Yuliang Jiao

Optimization of antioxidant exopolysaccharidess production by Bacillus licheniformis in solid state fermentation

Response surface methodology was applied to optimize physical and nutritional variables for the production of antioxidant exopolysaccharidess (EPSs) by Bacillus licheniformis UD061 in solid state fermentation with squid processing byproduct and maize cob meal used as a carbon and nitrogen source and solid matrix. The factors noted with Plackett-Burman design for optimization of EPSs production were NaCl, MgSO4·7H2O, and moisture level. These factors were further optimized using Box-Behnken design and response surface methodology. Using this methodology, the quadratic regression model of EPSs production was built. Maximum EPSs production was obtained under the optimal conditions of 4.08 g L(-1) NaCl, 0.71 g L(-1) MgSO4·7H2O, and 60.49% moisture level. A production of 14.68 mg gds(-1), which was well in agreement with the predicted value, was achieved by this optimized procedure.

Purification and characterization of a novel marine Arthrobacter oxydans KQ11 dextranase.

Dextranases can hydrolyze dextran deposits and have been used in the sugar industry. Microbial strains which produce dextranases for industrial use are chiefly molds, which present safety issues, and dextranase production from them is impractically long. Thus, marine bacteria to produce dextranases may overcome these problems. Crude dextranase was purified by a combination of ammonium sulfate fractionation and ion-exchange chromatography, and then the enzyme was characterized. The enzyme was 66.2 kDa with an optimal temperature of 50°C and a pH of 7. The enzyme had greater than 60% activity at 60°C for 1h. Moreover, 10mM Co(2+) enhanced dextranase activity (196%), whereas Ni(2+) and Fe(3+) negatively affected activity. 0.02% xylitol and 1% alcohol enhanced activity (132.25% and 110.37%, respectively) whereas 0.05% SDS inhibited activity (14.07%). The thickness of S. mutans and mixed-species oral biofilm decreased from 54,340 nm to 36,670 nm and from 64,260 nm to 43,320 nm, respectively.

A novel method for promoting antioxidant exopolysaccharidess production of Bacillus licheniformis

A novel method was described for improving the production of antioxidant extracellular polysaccharides from Bacillus licheniformis. Firstly, the tolerances of the strains to the organic solvents were investigated. Wild type strain of B. licheniformis OSTK95 and mutant strain UD061 can grow in a liquid medium in the presence of organic solvents with the logP value equal to or higher than 3.5 and 3.1, respectively. Secondly, the effects of different concentrations of n-hexane and xylene treatment on the extracellular polysaccharides excretion of both strains were studied. The maximum yield of the extracellular polysaccharides of B. licheniformis OSTK95 was 68.59 mg L(-1) after treated by 10% n-hexane or 1% xylene for 3h, while the maximum yield of the extracellular polysaccharides of strain UD061 was 185.01 mg L(-1) after treated by 12.5% n-hexane or 5% xylene for 3h. Finally, the continuous passage experiment showed that the strains have high genetic stability.

Discovery a novel organic solvent tolerant esterase from Salinispora arenicola CNP193 through genome mining.

An esterase gene, encoding a 325-amino-acid protein (SAestA), was mined form obligate marine actinomycete strain Salinispora arenicola CNP193 genome sequence. Phylogenetic analysis of the deduced amino acid sequence showed that the enzyme belonged to the family IV of lipolytic enzymes. The gene was cloned, expressed in Escherichia coli as a His-tagged protein, purified and characterized. The molecular weight of His-tagged SAestA is ∼38 kDa. SAestA-His6 was active in a temperature (5-40 °C) and pH range (7.0-11.0), and maximal activity was determined at pH 9.0 and 30 °C. The activity was severely inhibited by Hg(2+), Cu(2+), and Zn(2+). In particular, this enzyme showed remarkable stability in presence of organic solvents (25%, v/v) with log P>2.0 even after incubation for 7 days. All these characteristics suggested that SAestA may be a potential candidate for application in industrial processes in aqueous/organic media.

Purification and characterization of iron-cofactored superoxide dismutase from Enteromorpha linza

A superoxide dismutase was purified from Enteromorpha linza using a simple and safe procedure, which comprised phosphate buffer extraction, ammonium sulphate precipitation, ion exchange chromatography on Q-sepharose column, and gel filtration chromatography on Superdex 200 10/300GL. The E. linza superoxide dismutase (ElSOD) was purified 103.6-fold, and a yield of 19.1% and a specific activity of 1 750 U/mg protein were obtained. The SDS-PAGE exhibited ElSOD a single band near 23 kDa and the gel filtration study showed ElSOD’s molecular weight is near 46 kDa in nondenatured condition, indicating it’s a homodimeric protein. El SOD is an iron-cofactored superoxide dismutase (Fe-SOD) because it was inhibited by hydrogen peroxide, insensitive to potassium cyanide. The optimal temperature for its maximal enzyme activity was 35°C, and it still had 29.8% relative activity at 0°C, then ElSOD can be classified as a cold-adapted enzyme. ElSOD was stable when temperature was below 40°C or the pH was within the range of 5–10. The first 11 N-terminal amino acids of ElSOD were ALELKAPPYEL, comparison of its N-terminal sequence with other Fe-SOD N-terminal sequences at the same position suggests it is possibly a chloroplastic Fe-SOD.

