一种海洋琼胶酶的分离纯化、酶学性质研究及降解产物分析

摘要：【目的】对海洋Agarivorans albus QM38菌株所产琼胶酶的纯化工艺和酶学性质进行了研究。【方法】发酵液通过离心、(NH4 ) 2SO4盐析、DEAE-Sepharose Fast Flow 阴离子交换层析、Sephacry S-100 凝胶过滤等纯化步骤得到SDS-PAGE电泳级纯酶,并用质谱对酶的降解产物进行分析。【结果】得到琼胶酶A,纯化倍数为17.6倍,收率为15.21 %,SDS-PAGE测定其分子量为127.8 kDa。对琼胶酶A进行了进一步的性质分析,其最适反应温度为35 ℃,最适反应pH为7.6,最适底物浓度为0.9 %,多数金属离子为其活性抑制剂。琼胶酶A的降解产物经质谱分析主要为四糖和六糖。【结论】从菌株QM38的发酵液中纯化得到的琼胶酶A具有降解凝胶态琼胶的能力,其分子量与以往报道过的琼胶酶不同。     

1株产琼脂酶细菌的分离鉴定及其胞外酶活性测定

从四川省成都市青城山采集土壤,以琼脂作为唯一碳源,筛选到产琼脂酶细菌CMCK136;通过形态观察、生化鉴定、16S r DNA测序及序列分析鉴定其种属;随后测定了菌株CMCK136的胞外酶活性。菌株CMCK136被鉴定为芽胞杆菌属细菌,命名为Bacillus sp.CMCK136。菌株CMCK136的胞外琼脂酶的最适酸碱度为p H 7.0,最适温度为35℃。菌株CMCK136是产琼脂酶细菌家族的新成员,该菌株的发现进一步提示芽胞杆菌属很可能蕴含有尚待开发的琼脂酶资源。     

Optimization of Culture Conditions for the Production of Extracellular Agarases from Newly Isolated Pseudomonas Aeruginosa AG LSL-11

Summary An agar-degrading bacterium capable of utilizing agar as sole source of carbon and energy was isolated from sea water by enrichment culture technique. The bacterium was identified as Pseudomonas aeruginosa and the culture conditions were standardized for the maximal production of extracellular agarases. The bacterium grew in the pH range 5.0–11.0, optimal between pH 7.0 and 8.0; temperature between 25 °C and 37 °C, optimal at 30 °C and sodium chloride concentration 0–8% and optimal at 2% respectively. The agarases secreted by Pseudomonas aeruginosa AG LSL-11 were inducible by agar and not by any other simple sugars tested. Maximal agarase production was observed at pH 8.0, and temperature 30 °C. The bacterium had no requirement for NaCl for both growth and production of agarases. The bacterium did not utilize other polysaccharides like ĸ-carrageenan, alginate, cellulose and CMC. The activity staining of partially purified agarase preparation after native-PAGE revealed the presence of three different agarases, agarase LSL-11a, LSL-11b and LSL-11c, whose molecular weights were estimated to be 76, 64 and 46 kDa respectively.     

Isolation and characterization of a novel agarase-producing Pseudoalteromonas spp. bacterium from the guts of spiny turban shells

An agar-degrading bacterium was isolated from the guts of spiny turban shells. It was identified as a Pseudoalteromonas species and named Pseudoalteromonas sp. JYBCL 1. The viscosity of the inoculated agar medium decreased by more than 60% after 20 h cultivation. The agarase produced by the isolate had optimal activities at 35 degrees C and pH 7. The enzyme had extremely strong resistance to ionic stress compared with other known agarases. Its molecular mass was estimated at about 60 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The agarase could saccharify Gelidium amansii directly, with an efficiency about half that compared with agar saccharification.     

