Matrix of the isolation and cultivation media
The matrix of isolation was sampled from a fishpond near the Bohai Sea(Tianjin, China). The enriched medium contained (in g L-1) the following: Chitosan powder, 10 g; yeast powder, 2.5 g; sodium chloride, 5 g. The pH was not artificially regulated. Sterilization was executed at 121 oC for 20 min.
The seed medium contains peptone 10g, yeast extract 5g, NaCl 5g L-1 and glucose 10g. The pH was adjusted to 6.5. Sterilization was executed at 121 oC for 20 min.
The preliminary screening medium consisted of solution A and B. Solution A contained 1% (w/w) colloidal Chitosan and Solution B contained (in g L-1) 5 g peptone, 5 g (NH4)2SO4, 1.4 g K2HPO4, 0.6 g KH2PO4, 5 g NaCl, 1 g MgSO4·7H2O, and 30 g agar power 30. The pH was adjusted to 6.5. Solutions A and B were sterilized at 115 ℃ for 30 min. Upon cooling to 60 ℃, Solutions A and B were mixed with a ratio of 1:1. Colloidal Chitosan (1%) was prepared according to by adding 1 g of Chitosan powder (deacetylation above 90%) in a 250 ml beaker containing 20 ml purified water for 2 min followed by the addition of 30 ml 0.2 Mol L-1 acetic acid. The solution was constantly stirred until the Chitosan presented as a transparent gel. The pH of colloid Chitosan was adjusted to 5.5 with 1% sodium hydroxide solution and the capacity to 100 ml with distilled water.
The re-screening medium A contained (g L-1) 5 g Chitosan powder, 5 g peptone, 5 g (NH4)2SO4, 1.4 g K2HPO4, 0.6 g KH2PO4, 5 g NaCl, and 1 g MgSO4·7H2O. The pH of the media was adjusted to 6.5 with 2 mol L-1 NaOH and was sterilized at 121 ℃ for 20 min.
The re-screening medium B contained glucose instead of Chitosan powder from the re-screening medium A. Meanwhile, the seed culture medium contained (g L-1) 10 g peptone, 10 g glucose, 5 g yeast extract powder, and 10 g NaCl. The pH of the medium was adjusted to 6.5 with 2 mol L-1 NaOH and then sterilized at 121 ℃ for 20 minutes.
Basic fermentation medium contained (g L-1) glucose 5 g, yeast extract 5 g, K2HPO4 1.4 g, KH2PO4 0.6 g, NaCl 5 g, MgSO4·7H2O 1 g, and Tween-80 1mL. The pH of the medium was adjusted to 6.0 with 2 mol L-1 NaOH and was then sterilized at 121 ℃ for 20 minutes.
Enrichment and screening of chitosanase-producing strain
The collected soil sample was used as the inoculums to enrich chitosanase-producing bacteria in a 250 ml conical flask containing 100 ml of the enrichment medium at 30℃ for 48 h, with constant shaking at 160 rpm. Using a sterile saline solution, 1 ml bacterial solution in the enrichment medium was successively diluted from 10-1 to 10-6. Approximately 0.1 ml of each diluent was plated on the preliminary screening medium plate and cultured at 30 ℃ for 3 d. Each sample was done with three replications. Single colonies with good growth and transparent circle were selected for further purification. These colonies were inoculated by a sterile toothpick on preliminary screening medium plates and incubated at 30 ℃ for 3 d. The diameter (d) of the colony and size of the transparent circle around each colony (D) was measured. The five largest strains on the specific value of D/d were selected for further fermentation screening. The inoculated seed medium was incubated at 30 ℃ for about 8 h, under constant shaking at 160 rpm. The inoculation volume of the seed broth was 2% when the concentration of bacteria in seed broth was 1.0 as determined by OD600. The fermentation process was executed in 30 ℃ for 40 h under 160 rpm conditions. Then crude enzyme liquid was obtained by subjecting the fermentation broth to centrifugation, after which enzyme activity was measured. The strain with the highest enzyme activity was used in the next step.
