,87,436–441.
[9] Rochas, C., Potin, P., and Kloareg, B., NMR spectroscopic investigation of agarose oligomersproduced by an α-agarase. Carbohyd Res.1994,253:69–77.
[10] Morrice, L.M., Mclean, M.W., Williamson, F.B., and Long W.F., Beta-agarases I and II fromPseudomonas atlanticaa: Purification and some properties. Eur J Biochem.1983,135:553–558.
[11] Morrice, L.M., Maitland, W.M., Long, W.F., and Williamson, F.B. Beta-agarases I and II fromPseudomonas atlanticaa: Substrate specificities. Eur J Biochem,1983,137:149–154.
[12] Bibb, M.J., Jones, G.H., Joseph, R., Buttner, M.J., Ward, J.M. The agarase gene (dag A) ofStreptomyces coelicolor A3(2): Affinity purification and characterization of the cloned geneproduct. J Gen Microbiol.1987,133:2089–2096.
[13] Hosoda, A., Sakai, M., Kanazawa S. Isolation and characterization of agar-degradingPaenibacillus spp. Associated with the rhizosphere of spinach. Biosci Biotechnol Biochem.2003,67(5):1048–1055.
[14]韩文君,古静燕,李天东,罗英,阮期平.一株产琼胶酶土壤细菌Paenibacillus sp.MY03的筛选与分子鉴定.绵阳师范学院学报.2007,26(2):86–90.
[15] Fu, X.T., Kim, S.M. Agarase: Review of major sources, categories, purification method,enzyme characteristics and applications. Mar Drugs.2010,8:200–218.
[16] Voget, S., Leggewie, C., Uesbeck, A., Raasch, C., Jaeger, K.-E., Streit, W.R. Prospecting fornovel biocatalysts in soil metagenome. Appl Environ Microbiol,2003,69(10):6235–6242.
[17] Mello, L.V., Chen, X., Rigden, D.J. Mining metagenomic data for novel domains: BACON, anew carbohydrate-binding module. FEBS Lett.2010,584(11):2421–2426.
[18] Sim, S.J., Baik, S.K., Park, S.C., Choe, H.N., Seong, C.N., Shin, T.S., Woo, H.C., Cho, J.Y.,Kim, D. Characterization of alginate lyase gene using a mentagenomic library constructedfrom the gut microflora of abalone. J Ind Microbial Biotechnol.2011, Doi:10.1007/s10295-011-1054-0.
[19] Li, J.G., Zhang, K.G., Han, W.J. Cloning and biochemical characterization of a novel lipolyticgene from activated sludge metagenome, and its gene product. Microbial Cell Factories.2010,9:83.
[20] Glogauer, A., Martini, V.P., Faoro, H., Couto, G.H., Muller-Santos, M., Monteiro, R.A.,Mitchell, D.A., Souza, E.M., Pedrosa, F.O., Krieger, N. Identification and characterization ofa new true lipase isolated through metagenomic approach. Microbial Cell Factories.2011,10:54.
[22] Ohta, Y., Hatada, Y., Miyazaki, M., Nogi, Y., Ito, S., Horikoshi, K. Purification andcharacterization of a novel α-agarase from a Thalassomonas sp. Curr Microbiol.2005,50:212–216.
[23] Lahaye, M., Yaphe, W., Phan, Viet, M.T., and Rochas, C.,1989.13C-NMR spectroscopicinvestigation of metylated and charged agarose oligosaccharides and polysaccharides.Carbohydrate Research,90:249-265. Li, J.B., Han, F., Lu, X.Z., Fu, X.Y., Ma, C.P., Chu, Y.,and Yu, W.G.,2007. A simple method of preparing diverse neoagaro-oligosaccharides withβ-agarase. Carbohyd Res.342:1030–1033.
[24] Mei, J.F., Shao, J.T., Wang, Qi., Wang, H., Yi, Y., Ying, G.Q. Separation and quantification ofneoagaro-oligosaccharides. J Food Sci Technol.2011, Doi:10.1007/sl3197-011-0448-3.
[25] Michel, G., Barbeyron, T., Kloareg, B., Czjzek. The famly6carbohydrate-binding moduleshave coevolved with their appended catalytic modules toward similar substrate specificity.Glycobiol.2009,19(6):615–623.
[26] Flament, D., Barbeyron, T., Jam, M., Potin, P., Czjzek, M., Kloareg, B., Michel, G.Alpha-agarases define a new family of glycoside hydrolases, distinct from beta-agarasesfamilies. Appl Environ Microbiol.2007,73(14):4691–4694.