Purification and characterization of agarases from a marine bacterium <Emphasis Type="Italic">Vibrio</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> F-6

Marine bacterium Vibrio sp. F-6, utilizing agarose as a carbon source to produce agarases, was isolated from seawater samples taken from Qingdao, China. Two agarases (AG-a and AG-b) were purified to a homogeneity from the cultural supernatant of Vibrio sp. F-6 through ammonium sulfate precipitation, Q-Sepharose FF chromatography, and Sephacryl S-100 gel filtration. Molecular weights of agarases were estimated to be 54.0 kDa (AG-a) and 34.5 kDa (AG-b) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH values for AG-a and AG-b were about 7.0 and 9.0, respectively. AG-a was stable in the pH range of 4.0-9.0 and AG-b was stable in the pH range of 4.0-10.0. The optimum temperatures of AG-a and AG-b were 40 and 55 degrees C, respectively. AG-a was stable at temperature below 50 degrees C. AG-b was stable at temperature below 60 degrees C. Zn(2+), Mg(2+) or Ca(2+) increased AG-a activity, while Mn(2+), Cu(2+) or Ca(2+) increased AG-b activity. However, Ag(+), Hg(2+), Fe(3+), EDTA and SDS inhibited AG-a and AG-b activities. The main hydrolysates of agarose by AG-a were neoagarotetraose and neoagarohexaose. The main hydrolysates of agarose by AG-b were neoagarooctaose and neoagarohexaose. When the mixture of AG-a and AG-b were used, agarose was mainly degraded into neoagarobiose.     

Purification and Properties of an Extracellular Agarase from Alteromonas sp. Strain C-1

A marine bacterial strain isolated from the Bay of San Vicente, Chile, was identified as Alteromonas sp. strain C-1. In the presence of agar, this strain produced high levels of an extracellular agarase. The production of agarase was repressed by glucose, with a parallel decrease in bacterial growth. The enzyme was purified to homogeneity by anion-exchange chromatography and gel filtration, with an overall yield of 45%. The enzyme has a molecular weight of 52,000, is salt sensitive, and hydrolyzes agar, yielding neoagarotetraose as the main product, with an optimum pH of about 6.5.     

Characterization of a novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39

Aims:  The aims of this study were to identify and characterize the novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39.
Methods and Results:  Strain B39 was closely related to Paenibacillus cookii in 16S rRNA gene sequence. Nonetheless, this isolate can be identified as a novel Paenibacillus sp. with respect to its physiological characteristics, biochemical reactions, and profiles of fatty acid compositions. A cellulase with both CMCase and avicelase activities was secreted from strain B39 and purified by ion-exchange chromatography. By sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis, the molecular weight of B39 cellulase was determined as 148 kDa, which was much higher than other cellulases currently reported from Paenibacillus species. The enzyme showed a maximum CMCase activity at 60°C and pH 6·5. Addition of 1 mmol l⁻¹ of Ca²⁺ markedly enhanced both CMCase and avicelase activities of the enzyme.
Conclusions:  We have identified and characterized a novel thermophilic Paenibacillus sp. strain B39 which produced a high-molecular weight cellulase with both CMCase and avicelase activities.
Significance and Impact of the Study:  Based on the ability to hydrolyse CMC and avicel, the cellulase produced by Paenibacillus sp. strain B39 would have potential applications in cellulose biodegradation.     

Cloning and expression of an agarase gene from a marine bacterium Pseudomonas sp. w7

Two different agarase genes (pSW1, pSW3) were cloned from a marine bacterium Pseudomonas sp. W7 into E. coli JM83 using the multicopy plasmid vector pUC19. Two cloned strains of recombinant E. coli which showed the agarase activity were obtained and were named E. coli JM83/pSW1 and E. coli JM83/pSW3. These strains had the insert fragment of 3.7kb and 3.0kb, respectively. The N-terminal amino acid sequence of the agarase containing the recombinant plasmid pSW3 was determined and the sequence did not show homology to any other known agarases. The optimum pH and temperature of the agarases from the cloned strains, E. coli JM83/pSW1 and pSW3, were 6.0, 7.0 and 30°C, 40°C, respectively.     

Production and structural elucidation of trehalose tetraesters (biosurfactants) from a novel alkanothrophic Rhodococcus wratislaviensis strain

Aims:  To isolate a biosurfactant-producing bacterial strain and to identify and characterize the chemical structure and properties of its biosurfactants.
Methods and Results:  The bacterium Rhodococcus wratislaviensis BN38, isolated from soil, was found to produce glycolipid biosurfactants when grown on 2% n -hexadecane. The glycolipids were isolated by chromatography on silica gel columns and their structures elucidated using a combination of multidimensional NMR and ESI-MS/MS techniques. The main product was identified as 2,3,4,2'-trehalose tetraester with molecular mass of 876 g mol⁻¹. It was also noted that the biosurfactant was produced under nitrogen-limiting conditions and could not be synthesized from water-soluble substrates. The purified product showed extremely high surface-active properties.
Conclusions:  The glycolipid biosurfactant produced by the alkanothrophic strain R. wratislaviensis BN38 was characterized to be 2,3,4,2'-trehalose tetraester which exhibited high surfactant activities.
Significance and Impact of the Study:  Strain BN38 of R. wratislaviensis is a potential candidate for use in bioremediation applications or in biosurfactant exploration.     

