Yaowei Fang

Optimization of the production of organic solvent-stable protease by Bacillus sphaericus DS11 with response surface methodology

Response surface methodology (RSM) was employed to enhance the production of organic solvent-stable protease by Bacillus sphaericus DS11. A significant influence of glycerol, MgSO₄·7H₂O, and pH on organic solvent-stable protease production was noted with Plackett-Burman design. Then, a three-level Box-Behnken design was employed to optimize the medium composition and culture conditions for the production of the protease in shake-flask. Using this methodology, the quadratic regression model of producing organic solvent-stable protease was built and the optimal combinations of media constituents and culture conditions for maximum protease production were determined as glycerol 12.47 g/L, MgSO₄·7H₂O 0.73 g/L, and pH 8.25. Protease production obtained experimentally coincident with the predicted value and the model was proven to be adequate. The enhancement of protease from 465.06 U/mL to 1182.68 U/mL was achieved with the optimization 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.

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.

Screening and identification of a novel organic solvent-stable lipase producer

A strain named DS9 excreting organic solvent-stable lipase was screened and later identified asBacillus subtilis based on its phenotypes, biochemical test, and 16S rRNA gene sequence. Strain DS9 grows well on the medium with 10% (v/v) organic solvent with log P values equal to or above 2.5. The organic solvent-tolerant lipase excreted by strain DS9 had a wider tolerance for organic solvents. The relative activity of the lipase was above 60% at 37 °C, 200 rpm, 30 min in the present of 25% (v/v) organic solvents such as 1-butanol, hexanol, benzene, and toluene. The lipase was not only stable but also activated by n-hexane, xylene, heptane, isooctane, and n-decane. The optimal pH and temperature were 8.0 and 40 °C, respectively. Both the organic solvent-tolerant microorganism and the organic solvent-stable lipase produced by this strain could be used as a biocatalyst for application in non-aqueous biocatalysis.

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.