Expansion and diversity of caspases in Mytilus coruscus contribute to larval metamorphosis and environmental adaptation.

BACKGROUND: Apoptosis is involved (directly and indirectly) in several physiological processes including tissue remodeling during the development, the turnover of immune cells, and a defense against harmful stimuli. The disordered apoptotic process participates in the pathogenesis of various diseases, such as neoplasms, and chronic inflammatory or systemic autoimmune diseases, which are associated with its inadequate regulation. Caspases are vital components of the apoptotic pathway that are involved in developmental and immune processes. However, genome-wide identification and functional analysis of caspase have not been conducted in Mytilus coruscus, which is an economically important bivalve. RESULTS: Here, 47 caspase genes were identified from the genomes of M. coruscus, and the expansion of caspase-2/9 and caspase-3/6/7 genes were observed. Tandem duplication acts as an essential driver of gene expansion. The expanded caspase genes were highly diverse in terms of sequence, domain structure, and spatiotemporal expression profiles, suggesting their functional differentiation. The high expression of the expanded caspase genes at the pediveliger larvae stage and the result of apoptosis location in the velum suggest that the apoptosis mediated by them plays a critical role in the metamorphosis of M. coruscus larvae. In gill, caspase genes respond differently to the challenge of different strains, and most caspase-2/9 and caspase-3/6/7 genes were induced by copper stress, whereas caspase-8/10 genes were suppressed. Additionally, most caspase genes were upregulated in the mantle under ocean acidification which could weaken the biomineralization capacity of the mantle tissue. CONCLUSIONS: These results provide a comprehensive overview of the evolution and function of the caspase family and enhanced the understanding of the biological function of caspases in M. coruscus larval development and response to biotic and abiotic challenges.

Expression, purification and characterization of a cold-adapted dextranase from marine bacteria and its ability to remove dental plaque.

Dental plaque is a high-incidence health concern, and it is caused by Streptococcus mutans. Dextranase can specifically hydrolyze ɑ-1,6-glycosidic linkages in dextran. It is commonly used in the sugar industry, in the production of plasma substitutes, and the treatment and prevention of dental plaque. In this research work, we successfully cloned and expressed a cold-adapted dextranase from marine bacteria Catenovulum sp. DP03 in Escherichia coli. The recombinant dextranase named Cadex2870 contained a 2511 bp intact open reading frame and encoded 836 amino acids. The expression condition of recombinant strain was 0.1 mM isopropylthio-galactoside (IPTG), and the reduced temperature was 16 °C. The purified enzyme activity was 16.2 U/mg. The optimal temperature and pH of Cadex2870 were 45 °C and pH 8, and it also had catalytic activity at 0 °C. The hydrolysates of Cadex2870 hydrolysis Dextran T70 are maltose, maltotetraose, maltopentose, maltoheptaose and higher molecular weight maltooligosaccharides. Interestingly, 0.5% sodium benzoate, 2% xylitol, 0.5% sodium fluoride, 5% propanediol, 5% glycerin and 2% sorbitol can enhance stability Cadex2870, which are additives in mouthwashes. Additionally, Cadex2870 reduced the formation of dental plaque and effectively degraded formed plaque. Therefore, Cadex2870 shows great promise in commercial applications.

Identification of substituent groups and related genes involved in salecan biosynthesis in Agrobacterium sp. ZX09.

Salecan, a soluble ß-1,3-D-glucan produced by a salt-tolerant strain Agrobacterium sp. ZX09, has been the subject of considerable interest in recent years because of its multiple bioactivities and unusual rheological properties in solution. In this study, both succinyl and pyruvyl substituent groups on salecan were identified by an enzymatic hydrolysis following nuclear magnetic resonance (NMR), HPLC, and MS analysis. The putative succinyltransferase gene (sleA) and pyruvyltransferase gene (sleV) were determined and cloned. Disruption of the sleA gene resulted in the absence of succinyl substituent groups on salecan. This defect could be complemented by expressing the sleA cloned in a plasmid. Thus, the sleA and sleV genes located in a 19.6-kb gene cluster may be involved in salecan biosynthesis. Despite the lack of succinyl substituents, the molecular mass of salecan generated by the sleA mutant did not substantially differ from that generated by the wild-type strain. Loss of succinyl substituents on salecan changed its rheological characteristics, especially a decrease in intrinsic viscosity.

