Mingsheng Lu

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.

The atmospheric and room-temperature plasma (ARTP) method on the dextranase activity and structure

A novel atmospheric and room-temperature plasma (ARTP) method was used to breed high-yielding mutations of Arthrobacter KQ11. Mutagenesis produced two mutations, 4-1 and 4-13, which increased enzyme activity by 19 and 30%, respectively. Dents on the cell envelope were observed under scanning electron microscopy (SEM). The optimal temperature and pH of the wild strain were 45°C and 5.5 and those of the mutant strains were 45°C, pH 6.0 (4-1) and 50°C, pH 6.0 (4-13). Under optimal enzyme production conditions of the wild and mutant strains, the dextranase activity of 4-13 was 50% higher than that of the wild strain. Through amino acid alignment, several nucleotides of the mutant strains were found to have changed. Experiments performed in vitro suggested that this endo-dextranase may inhibit biofilm formation by Streptococcus mutans.

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.

Production of pullulan from raw potato starch hydrolysates by a new strain of Auerobasidium pullulans.

In the present study, hydrolysis of potato starch with marine cold-adapted α-amylase and pullulan production from the hydrolysates by a new strain of Auerobasidium pullulans isolated from sea mud were conducted. The hydrolysis conditions were optimized as follows: reaction time 2h, pH 6.5, temperature 20°C, and α-amylase amount 12 U/g. Under these optimum hydrolysis conditions, the DE value of the potato starch hydrolysates reached to 49.56. The potato starch hydrolysates consist of glucose, maltose, isomaltose, maltotriose, and trace of other maltooligosaccharides with degree of polymerization ranged 4-7. The maximum production of pullulan at 96 h from the hydrolysate of potato starch was 36.17 g/L, which was higher than those obtained from glucose (22.07 g/L, p<0.05) and sucrose (31.42 g/L, p<0.05). Analysis of the high performance liquid chromatography of the hydrolysates of the pullulan product with pullulanase indicated that the main composition is maltotriose, thus confirming the pullulan structure of this pullulan product.

Effect of oligosaccharides derived from Laminaria japonica-incorporated pullulan coatings on preservation of cherry tomatoes.

Laminaria japonica-derived oligosaccharides (LJOs) exhibit antibacterial and antioxidant activities, and pullulan is a food thickener that can form impermeable films. The ability of pullulan coatings with various LJO concentrations (1% pullulan+0.1%, 0.2% or 0.3% LJOs) to preserve cherry tomatoes during storage at room temperature was investigated. The LJO-incorporated pullulan coatings were found to effectively reduce respiratory intensity, vitamin C loss, weight loss and softening, as well as to increase the amount of titratable acid and the overall likeness of fruit compared with the control. These effects were observed to be dose-dependent. Therefore, using LJO-incorporated pullulan coatings can extend the shelf life of cherry tomatoes.

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.

The hyperthermophilic α-amylase from Thermococcus sp. HJ21 does not require exogenous calcium for thermostability because of high-binding affinity to calcium

The hyperthermophilic α-amylase from Thermococcus sp. HJ21 does not require exogenous calcium ions for thermostability, and is a promising alternative to commercially available α-amylases to increase the efficiency of industrial processes like the liquefaction of starch. We analyzed the amino acid sequence of this α-amylase by sequence alignments and structural modeling, and found that this α-amylase closely resembles the α-amylase from Pyrococcus woesei. The gene of this α-amylase was cloned in Escherichia coli and the recombinant α-amylase was overexpressed and purified with a combined renaturation-purification procedure. We confirmed thermostability and exogenous calcium ion independency of the recombinant α-amylase and further investigated the mechanism of the independency using biochemical approaches. The results suggested that the α-amylase has a high calcium ion binding affinity that traps a calcium ion that would not dissociate at high temperatures, providing a direct explanation as to why the addition of calcium ions is not required for thermostability. Understanding of the mechanism offers a strong base on which to further engineer properties of this α-amylase for better potential applications in industrial processes.

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.