Materials
Raw material: The composition of wheat straw is shown in Table 1. After crushed, wheat straw was sieved with a 40-mesh sieve and stored in dry environment.
Table 1
Composition of raw stover
Raw material | Cellulose(%) | Hemicellulose(%) | Lignin(%) |
Wheat straw | 40.83± 0.43 | 31.47 ± 0.57 | 8.7± 0.48 |
Enzymes: Lac (10,000 U/g), glucanase (50,000 U/g), xylanase (180,000 U/g), acidic cellulase (10,000 U/g), cellulobiase and GOD are purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd.
Experimental
Obtainment of the LiP
LiP was produced by E.coli pET21-lip recombinant engineering strain, and the pure LiP was obtained. The E.coli pET21-lip recombinant engineering strain was constructed and preserved by our laboratory.
The effect of adding cellulase on the lignin degradation rate
Taking 5 g of wheat straw, 1 U LiP, 0.1% glucanase, 0.1% acid cellulase, 0.1% xylanase, and 0.1% cellobiase was added to the experimental group; Adding only 1 U LiP in the control group, the water content is 50%, stir evenly and place it in a constant temperature and humidity incubator with a humidity of 80%, and enzymatic hydrolysis at 37°C for 72 h. After enzymatic hydrolysis, the lignin content of the product was determined by the filter bag method,9 lignin degradation rate was used to evaluate the effect of cellulase on the degradation rate of lignin.
Effect of Lac, GOD content on the degradation rate of lignin
On the basis of the experimental group described above in part I, Lac, GOD was added with the different concentration of 0%, 0.1%, 0.5%, 1%, 2%, and 3%, respectively. Other enzymatic hydrolysis conditions is same as the section discribed above, determine the lignin concentration of the product, lignin degradation rate was used to evaluate the effect of Lac, GOD on the degradation rate of lignin.
Effect of Some organic acid on the lignin degradation rate
On the basis of the experimental group described above in part I, malonic acid, citric acid, acetic acid, glutamic acid, and malic acid in each straw was added with the different concentration of 0.1%, 0.5%, 1%, 2%, and 4%, respectively. The enzymatic hydrolysis conditions are same as the section discribed above, the lignin concentration of the product is measured, and the lignin degradation rate is used to evaluate the influence of the above factors on the enzymatic hydrolysis of lignin.
Effect of ZnSO4, Na2SO4, CaCl2, KCl on the lignin degradation rate
On the basis of the experimental group described above in part I, ZnSO4, Na2SO4, CaCl2 and KCl in each straw was added with the different concentration of 0.1%, 0.5%, 1%, 2%, and 4%, respectively. Other enzymatic hydrolysis conditions are same as the section discribed above, the lignin concentration of the product was determined, and the lignin degradation rate was used to evaluate the influence of the above factors on the enzymatic hydrolysis of lignin.
Compound experiment
On the basis of the experimental group described above in part I, select the factors which can promote the lignin degradation rate, and explore the effect of multi-factor compound on the degradation of lignin. The experimental design is as follows:
The first group: (1) 0.5% malonic acid; (2) 2% GOD; (3) 2% CaCl2; (4) 2% GOD + 0.5% malonic acid; (5) 2% GOD + 0.5% malonic acid + 2% CaCl2;
The second group: (1) 0.5% malonic acid; (6) 2% citric acid; (7) 2% ZnSO4; (8) 0.5% malonic acid + 2% citric acid; (9) 0.5% malonic acid + 2% citric acid + 2% ZnSO4;
The water content was controlled to 50%, the enzymatic hydrolysis was carried out at 37℃ for 72 h, and the lignin concentration of the products was determined by filter bag method to evaluate the influence of multiple factors on lignin degradation.
Box-Behnken Design (BBD) experiment
Based on the above experiments, three factors: CaCl2, malonic acid and Lac were selected to explore the optimal conditions of multi-factors synergistically promoting the degradation of lignin by LiP. Taking the lignin degradation rate as the response value, the experiment designed according to Design Expert is as follows, and the enzymatic hydrolysis conditions are the same as the compound experiment. After degradation, the lignin concentration of the product was determined.
Table 2
BBD experimental factors and codes
Variable | -1 | 0 | + 1 |
CaCl2 (%)(X1) | 1 | 2.5 | 4 |
Malonic acid (%)(X2) | 0.10 | 0.55 | 1.00 |