Oligosaccharide elicitor prepared from Salecan triggers the defense responses of Arabidopsis thaliana Col0 against Botrytis cinerea infection.

Oligosaccharides from the water-soluble ß-glucan, Salecan, were investigated to evaluate the activation effect on the defense responses of Arabidopsis thaliana Col0. Salecan oligosaccharides (ScOs, DP 5-10) were prepared at first by acid hydrolysis and gel filtration chromatography and then employed for foliar spray on Arabidopsis seedlings and plants. After ScOs treatment, increase of hydrogen peroxide was histologically and analytically detected in leaves. Transcription levels of several indicator genes which correspond to the signaling pathways and pathogenesis-related proteins were up-regulated at different time by means of quantitative RT-PCR analysis. Importantly, the sequential activation of salicylic acid-mediated and jasmonate-mediated signaling pathway was observed in leaves. Furthermore, pot test and antimicrobial test gave the fact that pretreatment with ScOs restrained the leaf lesions caused by infection of Botrytis cinerea Bc0510 via the enhancement of defense responses of A. thaliana. In conclusion, Salecan oligosaccharides serve as an elicitor which can be used for biological control of plant pathogen.

Bacterial glucans: production, properties, and applications.

Bacterial glucans have aroused increasing interest in commercial applications in the food and pharmaceutical sectors. A number of bacterial glucans have been reported over recent decades, and their structure, production, and functional properties have been extensively studied. In this paper, we review recent researches on bacterial glucans, with emphasis on the production, physical and chemical properties, and the new developments in food, biomedical, pharmaceutical, and other industrial applications.

Recombinant expression and characterization of an acid-, alkali- and salt-tolerant ß-1,3-1,4-glucanase from Paenibacillus sp. S09.

A new ß-1,3-1,4-glucanase gene (PlicA) was cloned from Paenibacillus sp. S09. The ORF contained 717 bp coding for a 238 amino acid protein. PlicA, expressed in Escherichia coli and purified by Ni(2+)-affinity chromatography, had optimum activity at 55 °C and pH 6.2. The specific activity toward barley ß-glucan reached 7,055 U/mg. K m and V max values with barley ß-glucan were 3.7 mg/ml and 3.3 × 10(3) µmol/min mg, respectively. The enzyme exhibited acid- and alkali-tolerance with more than 80 % activity remaining after incubation for 4 h at pH 3.5-12. PlicA was salt-tolerant (>90 % activity retained in 4 M NaCl at 25 °C for 24 h) and salt-activated: activity rising 1.5-fold in 0.5 M NaCl. The thermostability was improved by NaCl and CaCl2. This is the first report of an acid-, alkali- and salt-tolerant bacterial ß-1,3-1,4-glucanase with high catalytic efficiency.

Optimization of α-L-arabinofuranosidase CcABF on clarification and beneficial active substances in fermented ginkgo kernel juice by artificial neural network and genetic algorithm.

This study aimed at using α-L-arabinofuranosidase CcABF to improve the clarity and active substances in fermented ginkgo kernel juice by artificial neural network (ANN) modeling and genetic algorithm (GA) optimization. A credible three-layer feedforward ANN model was established to predict the optimal parameters for CcABF clarification. The experiments proved the highest transmittance of 89.40% for fermented ginkgo kernel juice with this understanding, which exhibited a 25.56% increase over the unclarified group. With the clarification of CcABF, the antioxidant capacity in juice was enhanced with the increase of total phenolic and flavone contents, and the maximum DPPH and hydroxyl radical scavenging rates were increased by 89.71% and 26.65%, respectively. The contents of toxic ginkgolic acids declined markedly, while the active ingredients of ginkgetin and ginkgolide B showed a modest increase. Moreover, changes in free amino acids and volatile compounds improved the nutritive value and flavor of clarified fermented ginkgo kernel juice.