[27] Coutinho, P. M., and B. Henrissat. Carbohydrate-active enzymes: an integrated databaseapproach, p.3–12. In H. J. Gilbert, G. Davies, B. Henrissat, and B. Svensson (ed.), Recentadvances in carbohydrate bioengineering.1999. The Royal Society of Chemistry, Cambridge,United Kingdom.
[28] Sugano, Y., Matsumoto, T., Kodama, H., Noma, M. Cloning and sequencing of agaA, aunique agarase0107gene from a marine bacterium, Vibrio sp. strain JT0107. Appl EnvironMicrobiol.1993,59:3750–3756.
[29] Ohta, Y., Hatada, Y., Nogi, Y., Li, Z.J., Ito, S., Horikoshi, K. Cloning, expression, andcharacterization of a glycoside hydrolase family86β-agarase from a deep-seaMicrobulbifer-like isolate. Appl Microbiol Biotechnol.2004,66:266–275.
[30] Lu, X.Z., Chu, Y., Wu, Q.Q., Gu, Y.C., Han, F., Yu, W.G. Cloning, expression andcharacterization of a new agarase-encoding gene from marine Pseudoalteromonas sp..Biotechnol Lett.2009,31:1565–1570.
[31] Kim, H.T., Lee, S.Y., Lee, D.H., Kim, H.S., Bang, W.G., Kim, G.C. Overexpression andmolecular characterization of Aga50D from Saccharophagus degradans2-40: anexo-typeβ-agarase producing neoagarobiose. Appl Microbiol Biotechnol.2010,86:227–234.
[32] Jam, M., Flament, D., Allouch, J., Potin, P., Thion, L., Kloareg, B., Czjzek, M., Helbert, W.,Michel, G., Barbeyron, T. The endo-β-agarase AgaA and AgaB from the marine bacteriumZobellia galactanivorans: two paralogue enzymes with different molecular organizations andcatalytic behaviours. Biochem J.2005,385:703–713.
[33] Ma, C.P., Lu, X.Z., Shi, C., Li, J.B., Gu, Y.C., Ma, Y.M., Chu, Y., Han, F., Gong, Q.H., andYu, W.G. Molecular cloning and characterization of a novel beta-agarase, AgaB, frommarine Pseudoalteromonas sp. CY24. J Biol Chem.2007,282(6):3747–3754.
[34] Allouch, J., Jam, M., Helbert, W., Barbeyron, T., Kloareg, B., Henrissat, B., Czjzek, M. Thethree-deimensional structures of two β-agarases. J Biol Chem.2003,278(47):47171–47180.
[35] Shi, C., Lu, X.Z., Ma, C.P., Ma, Y.M., Fu, X.Y., Yu, W.G. Enhancing the thermostability of anovel β-agarase AgaB through directed evolution. Appl Biochem Biotechnol.2008,151:51–59.
[36] Jang, M.K., Lee, S.W., Lee, D.G., Kim, N.Y., Yu, K.H., Jang, H.J., Kim, S.K., Kim, A., Lee,S.H. Enhancement of the thermostability of a recombinant β-agarase, AgaB, from Zobelliagalactanivorans by random mutagenesis. Biotechnol Lett.2010,32:943–949.
[37] Lee, S.W., Lee, D.G., Jang, M.K., Jeon, M.J., Jang, H.J., Lee, S.H. Improvement in thecatalytic activity ofβ-agarase AgaA from Zobellia galactanivorans by site-directedmutagenesis. J Microbiol Biotechnol.2011,21(11):1116–1122.
[38] Henshaw, J., Horne-Bitschy, A., Lammerts van Bueren, A., Money, V.A., Bolam, D.N.,Czjzek, M., Ekborg, N.A., Weiner, R.M., Hutcheson, S.W., Davies, G.J., Boraston, A.B.,Gilbert, H.J. Family6carbohydrate binding modules in β-agarases display exquisiteselectivity for the non-reducing termini of agarose chains. J Biol Chem.2006,282(25):17099–17107.
[39] Allouch, J., Helbert, W., Henrissat, B., Czjzek, M. Parallel substrate binding sites in aβ-agarase suggest a novel mode of action on double-helical agarose. Structure,2004,12:622–632.
