Characterization and activity enhancement of a novel exo-type agarase Aga575 from Aquimarina agarilytica ZC1.

The novel ß-agarase gene aga575 from the agarolytic bacterium Aquimarina agarilytica ZC1 is composed of 2142 bp, and the encoded protein Aga575 has the highest amino acid sequence homology of only 65.2% with known agarases. Though carrying a domain of glycoside hydrolase family 42 in the C-terminal, Aga575 should belong to glycoside hydrolase family 50 according to the phylogenetic analysis. Gene aga575 was successfully cloned and overexpressed in Escherichia coli Rosetta (DE3) cells. The recombinant protein had the maximal agarase activity at pH 8.0 and 37 °C. The values Km and Vmax toward agarose were 8.4 mg/mL and 52.2 U/mg, respectively. Aga575 hydrolyzed agarose and neoagarooligosaccharides to yield neoagarobiose as the sole product. The agarose hydrolysis pattern of Aga575 indicated that it was an exo-type ß-agarase. Random mutagenesis was carried out to obtain two beneficial mutants M1 (R534G) and M2 (S4R-R424G) with higher activities. The results showed that the agarase activity of mutant M1 and M2 reached 162% and 192% of the wild-type agarase Aga575, respectively. Moreover, the activity of the mixed mutant M1/M2 (S4R-R424G-R534G) increased to 227%. KEY POINTS: • Aga575 is a novel exo-type ß-agarase degrading agarose to yield neoagarobiose as the sole product. • Though owning a domain of glycoside hydrolase family GH42, Aga575 should belong to family GH50. • The agarase activity of one mutant increased to 227% of the wild-type Aga575.

Research Progress on Strategies for Improving the Enzyme Properties of Bacteriophage Endolysins.

Bacterial resistance to commonly used antibiotics is one of the major challenges to be solved today. Bacteriophage endolysins (Lysins) have become a hot research topic as a new class of antibacterial agents. They have promising applications in bacterial infection prevention and control in multiple fields, such as livestock and poultry farming, food safety, clinical medicine and pathogen detection. However, many phage endolysins display low bactericidal activities, short half-life and narrow lytic spectrums. Therefore, some methods have been used to improve the enzyme properties (bactericidal activity, lysis spectrum, stability and targeting the substrate, etc) of bacteriophage endolysins, including deletion or addition of domains, DNA mutagenesis, chimerization of domains, fusion to the membrane-penetrating peptides, fusion with domains targeting outer membrane transport systems, encapsulation, the usage of outer membrane permeabilizers. In this review, research progress on the strategies for improving their enzyme properties are systematically presented, with a view to provide references for the development of lysins with excellent performances.

Characterization of a novel ß-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 ß-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.

Draft genome sequence of the novel agarolytic bacterium Aquimarina agarilytica ZC1.

The marine bacterium ZC1 is the type strain of the recently identified novel species Aquimarina agarilytica. It can produce multiple agarases. Here we report the draft genome sequence of strain ZC1 (4,253,672 bp, with a GC content of 32.8%) and major findings from its annotation. It is the first reported genome in the genus Aquimarina.

Aquimarina agarilytica sp. nov., an agarolytic species isolated from a red alga.

A novel yellow-pigmented, agarolytic bacterial strain, designated ZC1T, was isolated from the surface of the marine red alga Porphyra haitanensis collected near Nan Ao Island, Guangdong province, China. The isolate was Gram-stain-negative, strictly aerobic and rod-shaped and displayed ß-galactosidase, alkaline phosphatase, catalase and oxidase activities. The predominant cellular fatty acids were iso-C15:0, summed feature 3 (comprising C16:1ω7c and/or iso-C15:0 2-OH) and iso-C17:0 3-OH. The major menaquinone was menaquinone 6 (MK-6). The DNA G+C content was 32.8 mol%. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain ZC1T was closely related to members of the genus Aquimarina in the family Flavobacteriaceae, phylum Bacteroidetes. Based on phylogenetic and phenotypic evidence, strain ZC1T (=CCTCC AB 2010229T=NBRC 107695T) represents the type strain of a novel species in the genus Aquimarina, for which the name Aquimarina agarilytica sp. nov. is proposed.

Gene cloning, expression and characterization of a neoagarotetraose-producing ß-agarase from the marine bacterium Agarivorans sp. HZ105.

A ß-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 ß-agarase shared a gene sequence identity of 98.6% with the reported but much less characterized ß-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 ß-agarase agaB of Vibrio sp. JT0107. The agarose hydrolysis pattern suggested that rHZ2 was an endo-type ß-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(+), Mg(2+) and Ca(2+). 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 × 10(5) M(-1) s(-1), respectively. Agarase rHZ2 could have a potential application in the production of bioactive neoagarotetraose.

Processing of four different cooking methods of Oudemansiella radicata: Effects on in vitro bioaccessibility of nutrients and antioxidant activity.