Improving the thermostability of GH49 dextranase AoDex by site-directed mutagenesis.

As an indispensable enzyme for the hydrolysis of dextran, dextranase has been widely used in the fields of food and medicine. It should be noted that the weak thermostability of dextranase has become a restricted factor for industrial applications. This study aims to improve the thermostability of dextranase AoDex in glycoside hydrolase (GH) family 49 that derived from Arthrobacter oxydans KQ11. Some mutants were predicted and constructed based on B-factor analysis, PoPMuSiC and HotMuSiC algorithms, and four mutants exhibited higher heat resistance. Compared with the wild-type, mutant S357P showed the best improved thermostability with a 5.4-fold increase of half-life at 60 °C, and a 2.1-fold increase of half-life at 65 °C. Furthermore, S357V displayed the most obvious increase in enzymatic activity and thermostability simultaneously. Structural modeling analysis indicated that the improved thermostability of mutants might be attributed to the introduction of proline and hydrophobic effects, which generated the rigid optimization of the structural conformation. These results illustrated that it was effective to improve the thermostability of dextranase AoDex by rational design and site-directed mutagenesis. The thermostable mutant of dextranase AoDex has potential application value, and it can also provide references for engineering other thermostable dextranases of the GH49 family.

Characterization, production optimization, and fructanogenic traits of levan in a new Microbacterium isolate.

Levan is a high-valued ß-(2,6)-linked fructan with promising physicochemical and physiological properties and has diverse potential applications in the food, nutraceutical, pharmaceutical and cosmetic industry, but its commercial availability is still restricted to the relatively high costs of production. In this study, a strain identified as Microbacterium sp. XL1 was isolated from soil and highly produced exopolysaccharide (EPS). HPLC, FTIR and NMR spectroscopy revealed XL1-EPS is a levan-type fructan connected by ß-(2, 6) linkages. SEM, DLS and TGA-DSC analysis showed that XL1-EPS processed high morphological versatility, narrow size distribution in its solutions and excellent thermal stability. The levan yield reached 83.67 ± 4.06 g/L with corresponding productivity of 3.49 ± 0.17 g/L/h and a conversion yield of 39.8 ± 1.9 % using sucrose (210 g/L) as substrates under the optimal cultivation conditions concluded by the response surface methodology (RSM). More strikingly, the XL1 strain also has multi-type fructanases to generate levanbiose, kestose, DFA IV and other L-FOSs. These results suggest Microbacterium sp. XL1 is a promising strain to produce levan and can provide various levan/inulin-degrading enzymes to create a great diversity of FOSs.

Purification and characterization of cold-adapted and salt-tolerant dextranase from Cellulosimicrobium sp. THN1 and its potential application for treatment of dental plaque.

The cold-adapted and/or salt-tolerant enzymes from marine microorganisms were confirmed to be meritorious tools to enhance the efficiency of biocatalysis in industrial biotechnology. We purified and characterized a dextranase CeDex from the marine bacterium Cellulosimicrobium sp. THN1. CeDex acted in alkaline pHs (7.5-8.5) and a broad temperature range (10-50°C) with sufficient pH stability and thermostability. Remarkably, CeDex retained approximately 40% of its maximal activities at 4°C and increased its activity to 150% in 4 M NaCl, displaying prominently cold adaptation and salt tolerance. Moreover, CeDex was greatly stimulated by Mg2+, Na+, Ba2+, Ca2+ and Sr2+, and sugarcane juice always contains K+, Ca2+, Mg2+ and Na+, so CeDex will be suitable for removing dextran in the sugar industry. The main hydrolysate of CeDex was isomaltotriose, accompanied by isomaltotetraose, long-chain IOMs, and a small amount of isomaltose. The amino acid sequence of CeDex was identified from the THN1 genomic sequence by Nano LC-MS/MS and classified into the GH49 family. Notably, CeDex could prevent the formation of Streptococcus mutans biofilm and disassemble existing biofilms at 10 U/ml concentration and would have great potential to defeat biofilm-related dental caries.

