褐藻胶裂解酶分泌菌株的分离鉴定及Tamlana holothuriorum s12~T中褐藻胶裂解酶的研究
发布时间：2018-08-29 12:03
【摘要】：褐藻作为第三代可持续发展生物燃料的原料之一,其繁殖快、耗能低、不占用耕地和不消耗淡水,不产生粮食矛盾,与以谷物为代表的第一代生物能源原料和以秸秆等为代表的第二代生物能源原料相比,具有广阔的应用前景。尽管已有研究表明利用褐藻进行生物转化如发酵生产生物乙醇是切实可行的,但由褐藻生产生物乙醇的全部潜力还难以实现,其主要问题是工业微生物菌株难以有效地降解褐藻胶中L-古罗糖醛酸片段、D-甘露糖醛酸片段及其嵌合片段生成可被工业菌株吸收并利用的单糖,且降解产生的糖醛酸单体化合物也难以被转化利用。褐藻胶降解酶属于多糖裂解酶,多为聚D-甘露糖醛酸底物特异性的内切型褐藻胶裂解酶,降解产物为寡糖；而能够同时降解L-古罗糖醛酸片段、D-甘露糖醛酸片段及其嵌合片段的多功能酶较少,特别是缺乏广泛底物特异性的外切酶。因此,寻求新型高效且具有广泛底物特异性的褐藻胶裂解酶及酶系是褐藻胶生物转化生产所面临的重要任务。本项研究旨在筛选新型高效褐藻胶裂解酶分泌菌株,研究其产酶特性,并外源表征新型或高效褐藻胶裂解酶的基因,初步阐述高效褐藻胶降解菌的降解机制,为深入研究褐藻生物质降解转化机制积累基础数据,为褐藻胶的生物转化提供优良的菌种资源、酶制剂奠定生物学基础。在本项研究中取得的主要成果如下：一、对近海和陆地环境样品进行了褐藻胶裂解酶分泌菌株的广泛筛选,获得了多个新物种和多株高效褐藻胶降解菌。从十余种环境样品中分离获得182株褐藻胶裂解酶分泌菌株,这些菌株分布在52个属。发现其中22个属的细菌(Aestuariibacter sp., Idiomarina sp., Kushneria sp., Litorimonas sp., Paracoccus sp., Roseovarius sp., Salinivibrio sp., Thalassobius sp., Halobacillus sp., Planomicrobium sp., Saccharomonospora sp., Salegentibacter sp., Seonamhaeicola sp., Tenacibaculum sp., Agromyces sp., Brachybacterium sp., Cellulosimicrobium sp., Dietzia sp., Janibacter sp., Kytococcus sp., Micrococcus sp.和Flavohalobacter sp.)尚未见报道具有分泌褐藻胶裂解酶的功能。以16S rRNA目似度阈值98.7%为判断细菌新物种的标准,发现在所筛选的褐藻胶降解菌株中有13株为潜在新物种。4株细菌的16S rRNA最高相似度低于97%,新颖性好,其中3株海洋细菌属于拟杆菌门(菌株Dm15T和Gy8T属于拟杆菌门黄杆菌科,菌株Sy30T属于拟杆菌门嗜纤维菌科)。此外,筛选到的一株酶活高且传代稳定的褐藻胶降解菌为来自海参肠道的菌株s12T,与Tamlana agarivorans JW-26T最高相似度为99.1%,也属于拟杆菌门黄杆菌科。经多相分类学研究,对分离到的拟杆菌门4个新菌进行了鉴定。高效褐藻胶降解菌株s12T为黄杆菌科Tamlana属的新种,命名为Tamlana holothuriorum sp. nov.;菌株Dm15T为黄杆菌科的新属新种,命名为Flavohalobacter algicola gen. nov. sp. nov.;菌株Gy8T为黄杆菌科Seonamhaeicola属的新种,命名为Seonamhaeicola algicola sp. nov.;菌株Sy30T为拟杆菌门Hymenobacter科Pontibacter属的新种,命名为Pontibacter locisalis sp. nov.。二、完成了褐藻胶高效降解菌株s12T的基因组测序及分析,发现该菌具有新颖的褐藻胶降解酶和较完整的褐藻胶代谢途径。对菌株s12T进行了基因组测序及序列分析。菌株s12T基因组全长3.66 Mbp,共有3151个编码蛋白基因,其中1,729个蛋白可以匹配到同源蛋白聚簇数据库,其它大量功能未知的基因序列预示着该菌的新功能和新基因存在的可能。通过对基因组数据的生物信息分析,预测到该菌共有11条编码褐藻胶裂解酶基因(alg1-11),与数据库中已表征的褐藻胶裂解酶序列的相似度均低于73%,其中包括2个外切型褐藻胶裂解酶基因alg5和αlg9。对所预测的这些酶的氨基酸序列进行同源性分析,发现Alg9属于多糖解酶(Polysaccharide Lyase,PL) 17家族,其它10个褐藻胶裂解酶则均属于PL7家族,其中Alg1属于PL7第3亚家族,Alg2和Alg5属于PL7第5亚家族,Alg4、Alg6-8、Alg10、Alg11所属亚家族待定。Alg2、Alg5、Alg6和Alg9与其同源性高的褐藻胶裂解酶均具有保守的催化氨基酸残基,而Alg1、Alg3、Alg4、Alg7、Alg8、Alg10和Alg11对于催化位点关键氨基酸保守性差,说明后者可能具有新的催化特性。Alg1-4、Alg7、Alg9和Algl 1除了催化结构域外,还包含碳水化合物结合域、C端凝血因子结构域、凝集素结构域、信号肽、分泌系统、细菌免疫球蛋白样和类肝素酶结构域,根据结构域功能预测至少上述具有多个结构域的7个褐藻胶裂解酶为分泌性蛋白。总之,生物信息学分析显示菌株s12T中的褐藻胶裂解酶(酶系)具有多样性和新颖性。根据生物信息分析,同时预测出菌株s12T具有较完整的褐藻胶代谢途径。分析显示褐藻胶在胞外可被内切型和外切型褐藻胶裂解酶降解为双糖或不饱和单糖,自动形成开环单体4-脱氧-L-赤藓-糖醛酸后被吸收进入胞内,由还原酶催化生成2-酮-3-脱氧葡糖酸,进入Entner-Doudoroff途径生成三磷酸甘油醛和丙酮酸,三磷酸甘油醛通过糖酵解代谢途径转化为丙酮酸。然后,丙酮酸通过丙酮酸脱氢酶转化为乙酰辅酶A,进入三羧酸循环或乳酸发酵,完成褐藻胶的转化或彻底降解。三、完成了菌株s12T"的褐藻胶产酶特性和胞外蛋白组的测定分析,发现注释的11个褐藻胶裂解酶均为胞外蛋白,且蔗糖对褐藻胶裂解酶的表达具有诱导作用。菌株s12T可以在褐藻胶为唯一碳源的基础培养基中生长,添加EDTA、SDS和其它金属离子如MnSO4、CaCl2、CuSO4和ZnSO4后均显著抑制总酶活和总蛋白量；Na2CO3、NiSO4、KCl、纳米Si02、纳米Ti02和纳米A1304对产酶效应影响不显著。添加外源糖类时,果糖、麦芽糖和淀粉均降低该菌的产酶活性；葡萄糖的存在对产酶影响不显著,而较为特殊的是,蔗糖不能被该菌利用,不阻遏褐藻胶裂解酶的产生,反而能够显著提高酶的发酵活性,即蔗糖对该菌褐藻胶裂解酶的产生具有诱导作用。经优化得到该菌褐藻胶裂解酶的最适产酶条件为：培养温度28℃,氮源为0.5%NaNO3,初始pH值为7.5-8.0,褐藻胶初始浓度为2-2.5%,添加0.2%蔗糖。在该培养条件下,菌株s12T不利用培养基中的蔗糖,培养28 h后达到生长平台期,培养36 h后达到最高总酶活44.3 U/mL,44 h内可将2.0%海藻酸钠完全降解至产物中检测不出单糖。通过胞外蛋白组分析,发现菌株s12T基因组中所注释的9个褐藻胶裂解酶Alg1-4和Alg7-11为胞外蛋白,蔗糖的存在可以显著增加Alg1和Alg7的分泌量,从而提高菌株s12T褐藻胶裂解酶的总酶活。蔗糖促进微生物褐藻胶裂解酶的分泌和提高酶活的作用尚未见相关报道。四、外源表达了4个褐藻胶裂解酶基因,表征了酶活较高的内切型裂解酶rAlg2和外切型裂解酶rAlg5的酶学性质,发现该菌的褐藻胶裂解酶具有广泛的底物特异性。