褐藻膠裂解酶分泌菌株的分離鑒定及Tamlana holothuriorum s12~T中褐藻膠裂解酶的研究
發(fā)布時間：2018-08-29 12:03
【摘要】：褐藻作為第三代可持續(xù)發(fā)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,不產(chǎn)生糧食矛盾,與以谷物為代表的第一代生物能源原料和以秸稈等為代表的第二代生物能源原料相比,具有廣闊的應(yīng)用前景。盡管已有研究表明利用褐藻進行生物轉(zhuǎn)化如發(fā)酵生產(chǎn)生物乙醇是切實可行的,但由褐藻生產(chǎn)生物乙醇的全部潛力還難以實現(xiàn),其主要問題是工業(yè)微生物菌株難以有效地降解褐藻膠中L-古羅糖醛酸片段、D-甘露糖醛酸片段及其嵌合片段生成可被工業(yè)菌株吸收并利用的單糖,且降解產(chǎn)生的糖醛酸單體化合物也難以被轉(zhuǎn)化利用。褐藻膠降解酶屬于多糖裂解酶,多為聚D-甘露糖醛酸底物特異性的內(nèi)切型褐藻膠裂解酶,降解產(chǎn)物為寡糖；而能夠同時降解L-古羅糖醛酸片段、D-甘露糖醛酸片段及其嵌合片段的多功能酶較少,特別是缺乏廣泛底物特異性的外切酶。因此,尋求新型高效且具有廣泛底物特異性的褐藻膠裂解酶及酶系是褐藻膠生物轉(zhuǎn)化生產(chǎn)所面臨的重要任務(wù)。本項研究旨在篩選新型高效褐藻膠裂解酶分泌菌株,研究其產(chǎn)酶特性,并外源表征新型或高效褐藻膠裂解酶的基因,初步闡述高效褐藻膠降解菌的降解機制,為深入研究褐藻生物質(zhì)降解轉(zhuǎn)化機制積累基礎(chǔ)數(shù)據(jù),為褐藻膠的生物轉(zhuǎn)化提供優(yōu)良的菌種資源、酶制劑奠定生物學(xué)基礎(chǔ)。在本項研究中取得的主要成果如下：一、對近海和陸地環(huán)境樣品進行了褐藻膠裂解酶分泌菌株的廣泛篩選,獲得了多個新物種和多株高效褐藻膠降解菌。從十余種環(huán)境樣品中分離獲得182株褐藻膠裂解酶分泌菌株,這些菌株分布在52個屬。發(fā)現(xiàn)其中22個屬的細菌(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.)尚未見報道具有分泌褐藻膠裂解酶的功能。以16S rRNA目似度閾值98.7%為判斷細菌新物種的標準,發(fā)現(xiàn)在所篩選的褐藻膠降解菌株中有13株為潛在新物種。4株細菌的16S rRNA最高相似度低于97%,新穎性好,其中3株海洋細菌屬于擬桿菌門(菌株Dm15T和Gy8T屬于擬桿菌門黃桿菌科,菌株Sy30T屬于擬桿菌門嗜纖維菌科)。此外,篩選到的一株酶活高且傳代穩(wěn)定的褐藻膠降解菌為來自海參腸道的菌株s12T,與Tamlana agarivorans JW-26T最高相似度為99.1%,也屬于擬桿菌門黃桿菌科。經(jīng)多相分類學(xué)研究,對分離到的擬桿菌門4個新菌進行了鑒定。高效褐藻膠降解菌株s12T為黃桿菌科Tamlana屬的新種,命名為Tamlana holothuriorum sp. nov.;菌株Dm15T為黃桿菌科的新屬新種,命名為Flavohalobacter algicola gen. nov. sp. nov.;菌株Gy8T為黃桿菌科Seonamhaeicola屬的新種,命名為Seonamhaeicola algicola sp. nov.;菌株Sy30T為擬桿菌門Hymenobacter科Pontibacter屬的新種,命名為Pontibacter locisalis sp. nov.。二、完成了褐藻膠高效降解菌株s12T的基因組測序及分析,發(fā)現(xiàn)該菌具有新穎的褐藻膠降解酶和較完整的褐藻膠代謝途徑。對菌株s12T進行了基因組測序及序列分析。