[39] Ekborg, N.A., Taylor, L.E., Longmire, A.G., Henrissat, B., Weiner, R.M., Hutcheson, S.W.Genomic and proteomic analyses of the agarolytic system expressed by Saccharophagusdegradans2-40. Appl Environ Microbiol.2006,72(5):3396–3405.
[40] Shin, M.H., Lee, D.Y., Wohlgemuth, G., Choi, I.G., Fiehn, O., Kim, K.H. Global metaboliteprofiling of agarose degradation by Saccharophagus degradans2-40. New Biotechnol.2010,27(2):156–168.
[41] Yun, E.J., Shin, M.H., Yoon, J.J., Kim, Y.J., Choi, I.G., Kim, K.H. Production of3,6-anhydro-L-galactose from agaose by agarolytic enzymes of Saccharophagus degradans2-40. Process Biochem.2011,46:88–93.
[42] Cole KD, kerman B. Enhanced capacity for electrophoretic capture of plasmid DNA byagarase treatment of agarose gels. Biomacromolecules.2000,1:771–81.
[43] Yeong, H.Y., Khalid, N., Phang, S.M. Protoplast isolation and regeneration from Gracilariachangii (Gracilariales, Rhodophyta). J Appl Phycol.2008,20:641–51.
[44] Gupta, V., Kumar, M., Kumari, P., Reddy, C.R.K., Jha, B. Optimization of protoplast yieldsfrom the red algae Gracilaria dura (C. Agardh) J. Agardh and G. verrucosa (Huds.)Papenfuss. J Appl Phycol.2010, doi:10.1007/s10811-010-9579-6.
[45] Kujundzic, E., Greenberg, A.R., Fong, R., Moore, B., Kujundzic, D., Hernandez, M.Biofouling potential of industrial fermentation broth components during microfiltration. JMemb Sci.2010,349:44–55.
[46] Kim, H.T., Lee, S., Kim, K.H., Choi, I.G. The complete enzymatic saccharification of agaroseand its application to simultaneous saccharification and fementation of agaorse for ethanolproduction. Bioresource Technol.2011, doi:10.1016/j.biortech.2011.11.120.
[47] Wu, S.C., Wang, F.J., Chen, J.R., Pan, C.L. Study on wines produced from monostromanitidum hydrolysate solution. J Mar Sci Technol.2011,19(4):430–443.
[48] Wu, S.C., Wen, T.N., Pan, C.L. Algal-oligosaccharide-lysates prepared by two bacterialagarases stepwise hydrolyzed and their anti-oxidative properties. Fisher Sci.2005,71:1149–1159.
[49] Wang, J.X., Jiang, X.L., Mou, H.J., Guan, H.S. Anti-oxidation of agar oligosaccharidesproduced by agarase from a marine bacterium. J Appl Phycol.2004,16:333–340.
[50] Hu, B., Gong, Q.H., Wang, Y., Ma, Y.M., Li, J.B., Yu, W.G. Prebiotic effects ofneoagaro-oligosaccharides prepared by enzymatic hydrolysis of agarose. Anaerobe.2006,12:260–266.
[51] Oh, C.H., Nikapitiya, C.L., Lee, Y.D., Whang, I., Kim, S.J., Kang, D.H, Lee, J.H. Cloning,purification and biochemical characterization of beta agarase from the marine bacteriumPseudoalteromonas sp. AG4. J Ind Microbiol Biotechnol.2010,37:483–494.
[1] Zhong, Z.P., Toukdarian, A., Helinski, D., Knauf, V., Sykes, S., Wilkinson, J.E., O’Bryne, C.,Shea, T., DeLoughery, C., Caspi R. Sequence analysis of a101-kilobase plasmid required foragar degradation by a Microscilla isolate. Appl Environ Microbiol.2001,67(12):5771–5779.
[2]分子克隆实验指南(上、下册)/(美) J.萨姆布鲁克(Sambrook, J.)等著;黄培堂等译.–3版.–北京:科学出版社,2002.
[3] Thompson, J.D., Gibson,T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G. The ClustalXwindows interface: flexible strategies for multiple sequence alignment aided by qualityanalysis tools. Nucl Acids Res.1997,25:4876–4882.
[4] Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S. MEGA5: Molecularevolutionary genetics analysis using maximum likelihood, evolutionary distance, andmaximum parsimony methods. Mol Biol Evol.2011,28(10):2731–2739.
[5] Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities ofprotein utilizing the principle of protein-dye binding. Anal Biochem.1976,72:248–254.
