| As one of sustainable third-generation biomass for bio-ethanol production, brown algae has many advantages over first- and second-generation biomass, such as grain and straw. Brown algae has the features of fast breeding, low energy consumption, no occupation of plantation, no consumption of fresh water, no grain contradiction. It is expected that brown algae has broad application prospects. Alginate is the most abundant polysaccharides of brown algae, which contains poly-L-guluronic acid, poly-D-mannuronic acid and their chimeric blocks. It was shown that bio-ethanol production from brown algae is feasible, but it is hard to achieve the full potential of bio-ethanol production from brown algae because of the difficulty of effectively simultaneous degradation of poly-L-guluronic acid, poly-D-mannuronic acid and their chimeric blocks. The products, monosaccharide acids, should be obtained in order to further fermentation in engineering strains. Alginate-degrading enzymes reported before are lyases, most of which are endolytic alginate lyasses perfering poly-D-mannuronic acid, and their degradation products are oligosaccharides. In other words, the multifunctional enzymes are rare, which can simultaneously degrade L-guluronic acid, D-mannuronic acid and their chimeric blocks. Especially, there is a lack of excision enzymes with broad substrate specificity. Therefore, seeking new efficient alginate lyases with broad substrate specificity is an important mission of alginate bioconversion.This study aimed to screen novel bacteria excreting alginate lyase efficiently, to study the traits of enzyme producing, to characterize new or efficient alginate lyases, and to explain efficient degradation mechanism of alginate-degrading bacteria, which can accumulate essential data to elucidate the bioconversion mechanism of brown algae, and provide good bacteria resources to biotransformation of alginate and establish biological foundation for enzymic preparations. The main results achieved in this study are as follows:1. Isolation and identification of several new species and marine bacteria with high alginate lyase activities by the extensively screening of alginate lyase-excreting strains from coastal and terrestrial environmental samples.A total of 182 isolates excreting alginate lyase were obtained from more than ten kinds of environmental samples, and these isolates belonged to 52 genera, of which bacteria belonged to 22 genera (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. and Flavohalobacter sp.) have not been reported to have the function of excreting alginate lyase.Considering the threshold of 16S rRNA similarity for new species is 98.7%,13 alginate-degrading strains were found to be potential new species. Among these strains,4 strains with the highest similarity of 16S rRNA below 97% were more novel, and 3 stains of these bacteria were members of the phylum Bacteroidetes (Strains Dm15T and Gy8T belong to the family Flavobacteriaceae; Strain Sy30T belonged to cytophaga). In addition, an high-effectively degrading strain, named s12T, remained stable and the most highly efficient degradition through generation, which was from gut of trepang and had the highest 16S rRNA similarity (99.1%) to that of Tamlana agarivorans JW-26T. Strain s12T belonged to the family Flavobacteriaceae, the phylum Bacteroidetes as well.Further identification of 4 new isolates were carried out by polyphasic taxonomy. High-efficiency strain s12Twas considered to represent a novel species of the genus Tamlana within the family Flavobacteriaceae, for which the name Tamlana holothuriorum sp. nov. is proposed.strain Dm15Twas considered to represent a novel species of a new genus within the family Flavobacteriaceae, for which the name Flavohalobacter algicola gen. nov. sp. nov. is proposed. Strain Gy8T was considered to represent a novel species of the genus Seonamhaeicola within the family Flavobacteriaceae, for which the name Seonamhaeicola algicola sp. nov. is proposed. Strain Sy30T was considered to represent a novel species of the genus Pontibacter within the family Hymenobacter, for which the name Pontibacter locisalis sp. nov. is proposed.2. Analysis of the genomic sequence and alginate metabolic pathway of strain s12T.Genome sequencing and sequence analysis of strain s12T were carried out. The assembled genome sequence comprised 3.66 Mbp containing 3151 coding protein genes, and 1,729 proteins can be assigned to Clusters of Orthologous Groups families. As a result, the functions of other vast gene sequences were unknown. These indicate the existence of new functions and new genes in the bacterium. This isolate had 11 alginate lyases annotated (alg1-11), which exhibited similarities below 73% to alginate lyases characterized. By homology analysis for amino acid sequence of alginate lyases annotated, it was found that Alg9 belonged to 17th family of polysaccharide Lyase (PL), and that others belonged to PL7 family, in which Alg1 belonged to 3rd subfamily, Alg2 and Alg5 belonged to 5th subfamily, and Alg4, Alg6-8, Alg10, Algl 1 belonged to uncertain subfamilies. Furthermore, Alg2, Alg5, Alg6 and Alg9 and their homologous alginate lyases had conservative catalytic amino acid residue, while Algl, Alg3, Alg4, Alg7, Alg8, AlglO and Algl 1 had rare catalytic amino acids compared with sequences of alginate lyases reported, which showed that the latter may have new catalytic characteristics. Besides catalytic structural domain, there were also carbohydrate binding module, the fucose-binding lectins, Coagulation factor 5/8 C-terminal