根据前期预测,将菌株s12T中片段较小的褐藻胶裂解酶基因algl、alg2、alg5和αlg6在大肠杆菌中进行外源表达,并对酶活较高的rAlg2和rAlg5进行了酶学性质的表征。研究发现重组酶rAlg2降解产物为2、3和4糖,故重组酶rAlg2为内切酶；重组酶rAlg5的降解产物中有单糖,故重组酶,rAlg5为外切酶。重组酶rAlg2和rAlg5的最适反应温度均为40-45℃,温度稳定范围分别为4-40℃和4-20℃；两者的最适反应pH值分别为6.0-6.5和7.0-8.0,pH稳定范围为6.0-7.0和7.0-8.0。研究发现KC1和NaCl能显著提高重组酶rAlg2勺酶活,而对重组酶rAlg5酶活影响不显著；SDS、EDTA、NH4Cl、FeCl3、FeSO4、MnCl2、CaCl2和MgCl2显著抑制重组酶rAlg2和rAlg5的酶活。结果显示重组酶rAlg2和rAlg5都具有广泛的底物特异性,底物偏好性分别为聚L-古罗糖醛酸片段和聚D-甘露糖醛酸片段。在以海藻酸钠为底物时,重组酶rAlg2和rAlg5的比酶活分别为2350和1350U/mg,Km分别为0.03和0.20 mM, Kcat分别为13.4和4.4 S-1,Kcat/Km分别为45.4和220.5 S-1mM-1。褐藻胶裂解酶Alg5是在PL7家族发现的第二个外切酶,有独特的特点。经氨基酸序列分析,发现Alg5和来自菌株Zobellia galactanivorans DsiJT的AlyA5同源性最高,在催化腔内具有保守的关键氨基酸。功能表征发现两者均为广泛底物特异性的外切酶,但两者底物偏好性不同,据报道AlyA5偏好聚L-古罗糖醛酸,而本实验中重组酶rAlg5偏好聚D-甘露糖醛酸。这也说明重组酶rAlg5的底物识别与催化腔内保守氨基酸关系不大,可能与围绕在催化腔周围的loop构象或其非保守的氨基酸有关。此外,本研究中发现重组酶rAlg5在低温4℃下其酶活可达到最高酶活时的51.2%,热稳定性差,生理系数Kcat/Km高,对有机溶剂SDS和EDTA敏感,说明重组酶rAlg5具有适冷酶的特性。通过褐藻胶裂解酶的功能表征,可以发现菌株s12T中至少有2个褐藻胶裂解酶(Alg2和Alg5)具有广泛底物特异性,其中Alg5为适冷外切酶。这些结果说明菌株s12T可以适应低温以及不同底物条件而有效地降解褐藻胶,能在胞外将褐藻胶降解成糖醛酸单体化合物。
[Abstract]:Brown algae, as one of the third-generation biofuels for sustainable development, has the advantages of rapid propagation, low energy consumption, no occupation of arable land and no consumption of fresh water, and no food contradiction. Compared with the first-generation bioenergy raw materials represented by grain and the second-generation bioenergy raw materials represented by straw, brown algae has broad application prospects. The results showed that bioconversion by brown algae such as fermentation to produce bioethanol was feasible, but the full potential of bioethanol production by brown algae was still difficult to realize. The main problem was that industrial microorganisms could not effectively degrade L-guluronic acid fragments in alginate, D-mannosuric acid fragments and their chimeric fragments could be manufactured. Alginate degrading enzymes belong to polysaccharide lyases, mostly endo-alginate lyases with poly-D-mannite substrate specificity, and their degradation products are oligosaccharides; and they can degrade L-guluronic acid fragments, D-mannite alginate acid fragments simultaneously. There are few multifunctional enzymes in fragments and chimeric fragments, especially those lacking broad substrate-specific enzymes. Therefore, it is an important task for alginate biotransformation to search for novel alginate lyases and enzymes with broad substrate-specific properties. The strain was used to study the enzyme-producing characteristics and characterize the genes of novel or high-efficient alginate lyase. The degradation mechanism of high-efficient Alginate-degrading bacteria was preliminarily expounded. The basic data were accumulated for the further study of the mechanism of alginate biomass degradation and transformation, which provided excellent strain resources for alginate biotransformation and laid a biological foundation for enzyme preparation. The main results obtained in this study are as follows: 1. Several new species and strains of Alginate-degrading bacteria were obtained by extensive screening of alginate lyase-secreting strains from coastal and terrestrial environmental samples. 