[6] Miller, G.L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. AnalChem.1959,31:426–428.
[7]精编蛋白质科学实验指南/(美) JE.科林根(Coligan, J.E.)等著;李慎等译.–北京:科学出版社,2007.
[8] Zhang, M., Jiang, Z., Yang, S., Hua, C., Li, L. Cloning and expression of a Paecilomycesthermophila xylanase gene in E. coli and characterization of the recombinant xylanase.Bioresour Technol.2010,101:688–695.
[9] Lahaye, M., Yaphe, W., Phan, V.M.T., Rochas, C.13C-NMR spectroscopic investigation ofmetylated and charged agarose oligosaccharides and polysaccharides. Carbohydr Res.1989,90:249–265.
[10] Nakagawa, Y., Hamana, K., Sakane, T., Yamasato, K. Reclassification of Cytophaga aprica(Lewin1969) Reichenbach1989in Flammeovirga gen. nov. as Flammeovirga aprica comb.nov. and of Cytophaga diffluens (ex Stanier1940; emend. Lewin1969) Reichenbach1989inPersicobacter gen. nov. as Persicobacter diffluens comb. nov. Int J Syst Evol Microbiol.1997,47:220–223.
[11] Takahashi, M., Suzuki, K.C., Nakagawa, Y. Emendation of the genus Flammeovirga andFlammeovirga aprica with the proposal of Flammeovirga arenaria nom. rev., comb. nov. andFlammeovirga yaeyamensis sp. nov. Int J Syst Evol Microbiol.2006,56:2095–2100.
[12] Hosoya, S., Yokota, A. Flammeovirga kamogawensis sp. nov., isolated from coastal seawaterin Japan. Int J Syst Evol Microbiol.2007,57:1327–1330.
[13] Xu, H., Fu, Y.Y., Yang, N., Ding, Z.X., Lai, Q.L., Zeng, R.Y. Flammeovirga pacifica sp. nov.,isolate from deep sea sediment of west Pacific Ocea. J Syst Evol Microbiol.2011, doi:10.1099/ijs.0.030676-0
[14] Yang, J.L., Chen, L.C., Shih, Y.Y., Hsieh, C.Y., Chen, C.Y., Chen, W.M., Chen, C.C. Cloningand characterization of β-agarase AgaYT from Flammeovirga yaeyamensis strain YT. JBiosci Bioeng.2011,112:225–232.
[1]分子克隆实验指南(上、下册)/(美) J.萨姆布鲁克(Sambrook, J.)等著;黄培堂等译.–3版.–北京:科学出版社,2002.
[1]分子克隆实验指南(上、下册)/(美) J.萨姆布鲁克(Sambrook, J.)等著;黄培堂等译.–3版.–北京:科学出版社,2002.
[2] Hall, T.A. BioEdit: a user-friendly biological sequence alignment editor and analysis programfor Windows95/98/NT. Nucl Acids Symp Ser.1999,41:95–98.
[3] Zhong, Z.P., Toukdarian, A., Helinski, D., Knauf, V., Sykes, S., Wilkinson, J.E., O’Bryne, C.,Shea, T., DeLoughery, C., Caspi R. Sequence analysis of a101-kilobase plasmid required foragar degradation by a Microscilla isolate. Appl Environ Microbiol.2001,67(12):5771–5779.
[4] Allouch, J., Jam, M., Helbert, W., Barbeyron, T., Kloareg, B., Henrissat, B., Czjzek, M. Thethree-deimensional structures of two β-agarases. J Biol Chem.2003,278(47):47171–47180.
[5] Yang, J.L., Chen, L.C., Shih, Y.Y., Hsieh, C.Y., Chen, C.Y., Chen, W.M., Chen, C.C. Cloningand characterization of β-agarase AgaYT from Flammeovirga yaeyamensis strain YT. JBiosci Bioeng.2011,112:225–232.
[6] Voget, S., Leggewie, C., Uesbeck, A., Raasch, C., Jaeger, K.-E., Streit, W.R. Prospecting fornovel biocatalysts in soil metagenome. Appl Environ Microbiol,2003,69(10):6235–6242.
[7] Mello, L.V., Chen, X., Rigden, D.J. Mining metagenomic data for novel domains: BACON, anew carbohydrate-binding module. FEBS Lett.2010,584(11):2421–2426.