Gene cloning,expression and characterization of a neoagarotetraose-producing β-agarase from the marine bacterium <Emphasis Type="Italic">Agarivorans</Emphasis> sp. HZ105

A β-agarase gene hz2 with 2,868 bp was cloned from the marine agarolytic bacterium Agarivorans sp. HZ105. It encoded a mature agarase HZ2 of 102,393 Da (920 amino acids). Based on the amino acid sequence similarity, agarase HZ2 was assigned to the glycoside hydrolase family 50. The β-agarase shared a gene sequence identity of 98.6% with the reported but much less characterized β-agarase agaB from Vibrio sp. JT0107. Its recombinant agarase rHZ2 was produced in E. coli cells and purified to homogeneity. The agarase rHZ2 degraded agarose and neoagarooligosaccharides with degrees of polymerization above four, to yield neoagarotetraose as the dominant product, which was different from β-agarase agaB of Vibrio sp. JT0107. The agarose hydrolysis pattern suggested that rHZ2 was an endo-type β-agarase. Beta-mercaptoethanol (90 mM) and dithiothreitol (9 mM) increased the agarase activity of rHZ2 by 72.9% and 17.3% respectively, while SDS (9 mM) inhibited the activity completely. The agarase activity was independent of Na⁺, K⁺, Mg²⁺ and Ca²⁺. The maximal enzyme activity was observed at 40°C and pH 7. The kinetic parameters K _m, V _max, K _cat, and K _cat/K _m values toward agarose of agarase rHZ2 were 5.9 mg ml⁻¹, 235 U mg⁻¹, 401 s⁻¹ and 6.8 × 10⁵ M⁻¹ s⁻¹, respectively. Agarase rHZ2 could have a potential application in the production of bioactive neoagarotetraose.     

Characterization of a novel β-agarase from an agar-degrading bacterium Catenovulum sp. X3

An agar-degrading bacterium, Catenovulum sp. X3, was isolated from the seawater of Shantou, China. A novel β-agarase gene agaXa was cloned from the strain Catenovulum sp. X3. The gene agaXa consists of 1,590 bp and encodes a protein of 529 amino acids, with only 40 % amino acid sequence identity with known agarases. AgaXa should belong to the glycoside hydrolase family GH118 based on the amino acid sequence similarity. The molecular mass of the recombinant AgaXa (rAgaXa) was estimated to be 52 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. It had a maximal agarase activity at 52 °C and pH 7.4 and was stable over pH 5.0?~?9.0 and at temperatures below 42 °C. The K _m and V _max for agarose were 10.5 mg/ml and 588.2 U/mg, respectively. The purified rAgaXa showed endolytic activity on agarose degradation, yielding neoagarohexaose, neoagarooctaose, neoagarodecaose, and neoagarododecaose as the end products. The results showed that AgaXa has potential applications in agar degradation for the production of oligosaccharides with various bioactivities.     

Isolation of a novel freshwater agarolytic Cellvibrio sp. KY-YJ-3 and characterization of its extracellular beta-agarase

A novel agarolytic bacterium KY-YJ-3, producing extracellular agarase, was isolated from the freshwater sediment of the Sincheon River in Daegu, Korea. On the basis of gram-staining data, morphology, and phylogenetic analysis of the 16S rDNA sequence, the isolate was identified as Cellvibrio sp. By ammonium sulfate precipitation followed by Toyopearl QAE-550C, Toyopearl HW-55F, and Mono-Q column chromatography, the extracellular agarase in the culture fluid could be purified 120.2-fold with yield of 8.1%. The specific activity of the purified agarase was 84.2 U/mg. The molecular mass of the purified agarase was 70 kDa as determined by dodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal temperature and pH of the purified agarase were 35 degrees C and pH 7.0, respectively. The purified agarase failed to hydrolyze the other polysaccharide substrates, including carboxymethyl (CM)-cellulose, dextran, soluble starch, pectin, and polygalacturonic acid. Kinetic analysis of the agarose-hydrolysis catalyzed by the purified agarase using thin layer chromatography (TLC) exhibited that the main products were neoagarobiose, neoagarotetraose, and neoagarohexaose. These results demonstrated that the newly isolated freshwater agarolytic bacterium KY-YJ-3 was a Cellvibrio sp., and could produce an extracellular beta-agarase, which hydrolyzed agarose to yield neoagarobiose, neoagarotetraose, and neoagarohexaose as the main products.     