182 strains of Alginate-degrading bacteria were isolated from more than ten environmental samples, which were distributed in 52 genera. Among them, 22 genera of bacteria (Aestuariibacter sp., Idiomarina sp., Kushneria sp., Litorimonas sp., Paracoccus sp., Roseovarius sp., Salinivibrio sp., Thalassobius sp., Halobacillus sp., Planomicrobium sp., Saccharnospora., Salebacter sp., Seonamhaeicola sp., Tenbacum sp., Agricultural sp., B. Rahybacterium sp., Cellulosimicrobium sp., Dietzia sp., Janibacter sp., Kytococcus sp., Micrococcus sp. and Flavohalobacter sp.) have not been reported to secrete alginate lyase. The 16S rRNA similarity threshold of 98.7% was used as a criterion for the identification of new species of bacteria, and 13 of the screened Alginate-degrading strains were found. The 16S rRNA similarity of four strains of bacteria was lower than 97%, and the novelty was good. Three marine bacteria belonged to the phylum Pseudomonas (strains Dm15T and Gy8T belonged to the family Pheudomonas, strain Sy30T belonged to the family Pheudomonas). In addition, one strain with high enzyme activity and stable passage of Alginate-degrading bacteria was isolated from the sea. The strain s12T from the intestinal tract of Panax ginseng had the highest similarity with Tamlana agarivorans JW-26T, which was 99.1%. It also belonged to the family Flavobacterioideae. Strain Dm15T is a new genus and species of Flavohalobacter algicola gen.nov.sp.nov. A highly efficient alginate degrading strain, s12T, was obtained by genome sequencing and analysis. It was found that the strain had novel alginate degrading enzymes and relatively complete alginate metabolic pathways. The genome of s12T was sequenced and sequenced. The total length of s12T genome was 3.66 Mbp. There were 3151 coding protein genes, of which 1,729 proteins could be matched. By analyzing the bioinformatics of the genome data, 11 alginate lyase genes (alg1-11) were predicted to be similar to the sequence of alginate lyase in the database. The amino acid sequences of these enzymes were analyzed. It was found that Alg9 belonged to the 17 family of polysaccharide lyase (PL), and the other 10 alginate lyases belonged to the PL7 family. Alg1 belonged to the third subfamily of PL7, Alg2 and Alg. Alg 2, Alg 5, Alg 6-8, Alg 10 and Alg 11 belong to the fifth subfamily of PL7. Alg 2, Alg 5, Alg 6 and Alg 9 all have conserved catalytic amino acid residues with high homology, while Alg 1, Alg 3, Alg 4, Alg 7, Alg 8, Alg 10 and Alg 11 have poor conservativeness for key amino acids at catalytic sites, indicating that the latter may have new catalytic properties. Alg 1-4, Alg 7, Alg 9 and Algl 1 contain not only the catalytic domain, but also the carbohydrate binding domain, C-terminal coagulation factor domain, lectin domain, signal peptide, secretory system, bacterial immunoglobulin-like and heparanase domain. According to the function of the domain, it is predicted that at least seven alginate lyases with multiple domains mentioned above are secretory. In conclusion, bioinformatics analysis showed that the alginate lyases (enzymes) in strain s12T were diverse and novel. According to bioinformatics analysis, it was predicted that strain s12T had a complete alginate metabolism pathway. The analysis showed that alginate could be degraded to disaccharides