[8] Sim, S.J., Baik, S.K., Park, S.C., Choe, H.N., Seong, C.N., Shin, T.S., Woo, H.C., Cho, J.Y.,Kim, D. Characterization of alginate lyase gene using a mentagenomic library constructedfrom the gut microflora of abalone. J Ind Microbial Biotechnol.2011, Doi:10.1007/s10295-011-1054-0.
[9] Michel, G., Barbeyron, T., Kloareg, B., Czjzek. The famly6carbohydrate-binding moduleshave coevolved with their appended catalytic modules toward similar substrate specificity.Glycobiol.2009,19(6):615–623.
[10] Lu, X.Z., Chu, Y., Wu, Q.Q., Gu, Y.C., Han, F., Yu, W.G. Cloning, expression andcharacterization of a new agarase-encoding gene from marine Pseudoalteromonas sp..Biotechnol Lett.2009,31:1565–1570.
[1] Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities ofprotein utilizing the principle of protein-dye binding. Anal Biochem.1976,72:248–254.
[2] Miller, G.L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. AnalChem.1959,31:426–428.
[3] Zhang, M., Jiang, Z., Yang, S., Hua, C., Li, L. Cloning and expression of a Paecilomycesthermophila xylanase gene in E. coli and characterization of the recombinant xylanase.Bioresour Technol.2010,101:688–695.
[4] Bigge, J.C., Patel, T.P., Bruce, J.A., Goulding, P.N., Charles, S.M., Parekh, R.B. Nonselectiveand efficient fluorescent labeling of glycans using2-amino benzamide and anthranilic acid.Anal Biochem.1995,230:229–238.
[5] Yang, J.L., Chen, L.C., Shih, Y.Y., Hsieh, C.Y., Chen, C.Y., Chen, W.M., Chen, C.C. Cloningand characterization of β-agarase AgaYT from Flammeovirga yaeyamensis strain YT. JBiosci Bioeng.2011,112:225–232.
[6] Zhong, Z.P., Toukdarian, A., Helinski, D., Knauf, V., Sykes, S., Wilkinson, J.E., O’Bryne, C.,Shea, T., DeLoughery, C., Caspi R. Sequence analysis of a101-kilobase plasmid required foragar degradation by a Microscilla isolate. Appl Environ Microbiol.2001,67(12):5771–5779.
[7] Dong, J.H., Tamaru, Y., Araki, T. A unique β-agarase, AgaA, from a marine bacterium, Vibriosp. strain PO-303. Appl Microbiol Biotechnol.2007,74:1248–1255.
[8] Lu, X.Z., Chu, Y., Wu, Q.Q., Gu, Y.C., Han, F., Yu, W.G. Cloning, expression andcharacterization of a new agarase-encoding gene from marine Pseudoalteromonas sp..Biotechnol Lett.2009,31:1565–1570.
[9] Long, M.X., Yu, Z.N., Xu, X. A novel β-agarase with high pH stability from marineAgarivorans sp. LQ48. Mar Biotechnol.2010,12:62–69.
[10] Zhang, W.W., Sun, L. Cloning, characterization, and molecular application of a beta-agarasegene from Vibrio sp. strain V134. Appl Environ Microbiol.2007,2825–2831.
[11] Wu, S.C., Wen, T.N., Pan, C.L. Algal-oligosaccharide-lysates prepared by two bacterialagarases stepwise hydrolyzed and their anti-oxidative properties. Fisher Sci.2005,71:1149–1159.
[12] Wang, J.X., Jiang, X.L., Mou, H.J., Guan, H.S. Anti-oxidation of agar oligosaccharidesproduced by agarase from a marine bacterium. J Appl Phycol.2004,16:333–340.
[13] Hu, B., Gong, Q.H., Wang, Y., Ma, Y.M., Li, J.B., Yu, W.G. Prebiotic effects ofneoagaro-oligosaccharides prepared by enzymatic hydrolysis of agarose. Anaerobe.2006,12:260–266.
[14] Oh, C.H., Nikapitiya, C.L., Lee, Y.D., Whang, I., Kim, S.J., Kang, D.H, Lee, J.H. Cloning,purification and biochemical characterization of beta agarase from the marine bacteriumPseudoalteromonas sp. AG4. J Ind Microbiol Biotechnol.2010,37:483–494.
[1] Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities ofprotein utilizing the principle of protein-dye binding. Anal Biochem.1976,72:248–254.
[2] Miller, G.L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. AnalChem.1959,31:426–428.