Phenotypic variation in Streptomyces sp. DSM 40537, a lipoteichoic acid producing actinomycete

Aims:  To reinvestigate the production of lipoteichoic acid (LTA) by the actinomycete strain Streptomyces sp. DSM 40537 (=ATCC 3351).
Methods and Results:  LTA was extracted and purified from strain Streptomyces sp. DSM 40537. The identification of the LTA was confirmed by Western blotting with a monoclonal antibody. During these studies, two stable phenotypic variants of DSM 40537 were obtained, one of which released a distinctive orange pigment. 16S rRNA gene sequencing of each variant yielded identical sequences and allowed phylogenetic analysis to be performed.
Conclusions:  Streptomyces sp. DSM 40537 was shown to exhibit stable morphological variation. The strain was confirmed to be a LTA-producing actinomycete and to belong to the Streptomyces albidoflavus cluster within the genus Streptomyces .
Significance and Impact of the Study:  These data provide important support for the hypothesis that the distribution of LTA is linked to that of wall teichoic acids and emphasizes the need to reinvestigate LTA distribution in actinomycetes.     

Purification and characterization of β-agarase from marine bacterium Bacillus cereus ASK202

Extracellular agarase of Bacillus cereus ASK202 was purified 32-fold, giving a single band on PAGE with activity staining. The M_r of purified agarase was determined as 90 kDa by SDS-PAGE. The N-terminal amino acid was sequenced and the sequence did not show homology to any other known agarases. The optimum pH and temperature were 7.0 and 40 °C, respectively. This enzyme was found to be a -agarase which catalyzed the hydrolysis of the -1,4 linkage of agarose to yield neoagarohexaose, neoagarotetraose and neoagarobiose.     

Optimization of 2D-PAGE protocols for proteomic analysis of two nonaxenic toxin-producing freshwater cyanobacteria: Cylindrospermopsis raciborskii and Raphidiopsis sp.

Aims:  To optimize a protocol for the extraction and an in-depth analysis of the soluble protein fraction of two nonaxenic toxin-producing cyanobacteria Cylindrospermopsis raciborskii (hepatotoxin-producing), and Raphidiopsis sp. (neurotoxin-producing), using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).
Methods and Results:  The soluble protein fractions from strains of C. raciborskii and Raphidiosis sp. with different toxicity phenotypes were analysed by 2D-PAGE. Specific protocols were optimized specifically for each strain. Between 500 and 700 sharp protein spots were distinguished in a single 4–7 pH range 2D-PAGE for each cyanobacterium. Comparison of the protein maps of C. raciborskii CS-505 (a cylindrospermopsin-producing strain) and Raphidiopsis sp. D9 (saxitoxin-producing strain) against the nontoxic C. raciborskii strain CS-509 revealed many unique proteins in each protein map. We confirmed that the resolved proteins were cyanobacterial by identifying three randomly chosen protein spots from a Raphidiopsis sp. strain D9 2D-PAGE, using high-performance liquid chromatography (HPLC) tandem mass spectrometry (MS).
Conclusions:  The 2D-PAGE conditions presented here provide a robust protocol for proteomic studies in two CYN- and STX-producing model organisms, C. raciborskii and Raphidiopsis sp.
Significance and Impact of the Study:  We present the first protocols for proteomic analyses of Cylindrospermopsis raciborskii and Raphidiopsis sp.     