or not by endo-and ex-alginate lyases in the extracellular. Saturated monosaccharides, which automatically form the ring-opening monomer 4-deoxy-L-erythrocytic-glucuronic acid, are absorbed into the cell, catalyzed by reductase to produce 2-keto-3-deoxygluconic acid, and then enter the Entner-Doudoroff pathway to produce glyceraldehyde triphosphate and pyruvic acid. Glyceraldehyde triphosphate is converted to pyruvic acid via glycolysis pathway. Then pyruvic acid passes through pyruvic acid. The dehydrogenase was converted into acetyl coenzyme A and then entered the tricarboxylic acid cycle or lactic acid fermentation to complete the transformation or complete degradation of alginate. 3. The characteristics of alginate production and extracellular proteome analysis of strain s12T were completed. It was found that the 11 alginate lyases were extracellular proteins and sucrose could induce the expression of alginate lyase. Strain s12T can grow in the basic medium with alginate as the sole carbon source. When EDTA, SDS and other metal ions such as MnSO4, CaCl2, CuSO4 and ZnSO4 are added, the total enzyme activity and total protein content are significantly inhibited; Na2CO3, NiSO4, KCl, nano-Si02, nano-Ti02 and nano-A1304 have no significant effect on the enzyme production. Both maltose and starch decreased the enzyme activity of the strain, and glucose had no significant effect on the enzyme production. In particular, sucrose could not be used by the strain and could not inhibit the production of alginate lyase. On the contrary, sucrose could significantly improve the fermentation activity of the enzyme, that is, sucrose could induce the production of alginate lyase. The optimum conditions for producing alginate lyase were as follows: incubation temperature 28 C, nitrogen source 0.5% NaNO3, initial pH 7.5-8.0, initial alginate concentration 2-2.5% and adding 0.2% sucrose. Under these conditions, strain s12T did not use sucrose in the medium, and reached the growth plateau after 28 h, and reached the highest total enzyme activity 44.5% after 36 h. 3 U/mL, 2.0% sodium alginate could be completely degraded into the product within 44 h without monosaccharide detection. Extracellular proteomic analysis showed that the nine alginate lyases Alg1-4 and Alg7-11 in the s12T genome were extracellular proteins. The presence of sucrose could significantly increase the secretion of Alg1 and Alg7, thereby increasing the secretion of alginate lyase s12T. Total enzymatic activity. Sucrose promotes the secretion and enzymatic activity of microbial alginate lyase. 4. Exogenous expression of four alginate lyase genes characterizes the enzymatic properties of the endo-cleavage enzymes rAlg2 and exo-cleavage enzymes rAlg5. It is found that the alginate lyase of the bacteria has a wide range of substrates. Specificity. According to previous prediction, alginate lyase genes algl, alg2, alg5 and alphalg6 with smaller fragments in strain s12T were exogenously expressed in E. coli, and the enzymatic properties of rAlg2 and rAlg5 with higher enzyme activity were characterized. The optimum reaction temperatures of rAlg2 and rAlg5 were 40-45 degrees C, and the temperature stability ranges were 4-40 degrees C and 4-20 degrees C, respectively. The