Inductivity of chitosanase produced by strains
The strain was inoculated into the re-screening media A and B and cultivated for 40 h at 30 ℃, with constant agitation at 160 rpm. The inductivity of the enzyme was confirmed based on its activity in different fermentation broths.
Identification of strain
The cellular morphologies of strains from the agar plate were observed under the scanning electron microscope (SEM, FEI Apreo HIVac, Czech). Pure culture preserved in a test tube was initially identified by the Biolog test (Biolog ML3420, USA) and by 16S-rDNA sequencing.
Chitosanase production
Chitosanase production was performed in 250 ml flasks containing 100 ml fermentation medium. According to 2% (v/v) seed inoculum, the broths were incubated at 30 ℃, 160 rpm for 30 h in a rotary shaking incubator. After fermentation, the inoculum pellets were separated from the broth by subjecting to a centrifuge at 10000 g for 20 min and the supernatant was treated as the crude chitosanase for enzyme activity measurements.
Detection of chitosanase activity
Chitosanase activity was determined as the rate at which reducing sugar molecules were generated. Enzyme activity was detected by the dinitrosalicylic acid (DNS) method according to a previous study (30) with some modifications. The enzyme activity was calculated by measuring optical density (OD) at a wavelength of 540nm, which was related to the content of reducing sugar in the solution using D-glucosamine as standard(29). In this experiment, an enzyme activity unit (U) is defined as the amount of enzyme required to produce 1 µmol of glucosamine per minute, 1 μmol/min. The enzyme activity unit per milliliter of fermentation broth (or per milligram of enzyme protein) is U/ml (or U/mg).
ARTP mutagenesis
The bacteria in the logarithmic phase were collected by centrifuging the fermentation broth and washed twice with sterilized saline solution. Then bacterial suspensions of about 2×105 CFU/ml were obtained through diluting them with sterilized saline solution. The slide of ARTP was coated evenly by 10 µL of suspension and was placed in a hole on the rotary table in the ARTP system, then the mutagenic procedure was executed. The operation parameters in the mutagenic procedure were as follows: irradiation distance was 2 mm; gas flow rate was 10 L min-1; mutagenic power was 100 W; the bacterial strains were treated with 0 s, 10 s, 20 s, 30 s, 40 s, 50 s, 60 s, and 70 s , respectively. Three parallel slides were treated under each mutagenic time. In this experiment, the mortality rate of the bacteria was calculated through a colony count and the mortality curve was plotted. The treated time with a lethality rate of 90% for mutagenesis operation was selected as the operation time of mutagenesis experiments.
Screening of mutagenic strains
According to the lethality curve, the optimal irradiation time was selected for the experiment, and the slide coated mutant was shaken with hand for 1 min in the 5mL fermentation medium. Then the mutated bacterial solution was inoculated in two 100-well microwell plates with 200 μL in each well. The microwell plates were placed in the bioscreen system (OY Growth Curves Biocsreen C, Finland) and cultured for 48 hours. The micropores with high increase in OD600 were selected for measuring the activity of chitosanase. Aand the hole with larger value of enzyme activity / OD600 was obtained.The remaining bacterial liquids in the micropores with high enzyme activity were evenly spread on the plate of seed medium and cultured for 24 hours, then some single colonies were selected and preserved according to the shape and size of colonies on the plate, and then the selected strains were tested for enzymatic activity verification again. The strains with high enzyme activity were obtained by culturing at 30 ℃ for 72 hours. The obtained strains were stored at 4 ℃.
Optimization of culture conditions
The optimal temperature of fermentation was evaluated by producing chitosanase under different temperatures, including 28℃, 30℃, 32℃, 35℃ and 37 ℃ with initial pH 6.0. The optimal initial pH of fermentation medium has been investigated by producing chitosanase in the presence of different pH at 5, 5.5, 6, 6.5, and 7, respectively, with 30℃. Chitosanase activities in fermentation broth were analyzed at the end of fermentation.