Extracellular β-agarase LSL-1 producing neoagarobiose from a newly isolated agar-liquefying soil bacterium, Acinetobacter sp., AG LSL-1

Summary An agar-liquefying Acinetobacter species capable of utilizing agar as sole source of carbon and energy was isolated from soil samples and the culture conditions were standardized for the maximal production of extracellular agarase. The bacterium was capable of liquefying an agar-plate within 3 days of incubation and produced extracellular agarase within a short period of time (16–18 h) when grown in defined mineral salts medium. Bacterium grew in the pH range 4.0–9.0, optimal at pH 7.0; temperature 25–40 °C and optimal at 37 °C. The agarase secreted by the Acinetobacter strain was inducible by agar and not repressed by other simple sugars when supplemented along with agar in the medium. The bacterium did not require NaCl for growth or production of agarase. The bacterium did not utilize other polysaccharides like κ-carrageenan, alginate, cellulose, and CMC. The activity staining of partially purified agarase preparations after native-PAGE and SDS PAGE revealed the presence of a single zone of clearance corresponding to the molecular weight 100 kDa, suggesting that it is a monomer. Neoagarobiose was the end product of agarose hydrolysis by this enzyme. The agarase was an endo-type glycosidase and belongs to Group-III β-agarase family.     

Genome Sequence of the Thermostable-Agarase-Producing Marine Bacterium Catenovulum agarivorans YM01T, Which Reveals the Presence of a Series of Agarase-Encoding Genes

Marine bacterium Catenovulum agarivorans YM01(T) can produce highly thermostable agarases. The draft genome of YM01(T) is about 5.36 Mb and harbors approximately 4,913 genes, including 15 agarase (2 α-agarase and 13 β-agarase)-encoding genes, which will provide references to functional characterization of various agarases from marine bacteria.     

New Sulfated Oligosaccharides Produced by Pseudomonas β-Agarase from Gracilaria verrucosa Polysaccharide

Polysaccharide (partially sulfated agarose) with macrophage-stimulation activity, derived from Gracilaria verrucosa, was decomposed by two types of β-agarase (agarases II and IV) from Pseudomonas sp. O-148. The hydrolysates were fractionated with ethanol precipitation and anion-exchange chromatography. The resulting anionic oligosaccharides with sulfate groups were investigated by ¹³C-NMR spectroscopy. While the spectra of oligosaccharides produced by agarase IV showed identical patterns with those by β-agarase I from Pseudomonas atlantica and indicated the location of a sulfated saccharide unit on the non-reducing end, another new type of saccharide was found in the products by agarase II. The novel oligosaccharides by agarase II had a neoagarobiose unit on their non-reducing end and had sulfated units internally. This indicated the novelty of agarase II in cleavage fashion.     

Purification and structural characterization of bacillomycin F produced by a bacterial honey isolate active against Byssochlamys fulva H25

Aims:  Isolate and characterize antifungal peptides exhibiting activity against Byssochlamys fulva H25, a spoilage mould associated with juices and beverages.
Methods and Results:  A bacterium (H215) isolated from honey showed high antifungal activity against B. fulva H25. The antifungal producer strain was identified as Bacillus subtilis using 16S rDNA sequencing. The antifungal peptide was purified by 20% ammonium sulfate precipitation of the bacterial culture supernatant, followed by Octyl-Sepharose CL-4B and reverse phase-high performance liquid chromatography. The five active fractions were lyophilized and subjected to mass, tandem mass spectrometry and amino acid analysis to deduce their corresponding molecular masses and structural characteristics. The five peaks were determined to be identical to bacillomycin F, varying in the length of the fatty acid chain moiety from C14 to C16.
Conclusions:  The broad-spectrum antifungal activity produced by a bacterium from honey was determined to be due to the production of bacillomycin F.
Significance and Impact of the Study:  The antifungal compound produced by a bacterial strain isolated from honey was determined to be stable over a broad pH range and was stable to heat treatments up to 100°C. This is the first report of honey microflora producing bacillomycin F or any antifungal compound.     

海洋细菌Agarivorans albus NBRC102603琼胶酶的分离纯化

利用盐析、分子筛和离子交换等方法对海洋细菌Agarivorans albus NBRC102603分泌的琼胶酶粗酶液进行分离纯化,得到琼胶酶A和琼胶酶B.琼胶酶A纯化倍数为17.51倍,酶比活力为881.82 U/mg;琼胶酶B纯化倍数为16.64倍,酶比活力为838.32 U/mg.纯化的琼胶酶经SDS-PAGE检测,显示为单一条带,其相对分子质量分别为酶A 8.36×104和酶B 3.68×104.