optimum reaction pH values of rAlg2 and rAlg5 were 6.0-6.5 and 7.0-8.0, respectively, and the pH stability ranges were 6.0-7.0 and 7.0-8.0 respectively. The recombinant enzyme rAlg2 and rAlg5 showed no significant effect on the activity of rAlg5. SDS, EDTA, NH4Cl, FeCl3, FeSO4, MnCl2, CaCl2 and MgCl2 significantly inhibited the activity of rAlg2 and rAlg5. The results showed that both rAlg2 and rAlg5 had broad substrate specificity, and their substrate preferences were poly-L-guluronic acid fragment and poly-D-mannosuric acid, respectively. The specific enzyme activities of recombinant rAlg2 and rAlg 5 were 2350 and 1350 U/mg, 0.03 and 0.20 mM, 13.4 and 4.4 S-1 for Kcat, 45.4 and 220.5 S-1 mM-1 for Kcat and 120.5 S-1 mM-1 for alginate, respectively. Alg5 was the second exonuclease found in PL7 family and had unique characteristics. AlyA5 and AlyA5 from Zobellia galactanivorans DsiJT were found to have the highest homology and conserved key amino acids in the catalytic chamber. Functional characterization showed that both were broad substrate-specific exoenzymes, but their substrate preferences were different. AlyA5 was reported to prefer poly L-guluronic acid, while rAlg5 preferred poly-L-guluronic acid in this experiment. D-mannituronic acid. This also indicates that the substrate recognition of rAlg5 is not related to the conserved amino acids in the catalytic cavity, and may be related to the loop conformation around the catalytic cavity or its non-conserved amino acids. High physical coefficient Kcat/Km and sensitivity to organic solvents SDS and EDTA indicated that recombinant rAlg5 had the characteristics of cold-adapted enzymes. By characterizing the function of alginate lyase, it was found that at least two alginate lyases (Alg2 and Alg5) in strain s12T had broad substrate specificity, and Alg5 was a cold-adapted exogenous enzyme. Alginate can be effectively degraded to glucuronic acid monomers by adapting to low temperature and different substrate conditions.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q939.9
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本文编号：2211143
[Abstract]:Brown algae, as one of the third-generation biofuels for sustainable development, has the advantages of rapid propagation, low energy consumption, no occupation of arable land and no consumption of fresh water, and no food contradiction. Compared with the first-generation bioenergy raw materials represented by grain and the second-generation bioenergy raw materials represented by straw, brown algae has broad application prospects. The results showed that bioconversion by brown algae such as fermentation to produce bioethanol was feasible, but the full potential of bioethanol production by brown algae was still difficult to realize. The main problem was that industrial microorganisms could not effectively degrade L-guluronic acid fragments in alginate, D-mannosuric acid fragments and their chimeric fragments could be manufactured. Alginate degrading enzymes belong to polysaccharide lyases, mostly endo-alginate lyases with poly-D-mannite substrate specificity, and their degradation products are oligosaccharides; and they can degrade L-guluronic acid fragments, D-mannite alginate acid fragments simultaneously. There