菌株s12T基因組全長3.66 Mbp,共有3151個編碼蛋白基因,其中1,729個蛋白可以匹配到同源蛋白聚簇數(shù)據(jù)庫,其它大量功能未知的基因序列預(yù)示著該菌的新功能和新基因存在的可能。通過對基因組數(shù)據(jù)的生物信息分析,預(yù)測到該菌共有11條編碼褐藻膠裂解酶基因(alg1-11),與數(shù)據(jù)庫中已表征的褐藻膠裂解酶序列的相似度均低于73%,其中包括2個外切型褐藻膠裂解酶基因alg5和αlg9。對所預(yù)測的這些酶的氨基酸序列進行同源性分析,發(fā)現(xiàn)Alg9屬于多糖解酶(Polysaccharide Lyase,PL) 17家族,其它10個褐藻膠裂解酶則均屬于PL7家族,其中Alg1屬于PL7第3亞家族,Alg2和Alg5屬于PL7第5亞家族,Alg4、Alg6-8、Alg10、Alg11所屬亞家族待定。Alg2、Alg5、Alg6和Alg9與其同源性高的褐藻膠裂解酶均具有保守的催化氨基酸殘基,而Alg1、Alg3、Alg4、Alg7、Alg8、Alg10和Alg11對于催化位點關(guān)鍵氨基酸保守性差,說明后者可能具有新的催化特性。Alg1-4、Alg7、Alg9和Algl 1除了催化結(jié)構(gòu)域外,還包含碳水化合物結(jié)合域、C端凝血因子結(jié)構(gòu)域、凝集素結(jié)構(gòu)域、信號肽、分泌系統(tǒng)、細菌免疫球蛋白樣和類肝素酶結(jié)構(gòu)域,根據(jù)結(jié)構(gòu)域功能預(yù)測至少上述具有多個結(jié)構(gòu)域的7個褐藻膠裂解酶為分泌性蛋白。總之,生物信息學(xué)分析顯示菌株s12T中的褐藻膠裂解酶(酶系)具有多樣性和新穎性。根據(jù)生物信息分析,同時預(yù)測出菌株s12T具有較完整的褐藻膠代謝途徑。分析顯示褐藻膠在胞外可被內(nèi)切型和外切型褐藻膠裂解酶降解為雙糖或不飽和單糖,自動形成開環(huán)單體4-脫氧-L-赤蘚-糖醛酸后被吸收進入胞內(nèi),由還原酶催化生成2-酮-3-脫氧葡糖酸,進入Entner-Doudoroff途徑生成三磷酸甘油醛和丙酮酸,三磷酸甘油醛通過糖酵解代謝途徑轉(zhuǎn)化為丙酮酸。然后,丙酮酸通過丙酮酸脫氫酶轉(zhuǎn)化為乙酰輔酶A,進入三羧酸循環(huán)或乳酸發(fā)酵,完成褐藻膠的轉(zhuǎn)化或徹底降解。三、完成了菌株s12T"的褐藻膠產(chǎn)酶特性和胞外蛋白組的測定分析,發(fā)現(xiàn)注釋的11個褐藻膠裂解酶均為胞外蛋白,且蔗糖對褐藻膠裂解酶的表達具有誘導(dǎo)作用。菌株s12T可以在褐藻膠為唯一碳源的基礎(chǔ)培養(yǎng)基中生長,添加EDTA、SDS和其它金屬離子如MnSO4、CaCl2、CuSO4和ZnSO4后均顯著抑制總酶活和總蛋白量；Na2CO3、NiSO4、KCl、納米Si02、納米Ti02和納米A1304對產(chǎn)酶效應(yīng)影響不顯著。添加外源糖類時,果糖、麥芽糖和淀粉均降低該菌的產(chǎn)酶活性；葡萄糖的存在對產(chǎn)酶影響不顯著,而較為特殊的是,蔗糖不能被該菌利用,不阻遏褐藻膠裂解酶的產(chǎn)生,反而能夠顯著提高酶的發(fā)酵活性,即蔗糖對該菌褐藻膠裂解酶的產(chǎn)生具有誘導(dǎo)作用。經(jīng)優(yōu)化得到該菌褐藻膠裂解酶的最適產(chǎn)酶條件為：培養(yǎng)溫度28℃,氮源為0.5%NaNO3,初始pH值為7.5-8.0,褐藻膠初始濃度為2-2.5%,添加0.2%蔗糖。在該培養(yǎng)條件下,菌株s12T不利用培養(yǎng)基中的蔗糖,培養(yǎng)28 h后達到生長平臺期,培養(yǎng)36 h后達到最高總酶活44.3 U/mL,44 h內(nèi)可將2.0%海藻酸鈉完全降解至產(chǎn)物中檢測不出單糖。