The purpose of this study is to evaluate the bioaccessibility of nutrients and antioxidant activity of O. radicata after subjecting to four types of domestic cooking and followed by in vitro digestion. The result demonstrated that the group with the lowest amino acid release and the degree of protein hydrolysis (5.6%) was frying, but both reducing sugar content and antioxidant activity were the highest. The composition of fatty acids was different than undigested samples, especially the relative content of linolenic acid was significantly decreased (e.g., 34.49 to 8.23%, boiled). The difference of the minerals bioaccessibility was slightly affected by the cooking method, but mainly related to their natural properties, such as the highest phosphorus (62.73%) and the lowest iron (21.53%) in the steaming. The above data provides a starting point for the design of processes at an industrial and gastronomic level.

Structural characterization of a polysaccharide from Suillellus luridus and its antidiabetic activity via Nrf2/HO-1 and NF-κB pathways.

A heteropolysaccharide designated SLPC-1S with the Mw of 9.4 kDa was purified from the caps of Suillellus luridus. Monosaccharide composition analysis revealed that SLPC-1S was composed of galactose, glucose, arabinose and mannose in a molar ratio of 44.9:27.6:14.7:12.8. Structural characterization indicated that SLPC-1S had a backbone principally composed of 1,3 linked α-D-Galp, 1,3 linked ß-D-Glcp and 1,6 linked ß-D-Glcp with the branches mainly composed of 1,3 linked ß-D-Glcp, 1,3 linked α-L-Arap, 1,3 linked α-D-Manp and T-linked α-D-Galp. Furthermore, SLPC-1S exhibited excellent antidiabetic activities in the streptozotocin-induced diabetic mice. Protein expression and mRNA levels in NF-kB and Nrf2/HO-1 signaling pathways were detected by western blots and real-time polymerase chain reaction (RT-PCR), respectively. The results strongly proved that SLPC-1S can be treated as a potential agent for preventing and treating diabetes via regulating Nrf2-mediated oxidative stress and NF-κB-mediated inflammatory responses.

Polysaccharides from Cordyceps miltaris cultured at different pH: Sugar composition and antioxidant activity.

In the study, the ß-glucan content, the primary structure and the antioxidant capacity of polysaccharides in Cordyceps militaris cultivated with different initial growth pH were evaluated. Meanwhile, the mechanism of ß-glucan biosynthesis was investigated by RNA-Seq. Based on the results, C. militaris growing at an initial growth pH of 5-7 (CMsA) was distinguished from C. militaris growing at an initial growth pH of 8-9 (CMsB) and their unigenes showed the comparable expression. The mean of ß-glucan content of CMsB group was 32.7% (w/w), 10% higher than that of CMsA. The results of RNA-seq showed 1088 differentially expressed genes between CMsA and CMsB groups. Furthermore, oxidative phosphorylation-related Gene ontology terms were up-regulated in CMsB groups. In addition, the results of structural analysis (FTIR spectrum, monosaccharide composition, periodate oxidation) and bioactivity evaluation speculated that C. militaris polysaccharides possessed higher ß-(1 â†’ 6)-glucan content and antioxidant activities in CMsB groups.

An agarase of glycoside hydrolase family 16 from marine bacterium Aquimarina agarilytica ZC1.

A novel ß-agarase gene aga672 was cloned from strain ZC1, the typical strain of agar-degrading Aquimarina agarilytica. Gene aga672 is composed of 2130 bp, and the encoded protein Aga672 showed an amino acid sequence identity of only 42% with reported agarases. Aga672 should belong to glycoside hydrolase family 16 according to the protein sequence similarity. The molecular mass of the recombinant Aga672 was estimated to be 98 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Aga672 decomposed agarose to produce neoagarotetraose, neoagarohexaose and neoagarooctaose as the main products. That is the main difference between Aga672 and other reported agarases of family GH16. The Km and Vmax for agarose degradation were 59.8 mg mL-1 and 154.3 U mg-1, respectively. The activity of Aga672 was stable at temperatures below 40°C and at pH 7.0-11.0 with the maximal agarase activity at 25°C and pH 7.0. The results showed that agarase Aga672 could be suitable to hydrolyze the gelated agarose. Thus, it has potential applications in the production of neoagarooligosaccharides directly from red alga.

Rapid determination of alkaloids in Macleaya cordata using ionic liquid extraction followed by multiple reaction monitoring UPLC-MS/MS analysis.

The ultrasonic-assisted extraction (UAE) and ionic liquid based dispersive liquid-liquid microextraction (IL-DLLME) have been successfully applied in extracting of six alkaloids from M. cordata. 1-hexyl-3-methylimidazolium tetrafluoroborate ([C6MIM][BF4]) aqueous solution was used as extraction solvent. The target analytes in raw material were deposited into a single drop of 1-hexyl-3-methylimidazolium hexafluorophosphate ([C6MIM][PF6]), which was in situ formed by mixing [C6MIM][BF4] and potassium hexafluorophosphate ([K][PF6]. Afterwards, the extract was analyzed by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) in multiple-reaction monitoring (MRM) mode. The proposed method was fully validated in terms of linearity (0.9983-0.9992), LOD (0.080ngmL-1), LOQ (0.25ngmL-1), intra-day precision (<5.46%), inter-day precision (<6.36%), and recovery (86.42-112.48%). The results indicate that the approach of combining IL-DLLME with UPLC-MS/MS is powerful and practical for analyzing alkaloids in M. cordata., and it also has great potential for comprehensive quality control of other herbal medicines.