[3] Zhang, M., Jiang, Z., Yang, S., Hua, C., Li, L. Cloning and expression of a Paecilomycesthermophila xylanase gene in E. coli and characterization of the recombinant xylanase.Bioresour Technol.2010,101:688–695.
[4] Bigge, J.C., Patel, T.P., Bruce, J.A., Goulding, P.N., Charles, S.M., Parekh, R.B. Nonselectiveand efficient fluorescent labeling of glycans using2-amino benzamide and anthranilic acid.Anal Biochem.1995,230:229–238.
[1] Guex, N., Peitsch, M.C. SWISS-MODEL and the Swiss-PdbViewer: An environment forcomparative protein modeling. Electrophoresis.1997,18:2714–2723.
[2] Guex, N., Peitsch, M.C. Swiss-PdbViewer: A fast and easy-to-use PDB Viewer for Macintoshand PC. Protein Data Bank Quaterly Newsletter.1996,77:7.
[3] Zhang, M., Jiang, Z., Yang, S., Hua, C., Li, L. Cloning and expression of a Paecilomycesthermophila xylanase gene in E. coli and characterization of the recombinant xylanase.Bioresour Technol.2010,101:688–695.
[1] Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S. MEGA5: Molecularevolutionary genetics analysis using maximum likelihood, evolutionary distance, andmaximum parsimony methods. Mol Biol Evol.2011,28(10):2731–2739.
[2] Allouch, J., Jam, M., Helbert, W., Barbeyron, T., Kloareg, B., Henrissat, B., Czjzek, M. Thethree-deimensional structures of two β-agarases. J Biol Chem.2003,278(47):47171–47180.
[3] Ekborg, N.A., Taylor, L.E., Longmire, A.G., Henrissat, B., Weiner, R.M., Hutcheson, S.W.Genomic and proteomic analyses of the agarolytic system expressed by Saccharophagusdegradans2-40. Appl Environ Microbiol.2006,72(5):3396–3405.
[4] Pagani, I., Chertkov, O., Lpidus, A., Lucas, S., Rio, T.G.D., Tice, H., Copeland, A., Cheng,J.F., Nolan, M., Saunders, E., Pitluck, S., Held, B., Goodwin, L., Liolios, K., Ovchinikova,G., Lvanova, N., Mavromatis, K., Pati, A., Chen, A., Palaniappan, K., Land, M., Hauser, L.,Jeffries, C.D., Detter, J.C., Han, C., Tapia, R., Ngatchou-Djao, O.D., Rohde, M., Goker, M.,Spring, S., Sikorski, J., Woyke, T., Bristow, J., Eisen, J.A., Markowitz, V., Hugenholtz, P.,Klenk, H.P., Kyrpides, N.C. Complete genome sequence of Marivirga tractuosa type strian(H-43)T. Stand Genomic Sci.2011,42(2):154–1622.
[5] Nakagawa, Y., Hamana, K., Sakane, T., Yamasato, K. Reclassification of Cytophaga aprica(Lewin1969) Reichenbach1989in Flammeovirga gen. nov. as Flammeovirga aprica comb.nov. and of Cytophaga diffluens (ex Stanier1940; emend. Lewin1969) Reichenbach1989inPersicobacter gen. nov. as Persicobacter diffluens comb. nov. Int J Syst Evol Microbiol.1997,47:220–223.
[6] Takahashi, M., Suzuki, K.C., Nakagawa, Y. Emendation of the genus Flammeovirga andFlammeovirga aprica with the proposal of Flammeovirga arenaria nom. rev., comb. nov. andFlammeovirga yaeyamensis sp. nov. Int J Syst Evol Microbiol.2006,56:2095–2100.
[7] Hosoya, S., Yokota, A. Flammeovirga kamogawensis sp. nov., isolated from coastal seawaterin Japan. Int J Syst Evol Microbiol.2007,57:1327–1330.
[8] Xu, H., Fu, Y.Y., Yang, N., Ding, Z.X., Lai, Q.L., Zeng, R.Y. Flammeovirga pacifica sp. nov.,isolate from deep sea sediment of west Pacific Ocea. J Syst Evol Microbiol.2011, doi:10.1099/ijs.0.030676-0
[9] Yang, J.L., Chen, L.C., Shih, Y.Y., Hsieh, C.Y., Chen, C.Y., Chen, W.M., Chen, C.C. Cloningand characterization of β-agarase AgaYT from Flammeovirga yaeyamensis strain YT. JBiosci Bioeng.2011,112:225–232.