domain, signal peptide, secretory system, bacterial Ig-like domain and Heparinase II/III like domain in Algl-4, Alg7, Alg9 and Algll. According to function prediction of structural domain,7 alginate lyases above were secretory proteins. In short, the result of bioinformatics analysis showed that the alginate lyases of strain s12T are diverse and novel.Based on the genomic sequence, the metabolic pathways of alginate were predicted. Alginate can be degraded to unsaturated monosaccharides by exo-and endo-lytic alginate lyases in extracellular, and then automatically converted to the open-loop monomer,4-deoxy-L-erythro-uronic acid, which can be absorbed in the cell and reduced to 2-keto-3-deoxy glucose acid by reductase. The product can be converted to glyceraldehyde-3-phosphate and pyruvate in the Entner doudoroff pathway, and they can transform each other in Embden-Meyerhof-Parnas pathway. At last, pyruvate can product acetyl CoA by pyruvate dehydrogenase, which enter the tricarboxylic acid cycle or lactic acid fermentation to complete degradation of alginate.3. Based on optimization of enzyme production and analysis of extracellular proteins from strain s12T,11 alginate lyase annotated were found to be extracellular proteins, and expression of 2 alginate lyases were differentially induced by sucrose.Strain s12T grew well in basal salt medium with alginate as the sole carbon source. EDTA, SDS and other metal ions, such as MnSO4, CaCl2, GuSO4, ZnSO4 significantly inhibited the total enzyme activity and protein content. In addtion, Na2CO3, NiSO4, KCl, nano-SiO2, nano-TiO2 and nano-Al3O4 had no significant influence on enzyme activity. Adding exogenous sugars, Fructose, maltose or starch significantly inhibited the total enzyme activity and protein content, and glucose did not have a significant effect on enzyme production. However, sucrose improved the enzyme activity, but it was not absorbed as nutrient. Namely, the expression of alginate lyases may be induced by sucrose. By optimizing the condition of enzyme production, the optimal temperature was 28 ℃; Initial pH was 7.5-8.0; The nitrogen source was 0.5% NaNO3; Initial concentration of alginate was 2-2.5%, adding 0.2% sucrose. Under the optimal conditions, strain s12T reached growth platform phase after 28 h, exhibited the highest enzyme activity of 44.3 U/ml after 36 h, and completely degraded the 2.0% sodium alginate within 44 h so that monosaccharides were not detected.The extracellular proteome showed that 11 alginate lyase annotated in the genome of strain s12T were extracellular proteins. Furthermore, sucrose could significantly increase secretion of Algl and Alg7, so as to improve the total enzyme activity of strain s12T. The influence of sucrose on secretion of microbial alginate lyases has not been reported.4. Heterologous expression of 4 alginate lyases and characterization of endolytic rAlg2 and exolytic rAlg5 with high enzyme activity were completed. And alginate lyases of strain s12T were found to be with broad substrate specificity.According to the early prediction, alginate lyase genes in small size, such as algl, alg2, alg5 and alg6 of strain s12T, were cloned and expressed in Escherichia coli. And then the enzymatic properties of rAlg2 and rAlg5 with high enzyme activity were characterized. The optimum reaction temperature of rAlg2 and rAlg5 were from 40 to 45 ℃; The optimum pH were 6.0-6.5 and 7.0-8.0; Stable range of rAlg2 and rAlg5 were 4-40℃,4-20 ℃, pH 6.0-7.0, and pH 7.0-8.0. KCl and NaCl can significantly improve the activity of rAlg2 but not rAlg5; SDS, EDTA, NH4C1, FeCl3, FeSO4, MnCl2, CaCl2 and MgCl2 can significantly inhibit their activity; rAlg2 and rAlg5 were with broad substrate specificity, but preferred poly-L-guluronic acid and poly-D-mannuronic acid, respectively. Using sodium alginate as substrate, rAlg2 and rAlg5 had enzyme activity of 2350 and 1350 U/mg, Km values of 0.03 and 0.20 mM, Kcat values of 13.4 and 4.4 S-1, and Kcat/Km of 45.4 and 220.5 S-1mM-1, respectively.Alg5 is the second exolytic alginate lyase with special characteristics in the PL7 family. On the one hand, the results of sequence analysis showed that Alg5 had 73% highest identity and conservative catalytic amino acids to AlyA5, a PL7 characterized alginate lyase, from Zobellia galactanivorans DsiJT. Based on functional characterization, both were exotype enzymes with broad substrate specificity, but their substrate preference is different. AlyA5 was reported to prefer poly-L-guluronic acid, and rAlg5 preferred poly-D-mannuronic acid. Hence, the conserved amino acids in catalytic cavity have little relations to substrate recognition of rAlg5. On the contrary, the loop around the catalytic cavity or the non conservative amino acids may play a role in substrate recognition. On the other hand, It was found that the enzyme activity of rAlg5 at a low temperature of 4℃ reached 51.2% of the highest enzyme activity, and that rAlg5 has poor thermal stability, high physiological coefficient kcat/km, the sensitivity to SDS and EDTA. These results indicated that rAlg5 has some characteristics of cold adapted enzyme.The characterization of alginate lyases showed that strain s12T containes at least eight alginate lyases (Alg2 and Alg5) with broad substrate specificity, of which Alg5 is a cold adapted enzyme with exolytic enzyme activity. These results suggestted that strain s12T could adapt to low temperature and different substrate conditions so as to effectively degrade alginate into monomer compounds in extracellular. |
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