are few multifunctional enzymes in fragments and chimeric fragments, especially those lacking broad substrate-specific enzymes. Therefore, it is an important task for alginate biotransformation to search for novel alginate lyases and enzymes with broad substrate-specific properties. The strain was used to study the enzyme-producing characteristics and characterize the genes of novel or high-efficient alginate lyase. The degradation mechanism of high-efficient Alginate-degrading bacteria was preliminarily expounded. The basic data were accumulated for the further study of the mechanism of alginate biomass degradation and transformation, which provided excellent strain resources for alginate biotransformation and laid a biological foundation for enzyme preparation. The main results obtained in this study are as follows: 1. Several new species and strains of Alginate-degrading bacteria were obtained by extensive screening of alginate lyase-secreting strains from coastal and terrestrial environmental samples. 182 strains of Alginate-degrading bacteria were isolated from more than ten environmental samples, which were distributed in 52 genera. Among them, 22 genera of bacteria (Aestuariibacter sp., Idiomarina sp., Kushneria sp., Litorimonas sp., Paracoccus sp., Roseovarius sp., Salinivibrio sp., Thalassobius sp., Halobacillus sp., Planomicrobium sp., Saccharnospora., Salebacter sp., Seonamhaeicola sp., Tenbacum sp., Agricultural sp., B. Rahybacterium sp., Cellulosimicrobium sp., Dietzia sp., Janibacter sp., Kytococcus sp., Micrococcus sp. and Flavohalobacter sp.) have not been reported to secrete alginate lyase. The 16S rRNA similarity threshold of 98.7% was used as a criterion for the identification of new species of bacteria, and 13 of the screened Alginate-degrading strains were found. The 16S rRNA similarity of four strains of bacteria was lower than 97%, and the novelty was good. Three marine bacteria belonged to the phylum Pseudomonas (strains Dm15T and Gy8T belonged to the family Pheudomonas, strain Sy30T belonged to the family Pheudomonas). In addition, one strain with high enzyme activity and stable passage of Alginate-degrading bacteria was isolated from the sea. The strain s12T from the intestinal tract of Panax ginseng had the highest similarity with Tamlana agarivorans JW-26T, which was 99.1%. It also belonged to the family Flavobacterioideae. Strain Dm15T is a new genus and species of Flavohalobacter algicola gen.nov.sp.nov. A highly efficient alginate degrading strain, s12T, was obtained by genome sequencing and analysis. It was found that the strain had novel alginate degrading enzymes and relatively complete alginate metabolic pathways. The genome of s12T was sequenced and sequenced. The total length of s12T genome was 3.66 Mbp. There were 3151 coding protein genes, of which 1,729 proteins could be matched. By analyzing the bioinformatics of the genome data, 11 alginate lyase genes (alg1-11) were predicted to be similar to the sequence of alginate lyase in the database. The amino acid sequences of these enzymes were analyzed. It was found that Alg9 belonged to the 17 family of polysaccharide lyase (PL), and the other 10 alginate lyases belonged to the PL7 family. Alg1 belonged to the third subfamily