Escherichia coli DH5α, BL21(DE3) were purchased from Takara (Dalian, China) and plasmid pET-28a was purchased from Novegen (Darmstadt, Germany). Chitosan (degree of deacetylation over 80%) and D(+)-glucosamine, hydrochloride were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Restriction enzymes BamH I and Hind III were purchased from Takara (Nanjing, China).The High Purity Plasmid Small Preparation Kit was purchased from Beijing Bioteke Corporation Co., Ltd, (Beijing, China).The Site-directed Mutagenesis Kit, the Modified Bradford Protein Assay Kit, Ni-NTA Sefinose(TM) Resin 6FF (Settled Resin) and Desalting Gravity Column were purchased from Sangong Biotech (Shanghai) Co., Ltd. (Shanghai, China). The chitosanase gene was from Bacillus amyloliquefaciens (GenBank: QEK97559.1). After optimizing the codon of the gene sequence (BaCsn46A), it was synthesized by Nanjing GenScript Biotechnology Co., Ltd. (Nanjing, China).
Three-dimensional model construction and homologous sequence comparison of chitosanase
The protein sequence of Bacillus amyloliquefaciens (BaCsn46A) was submitted to Swiss-Model (https://swissmodel.expasy.org/) online server to predict its three-dimensional structure, and the chitosanase molecular model from Bacillus subtilis MY002 was used as the template (PDB ID : 7C6C. 1. A). The three-dimensional model was analyzed on PyMOL 2.4.1 (https://pymol.org/2/). The amino acid sequence was compared and analyzed by DNAMAN 9.0. The molecular docking was performed between chitosanase and substrate chitotetraose by autodock 4.2 (http://autodock.scripps.edu/).
Design and synthesis of primers
The primers used for saturated site-directed mutagenesis of Ser196 in BaCsn46A were designed and sent to Sangong Biotech (Shanghai) Co., Ltd. (Shanghai, China) for synthesis. The designed primer sequences were shown in Table 1.
Construction of saturated mutation library
The saturated mutation library was constructed by whole plasmid PCR. The PCR system includes Pfu DNA polymerase and dNTP. This process takes plasmid pET-28a as template and the sequences in Table 1 as primers respectively. After PCR, Endonuclease Dpn I was added to the product and placed in a 37 °C water bath for 1 h to remove the methylated plasmid. The target plasmid in the PCR product was combined with competent cells made of E. coli DH5α and transformed. The above conversion solution which was added to 1 mL Luria-Bertani (LB) solution was incubated at 37 °C and 160 rpm for 1 h. After the bacterial solution was concentrated, it was evenly distributed on the LB solid medium plate containing kanamycin and incubated overnight at 37 °C and 160 rpm. Transformants were selected from the plate and cultured in LB liquid medium. The plasmids were extracted from the bacterial solution according to the kit, and the nucleic acid gel was run to test whether the PCR was successful. Finally, the bacterial solution was sent to Sangong Biotech (Shanghai) Co., Ltd. (Shanghai, China) for sequencing.
Expression and purification of recombinant enzymes
The sequenced recombinant enzymes were reincubated in LB liquid medium at 37 °C and 160 rpm for about 12 hours. Thereafter, 1 % of the above bacterial solution was transferred to a unused LB liquid medium and continued to incubate for 4 hours under the same conditions, and then isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to induce protein expression. After being incubated overnight at 16 °C and 160 rpm, the bacterial solution was centrifuged by high speed freezing centrifuge, and the cells were resuspended with buffer M0 (20 mM Tris-HCl solution, 500 mM NaCl solution, 10 % glycerol, pH 8.0, constant volume to 1L). Then, the resuspended solution was ultrasonically broken on ice, centrifuged, and the supernatant was taken out and stored at -20 °C. Ni-NTA column can adsorb proteins with histidine tags and imidazole solutions with different concentration gradients (on the basis of buffer M0, 20-300 mM imidazole solution was added respectively) can be used for elution. Ultimately, the purified chitosanase solution can be stored at -20 °C and used for subsequent determination.
SDS–polyacrylamide gel electrophoresis (SDS–PAGE)
One volume of protein sample was mixed with 4 times volume of SDS-PAGE loading buffer (2.5 mL 1 M Tris-HCl (pH 6.8), 1 g SDS, 50 mg bromophenol blue, 5 mL glycerol, 0.5 mL β-mercaptoethanol，10 ml in total). The mixture solution was boiled for 5 min and was added to the 12 % SDS-PAGE gel. The electrophoresis apparatus ran at 80V first, and then changed to run at 120V until the end. After electrophoresis, the gel was dyed and decolorizing. Finally, a picture containing protein bands was obtained on electrophoresis gel imaging system.
Determination of chitosanase activity and protein content
Chitosanase activity was measured by using 3,5-dinitrosalicylic acid (DNS) method with slight modification. The total reaction system of chitosan enzyme was 2 mL, including 50 mM phosphate buffer (pH 6.6), 1 % colloidal chitosan (chitosan is dissolved in dilute acid, w/v) and an appropriate amount of purified chitosanase solution, which is properly diluted. After being mixed completely, the mixture solution was incubated at 50 °C for 10 min. The reaction was stopped by adding 1.5 mL DNS to the above mixture solution and then the mixture solution was incubated in boiling water for 5 min to develop color. Next, the boiled solution was diluted to 25 mL and centrifuged. Finally, the absorbance of the supernatant was measured at 520 nm with those without enzyme as the control. One enzyme activity unit (U) is defined that the volume of enzyme required for producing 1 μmol sugar of glucosamine hydrochloride per minute under the experimental conditions (50 °C, reaction for 10 min). The protein content was determined according to the method in the Modified Bradford Protein Assay Kit.
Biochemical characterization of purified chitosanase
The chitosanase activity was determined according to the above method. In order to determine the optimum reaction pH value, the purified chitosanase was measured at 50 °C in different buffer of pH 3-7.2 (citric acid-citrate sodium buffer pH 3-6 and phosphate buffer pH 6-7.2). Under the optimum reaction pH condition, the effect of reaction temperature was studied by measuring the activity of chitosanase at different temperatures ranging from 35 °C to 70 °C. After knowing the optimum reaction pH and temperature, the specific activity of chitosanase can be calculated. The thermostability and pH stability of chitosanase were determined by measuring its activity after being maintained at 60 °C for 90 minutes and on ice at the optimum pH for 8 hours. The enzymatic properties of wild-type (WT, Ser196) and mutants were compared, and then better mutants were selected.
Kinetic parameters assay
Under the optimum reaction conditions, 1 % colloidal chitosan (w/v) was diluted into 1 g/L, 2 g/L, 4 g/L, 6 g/L and 8 g/L, different concentrations of colloidal chitosan were used as substrates for the enzymatic hydrolysis reactions. To ensure the similar hydrolysis degree of the substrate, the reaction time was shortened to 5 min. According to the calculated data, the values of maximum enzymatic reaction rate (Vmax) and Michaelis constant (Km) were obtained. The affinity of substrate to chitosanase can be judged from the value of Km and the catalytic efficiency of chitosanase is determined by the value of Kcat/Km.
Determination of chitosanase by circular dichroism (CD)
The imidazole in the purified enzyme solution was removed by Desalting Gravity Column. The treated enzyme solutions were sent to Jiangnan University for circular dichroism detection. The circular dichroism results of WT and mutant chitosanases were determined on Chirascan spectropolarimeter (Bio-Logic MOS405, France).