Purification, Characterization, and Hydrolysate Analysis of Dextranase From Arthrobacter oxydans G6-4B.

Dextran has aroused increasingly more attention as the primary pollutant in sucrose production and storage. Although enzymatic hydrolysis is more efficient and environmentally friendly than physical methods, the utilization of dextranase in the sugar industry is restricted by the mismatch of reaction conditions and heterogeneity of hydrolysis products. In this research, a dextranase from Arthrobacter oxydans G6-4B was purified and characterized. Through anion exchange chromatography, dextranase was successfully purified up to 32.25-fold with a specific activity of 288.62 U/mg protein and a Mw of 71.12 kDa. The optimum reaction conditions were 55°C and pH 7.5, and it remained relatively stable in the range of pH 7.0-9.0 and below 60°C, while significantly inhibited by metal ions, such as Ni+, Cu2+, Zn2+, Fe3+, and Co2+. Noteworthily, a distinction of previous studies was that the hydrolysates of dextran were basically isomalto-triose (more than 73%) without glucose, and the type of hydrolysates tended to be relatively stable in 30 min; dextranase activity showed a great influence on hydrolysate. In conclusion, given the superior thermal stability and simplicity of hydrolysates, the dextranase in this study presented great potential in the sugar industry to remove dextran and obtain isomalto-triose.

Characterization of an exopolysaccharide with distinct rheological properties from Paenibacillus edaphicus NUST16.

Microbial exopolysaccharides (EPSs) have been commercially used for decades owing to their distinct rheological properties. New EPSs with special traits are being explored continuously. The strain Paenibacillus edaphicus NUST16 was isolated in this study. NUST16 produces 12.5g/L EPS after 72h cultivation in shaker flask. Moreover, the purified EPS, POS16, has the molecular weight of 1.2×107Da and consists of five types of glycosides, namely, D-Glc, D-Man, L-Fuc, D-GlcA, and D-Gal. Methylation, NMR, and FT-IR results indicate that three glucosides and three mannosides make up the main chain of POS16 via 1→3 linkage. The Gal(1→3)Fuc(1→3)Fuc(1→ and GlcA(1→ branches link to the C6 positions of two mannosides, respectively. The other mannoside is partially derivatized by the acetyl group at C2 position. Rheological analysis shows that POS16 has typical shear-thinning pattern which fits the Hershel-Bulkley model well. More importantly, POS16 shows ideal saline tolerance as the apparent viscosity keeps high in solutions containing 100g/L salts, such as NaCl, KCl, MgCl2, and CaCl2. Furthermore, POS16 behaves more viscous than xanthan gum and sodium carboxymethylcellulose in divalent ion solutions. The results suggest that POS16 is a potential rheological modulator that can be used in operations where high concentrations of salts exist.

Salecan diet increases short chain fatty acids and enriches beneficial microbiota in the mouse cecum.

Salecan, a linear extracellular polysaccharide consisting of ß-(1,3)-D-glucan, has potential applications in the food industry due to its excellent toxicological profile and rheological properties. The aim of the present study was to evaluate the effects of dietary supplementation with 8% Salecan on the gastrointestinal microbiota in mice. In the Salecan group, the following significant differences (p<0.05) from the cellulose group were found: increased body weight gain, greater mass of cecum and cecal contents, and higher butyrate concentrations in the cecal and colonic contents at wk 4. Moreover, populations of Lactobacillus and Bifidobacterium increased 3- and 6-fold, respectively, in the cecal contents of mice consuming Salecan. These results suggest that the dietary incorporation of Salecan, by providing SCFAs and increasing beneficial microbiota, may be beneficial in improving gastrointestinal health, and have relevance to the use of Salecan as a dietary supplement for human consumption.