of PL7, Alg2 and Alg. Alg 2, Alg 5, Alg 6-8, Alg 10 and Alg 11 belong to the fifth subfamily of PL7. Alg 2, Alg 5, Alg 6 and Alg 9 all have conserved catalytic amino acid residues with high homology, while Alg 1, Alg 3, Alg 4, Alg 7, Alg 8, Alg 10 and Alg 11 have poor conservativeness for key amino acids at catalytic sites, indicating that the latter may have new catalytic properties. Alg 1-4, Alg 7, Alg 9 and Algl 1 contain not only the catalytic domain, but also the carbohydrate binding domain, C-terminal coagulation factor domain, lectin domain, signal peptide, secretory system, bacterial immunoglobulin-like and heparanase domain. According to the function of the domain, it is predicted that at least seven alginate lyases with multiple domains mentioned above are secretory. In conclusion, bioinformatics analysis showed that the alginate lyases (enzymes) in strain s12T were diverse and novel. According to bioinformatics analysis, it was predicted that strain s12T had a complete alginate metabolism pathway. The analysis showed that alginate could be degraded to disaccharides or not by endo-and ex-alginate lyases in the extracellular. Saturated monosaccharides, which automatically form the ring-opening monomer 4-deoxy-L-erythrocytic-glucuronic acid, are absorbed into the cell, catalyzed by reductase to produce 2-keto-3-deoxygluconic acid, and then enter the Entner-Doudoroff pathway to produce glyceraldehyde triphosphate and pyruvic acid. Glyceraldehyde triphosphate is converted to pyruvic acid via glycolysis pathway. Then pyruvic acid passes through pyruvic acid. The dehydrogenase was converted into acetyl coenzyme A and then entered the tricarboxylic acid cycle or lactic acid fermentation to complete the transformation or complete degradation of alginate. 3. The characteristics of alginate production and extracellular proteome analysis of strain s12T were completed. It was found that the 11 alginate lyases were extracellular proteins and sucrose could induce the expression of alginate lyase. Strain s12T can grow in the basic medium with alginate as the sole carbon source. When EDTA, SDS and other metal ions such as MnSO4, CaCl2, CuSO4 and ZnSO4 are added, the total enzyme activity and total protein content are significantly inhibited; Na2CO3, NiSO4, KCl, nano-Si02, nano-Ti02 and nano-A1304 have no significant effect on the enzyme production. Both maltose and starch decreased the enzyme activity of the strain, and glucose had no significant effect on the enzyme production. In particular, sucrose could not be used by the strain and could not inhibit the production of alginate lyase. On the contrary, sucrose could significantly improve the fermentation activity of the enzyme, that is, sucrose could induce the production of alginate lyase. The optimum conditions for producing alginate lyase were as follows: incubation temperature 28 C, nitrogen source 0.5% NaNO3, initial pH 7.5-8.0, initial alginate concentration 2-2.5% and adding 0.2% sucrose. Under these conditions, strain s12T did not use sucrose in the medium, and reached the growth plateau after 28 h, and reached the highest total enzyme activity 44.5% after 36 h. 3 U/mL, 2.0% sodium alginate could be completely degraded into the product within 44 h without monosaccharide detection. Extracellular proteomic analysis showed that the nine alginate lyases Alg1-4 and Alg7-11 in the s12T genome were extracellular proteins. The presence of sucrose could significantly increase the secretion of Alg1 and Alg7, thereby increasing the secretion of alginate lyase s12T. Total enzymatic activity. Sucrose promotes the secretion and enzymatic activity of microbial alginate lyase. 