Optimization of four types of antimicrobial agents to increase the inhibitory ability of marine Arthrobacter oxydans KQ11 dextranase mouthwash

We adopted the response surface methodology using single factor and orthogonal experiments to optimize four types of antimicrobial agents that could inhibit biofilm formation by Streptococcus mutans, which is commonly found in the human oral cavity and causes tooth decay. The objective was to improve the function of marine Arthrobacter oxydans KQ11 dextranase mouthwash (designed and developed by our laboratory). The experiment was conducted in a three-level, four-variable central composite design to determine the best combination of ZnSO4, lysozyme, citric acid and chitosan. The optimized antibacterial agents were 2.16 g/L ZnSO4, 14 g/L lysozyme, 4.5 g/L citric acid and 5 g/L chitosan. The biofilm formation inhibition reached 84.49%. In addition, microscopic observation of the biofilm was performed using scanning electron microscopy and confocal laser scanning microscopy. The optimized formula was tested in marine dextranase Arthrobacter oxydans KQ11 mouthwash and enhanced the inhibition of S. mutans. This work may be promoted for the design and development of future marine dextranase oral care products.

Improving stability of a novel dextran-degrading enzyme from marine Arthrobacter oxydans KQ11.

Dextranases can hydrolyze dextran, so they are used in the sugar industry to mitigate the milling problems associated with dextran contamination. Few studies have been carried out on the storage stability of dextranase, let alone the dextranase of Arthrobacter oxydans KQ11 isolated from sea mud samples. This study improved the storage stability of dextranase from marine A. oxydans KQ11 by adding enzymatic protective reagents (stabilizer and antiseptic). Initially, the conditions (55 °C and 30 min) for maintaining 50% dextranase activity were obtained. Then, the best stabilizers of dextranase were obtained, namely, glycerol (16%), sodium acetate (18%) and sodium citrate (20%). Results showed that p-hydroxybenzoic acid compound sodium acetate (0.05%), D-sodium isoascorbiate (0.03%), and potassium sorbate (0.05%) were the best antiseptics. Subsequent validation experiment showed that dextranase with enzymatic protective reagents maintained 70.8% and 28.96% activities at the 13th week at 25 and 37 °C, respectively.

An evolutionary analysis of the GH57 amylopullulanases based on the DOMON_glucodextranase_like domains

Thermostable amylopullulanase (TAPU) is valuable in starch saccharification industry for its capability to catalyze both α‐1,4 and α‐1,6 glucosidic bonds under the industrial starch liquefication condition. The majority of TAPUs belong to glycoside hydrolase family 57 (GH57). In this study, we performed a phylogenetic analysis of GH57 amylopullulanase (APU) based on the highly conserved DOMON_glucodextranase_like (DDL) domain and classified APUs according to their multidomain architectures, phylogenetic analysis and enzymatic characters. This study revealed that amylopullulanase, pullulanase, andα‐amylase had passed through a long joint evolution process, in which DDL played an important role. The phylogenetic analysis of DDL domain showed that the GH57 APU is directly sharing a common ancestor with pullulanase, and the DDL domains in some species undergo evolution scenarios such as domain duplication and recombination.

Solid-state fermentation of Acanthogobius hasta processing by-products for the production of antioxidant protein hydrolysates with Aspergillus oryzae

Functional properties and antioxidative activity of a protein hydrolysate prepared from Acanthogobius hasta processing by-product protein during solid-state fermentation with Aspergillus oryzae were investigated. Overall, protease activity increased with the degree of hydrolysis (DH) decreased during solid-state fermentation. All the protein hydrolysate had excellent solubility, possessed interfacial properties, and varying degrees of antioxidant activity which were governed by their concentrations and DH, molecular weight distribution and amino acid composition. After 5 days fermentation, the DH of the protein hydrolysate was 31.23%. The protein hydrolysate had the highest total hydrophobic amino acid content, the highest DPPH scavenging activity, reducing power, and the chelating activity. The radical-scavenging activity of the hydrolysates at 6 mg/mL was 78.6%. The reducing power of protein hydrolysate at the range of 0-6 mg/mL was lower than that of BHA at the range of 0-60 µg/mL, while the chelating activity of APs was similar to that of BHA at the range of 0-60 µg/mL. Moreover, the protein hydrolysate showed good emulsifying and foaming properties over a wide pH range from 2 to 12. Therefore, solid state fermentation provided a suitable and low-cost method for converting Acanthogobius hasta processing by-product protein into antioxidant protein hydrolysates.

Dextranase from Arthrobacter oxydans KQ11-1 inhibits biofilm formation by polysaccharide hydrolysis

Abstract Dental plaque is a biofilm of water-soluble and water-insoluble polysaccharides, produced primarily by Streptococcus mutans. Dextranase can inhibit biofilm formation. Here, a dextranase gene from the marine microorganism Arthrobacter oxydans KQ11-1 is described, and cloned and expressed using E. coli DH5α competent cells. The recombinant enzyme was then purified and its properties were characterized. The optimal temperature and pH were determined to be 60°C and 6.5, respectively. High-performance liquid chromatography data show that the final hydrolysis products were glucose, maltose, maltotriose, and maltotetraose. Thus, dextranase can inhibit the adhesive ability of S. mutans. The minimum biofilm inhibition and reduction concentrations (MBIC50 and MBRC50) of dextranase were 2 U ml−1 and 5 U ml−1, respectively. Scanning electron microscopy and confocal laser scanning microscope (CLSM) observations confirmed that dextranase inhibited biofilm formation and removed previously formed biofilms.