通過胞外蛋白組分析,發(fā)現(xiàn)菌株s12T基因組中所注釋的9個褐藻膠裂解酶Alg1-4和Alg7-11為胞外蛋白,蔗糖的存在可以顯著增加Alg1和Alg7的分泌量,從而提高菌株s12T褐藻膠裂解酶的總酶活。蔗糖促進微生物褐藻膠裂解酶的分泌和提高酶活的作用尚未見相關(guān)報道。四、外源表達了4個褐藻膠裂解酶基因,表征了酶活較高的內(nèi)切型裂解酶rAlg2和外切型裂解酶rAlg5的酶學(xué)性質(zhì),發(fā)現(xiàn)該菌的褐藻膠裂解酶具有廣泛的底物特異性。根據(jù)前期預(yù)測,將菌株s12T中片段較小的褐藻膠裂解酶基因algl、alg2、alg5和αlg6在大腸桿菌中進行外源表達,并對酶活較高的rAlg2和rAlg5進行了酶學(xué)性質(zhì)的表征。研究發(fā)現(xiàn)重組酶rAlg2降解產(chǎn)物為2、3和4糖,故重組酶rAlg2為內(nèi)切酶；重組酶rAlg5的降解產(chǎn)物中有單糖,故重組酶,rAlg5為外切酶。重組酶rAlg2和rAlg5的最適反應(yīng)溫度均為40-45℃,溫度穩(wěn)定范圍分別為4-40℃和4-20℃；兩者的最適反應(yīng)pH值分別為6.0-6.5和7.0-8.0,pH穩(wěn)定范圍為6.0-7.0和7.0-8.0。研究發(fā)現(xiàn)KC1和NaCl能顯著提高重組酶rAlg2勺酶活,而對重組酶rAlg5酶活影響不顯著；SDS、EDTA、NH4Cl、FeCl3、FeSO4、MnCl2、CaCl2和MgCl2顯著抑制重組酶rAlg2和rAlg5的酶活。結(jié)果顯示重組酶rAlg2和rAlg5都具有廣泛的底物特異性,底物偏好性分別為聚L-古羅糖醛酸片段和聚D-甘露糖醛酸片段。在以海藻酸鈉為底物時,重組酶rAlg2和rAlg5的比酶活分別為2350和1350U/mg,Km分別為0.03和0.20 mM, Kcat分別為13.4和4.4 S-1,Kcat/Km分別為45.4和220.5 S-1mM-1。褐藻膠裂解酶Alg5是在PL7家族發(fā)現(xiàn)的第二個外切酶,有獨特的特點。經(jīng)氨基酸序列分析,發(fā)現(xiàn)Alg5和來自菌株Zobellia galactanivorans DsiJT的AlyA5同源性最高,在催化腔內(nèi)具有保守的關(guān)鍵氨基酸。功能表征發(fā)現(xiàn)兩者均為廣泛底物特異性的外切酶,但兩者底物偏好性不同,據(jù)報道AlyA5偏好聚L-古羅糖醛酸,而本實驗中重組酶rAlg5偏好聚D-甘露糖醛酸。這也說明重組酶rAlg5的底物識別與催化腔內(nèi)保守氨基酸關(guān)系不大,可能與圍繞在催化腔周圍的loop構(gòu)象或其非保守的氨基酸有關(guān)。此外,本研究中發(fā)現(xiàn)重組酶rAlg5在低溫4℃下其酶活可達到最高酶活時的51.2%,熱穩(wěn)定性差,生理系數(shù)Kcat/Km高,對有機溶劑SDS和EDTA敏感,說明重組酶rAlg5具有適冷酶的特性。通過褐藻膠裂解酶的功能表征,可以發(fā)現(xiàn)菌株s12T中至少有2個褐藻膠裂解酶(Alg2和Alg5)具有廣泛底物特異性,其中Alg5為適冷外切酶。這些結(jié)果說明菌株s12T可以適應(yīng)低溫以及不同底物條件而有效地降解褐藻膠,能在胞外將褐藻膠降解成糖醛酸單體化合物。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q939.9
，
本文編號：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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q939.9
，
本文編號：2211143
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