4. Exogenous expression of four alginate lyase genes characterizes the enzymatic properties of the endo-cleavage enzymes rAlg2 and exo-cleavage enzymes rAlg5. It is found that the alginate lyase of the bacteria has a wide range of substrates. Specificity. According to previous prediction, alginate lyase genes algl, alg2, alg5 and alphalg6 with smaller fragments in strain s12T were exogenously expressed in E. coli, and the enzymatic properties of rAlg2 and rAlg5 with higher enzyme activity were characterized. The optimum reaction temperatures of rAlg2 and rAlg5 were 40-45 degrees C, and the temperature stability ranges were 4-40 degrees C and 4-20 degrees C, respectively. The optimum reaction pH values of rAlg2 and rAlg5 were 6.0-6.5 and 7.0-8.0, respectively, and the pH stability ranges were 6.0-7.0 and 7.0-8.0 respectively. The recombinant enzyme rAlg2 and rAlg5 showed no significant effect on the activity of rAlg5. SDS, EDTA, NH4Cl, FeCl3, FeSO4, MnCl2, CaCl2 and MgCl2 significantly inhibited the activity of rAlg2 and rAlg5. The results showed that both rAlg2 and rAlg5 had broad substrate specificity, and their substrate preferences were poly-L-guluronic acid fragment and poly-D-mannosuric acid, respectively. The specific enzyme activities of recombinant rAlg2 and rAlg 5 were 2350 and 1350 U/mg, 0.03 and 0.20 mM, 13.4 and 4.4 S-1 for Kcat, 45.4 and 220.5 S-1 mM-1 for Kcat and 120.5 S-1 mM-1 for alginate, respectively. Alg5 was the second exonuclease found in PL7 family and had unique characteristics. AlyA5 and AlyA5 from Zobellia galactanivorans DsiJT were found to have the highest homology and conserved key amino acids in the catalytic chamber. Functional characterization showed that both were broad substrate-specific exoenzymes, but their substrate preferences were different. AlyA5 was reported to prefer poly L-guluronic acid, while rAlg5 preferred poly-L-guluronic acid in this experiment. D-mannituronic acid. This also indicates that the substrate recognition of rAlg5 is not related to the conserved amino acids in the catalytic cavity, and may be related to the loop conformation around the catalytic cavity or its non-conserved amino acids. High physical coefficient Kcat/Km and sensitivity to organic solvents SDS and EDTA indicated that recombinant rAlg5 had the characteristics of cold-adapted enzymes. By characterizing the function of alginate lyase, it was found that at least two alginate lyases (Alg2 and Alg5) in strain s12T had broad substrate specificity, and Alg5 was a cold-adapted exogenous enzyme. Alginate can be effectively degraded to glucuronic acid monomers by adapting to low temperature and different substrate conditions.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q939.9
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本文编号：2211143
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