Characterization of cefalexin degradation capabilities of two Pseudomonas strains isolated from activated sludge.

Pharmaceuticals have recently been regarded as contaminants of emerging concern. To date, there is limited knowledge about antibiotic-degrading microorganisms in conventional activated sludge treatment systems and their characteristics toward antibiotic degradation especially in the presence of a pharmaceutical mixture. As such, antibiotic-degrading microorganisms were investigated and isolated from the activated sludge, and their degradation capabilities were evaluated. Two strains of cefalexin-degrading bacteria CE21 and CE22 were isolated and identified as Pseudomonas sp. in the collected activated sludge. Strain CE22 was able to degrade over 90% of cefalexin, while CE21 was able to remove 46.7% of cefalexin after incubation for 24h. The removal efficiency of cefalexin by CE22, different from that of CE21, was not significantly affected by an increase in cefalexin concentration, even up to 10ppm, however the presence of 1ppm of other pharmaceuticals had a significant effect on the degradation of cefalexin by CE22, but no significant effect on CE21. The degradation product of cefalexin by the two strains was identified to be 2-hydroxy-3-phenyl pyrazine. Our results also indicated that CE21 and CE22 were able to degrade caffeine, salicylic acid and chloramphenicol. Moreover, CE21 was found to be capable of eliminating sulfamethoxazole and naproxen.

Draft Genome Sequence of a Xylanase-Producing Bacterial Strain, Cellvibrio mixtus J3-8.

The xylanase-producing bacterial strain Cellvibrio mixtus J3-8 was isolated from grassland giant snails. The draft genome of strain J3-8 comprises 5,171,890 bp in 152 contigs with a G+C content of 46.66%. This is the first genome report about this bacterial species.

Gene cloning and characterization of an α-amylase from Alteromonas macleodii B7 for Enteromorpha polysaccharide degradation.

Enteromorpha polysaccharides (EP) extracted from green algae have displayed a wide variety of biological activities. However, their high molecular weight leads to a high viscosity and low solubility, and therefore, greatly restrains their application. To solve this problem, bacteria from the surface of Enteromorpha were screened, and an Alteromonas macleodii strain B7 was found to be able to decrease the molecular weight of EP in culture media. Proteins harvested from the supernatant of the A. macleodii B7 culture were subjected to native gel electrophoresis, and a band corresponding to the Enteromorpha polysaccharide lyase (EPL) was detected by activity staining. The enzyme identity was subsequently confirmed by MALDI-TOF/TOF mass spectrometry as the putative α-amylase reported in A. macleodii ATCC 27126. The amylase gene (amySTU) from A. macleodii B7 was cloned into Escherichia coli, resulting in highlevel expression of the recombinant enzyme with EP-degrading activity. AmySTU was found to be cold-adapted; however, its optimal enzyme activity was detected at 40°C. The α-amylase was highly stable over a broad pH range (5.5-10) with the optimal pH at 7.5-8.0. The highest enzyme activity was detected when NaCl concentration was 2%, which dropped by 50% when the NaCl concentration was increased to 16%, showing an excellent nature of halotolerance. Furthermore, the amylase activity was not significantly affected by tested surfactants or the presence of some organic solvents. Therefore, the A. macleodii strain B7 and its α-amylase can be useful in lowering EP molecular weight and in starch processing.

Affinity proteomic approach for identification of an IgA-like protein in Litopenaeus vannamei and study on its agglutination characterization.

An unknown protein reacted with anti-human IgA, namely, IgA-like protein, has been reported in shrimp, but information regarding its identification is not available. In the present study, an affinity proteomic strategy was applied to identify the IgA-like protein of shrimp Litopenaeus vannamei. The protein of 75 kDa was isolated and confirmed by affinity chromatography and Western blotting with goat anti-human IgA, respectively, and then identified as hemocyanin, a member of IgSF, by mass spectrometry. Moreover, our results showed that human IgA and L. vannamei hemocyanin could separately react with goat anti-human IgA or rabbit anti-shrimp affinity hemocyanin (a-hemocyanin). Further evidences indicated that the recombinant protein of the Ig-like conserved domain could react with anti-human IgA. Interestingly, our results indicated that L. vannamei hemocyanin could aggregate with eight species of shrimp pathogenic bacteria and four types of animal erythrocytes directly. These results indicate that L. vannamei hemocyanin, an IgA-like protein, has dual function of reaction with anti-human IgA as an antigen and of activity binding to bacteria and animal erythrocytes as an agglutinin, suggesting its characteristic role as an IgSF molecule. In addition, our approach suggests that affinity proteomics based on heterogeneous antibody can speed up the identification of Fossman antigens.