Studies On The Directed Evolution And Thermostability Of β-glucanase

p-glucanase is an important industrial enzyme and is often added to reduce negative effect of P-glucan in brewing and feedstuff industries, but the study on the β-glucanase began late and the thermostability of p-glucanase was poor when compared with foreign studies.The poor thermostability of p-glucanase does not satisfy the temperature requirement of malting and feedstuff preparation, so it is significant to improve the thermostability of β-glucanase.This project is aimed at the following aspects: Molecular cloning and expression of p-glucanase gene in E.coil;The development of high-throughput screening method for thermostable P-glucanase;Enhancing the thermostability of p-glucanase by directed evolution and screening with the developed method;Checking the genetic stability of the thermostable mutants;Optimizing the fermentation conditions of mutant EGs2;Screening and optimizing the complex protective agent for p-glucanase;Evaluating the storage period of P-glucanase by accelerated storage test. The main results are shown as following:The endo-p-glucanase gene from Bacillus subtilis ZJF-1A5 was cloned. The obtained 849bp DNA fragment was inserted into the vector pET28a (+). The sequence homology analysis showed that the deduced amino acids in open-reading frame had 1-3 amino acids substitutions compared with other amino acids sequence of p-glucanase determined by Borriss R, Sun, J, Hidetoshi, respectively. When the strain was induced by IPTG, it produced 27KD protein and accumulated the enzyme in the extracellular, periplasmic and intracellular space. The enzyme was salted out from the broth using (NH₄)SO₄ and purified with DEAE Cellulose 52.0ne clear band was shown on the SDS-PAGE and the molecular weight was about 27KD.The enzyme properties were measured,the results indicated that the melting temperature (T_m)of the enzyme is 62.5℃;the optimal temperature (T_opt) of enzyme activity is 55℃.The directed-site membrane transferring and visual screening method based on plate were used, and the factors, which affect bacterium growth, enzyme expression and the success rate of membrane transferring, were optimized to develop the high through-put screening method for thermo stable p-glucanase. The results showed: 4μl200 mg/ml IPTG per plate (90mm) was used;the IPTG should be plated previously;the colony density was about 100 per plate;the colonies were incubated for 15 hours at 37°C.the condition for inactivating the wild-type enzyme was 100℃ for 2 hours. The reliability of the method was measured. The results showed that the recurrence rate is 100%;the false positive rate is 0.16 %;the false negative rate is 12.22 %.Comparing with other methods, the developed method has many advantages, such as the clearer transparent halos, the lower false positive rate, the higher through-put of screening, the lower cost and the simpler operation. In a word, the developed method is effective to screen the thermo stable variants as a solid based enzyme screening method.In order to improve the thermo stability of p-glucanase, directed evolution was used.The evolution strategy involves one cycle of random mutation and one cycle of DNA shuffling followed by screeningon the filter-based high through-put screening method. Through mutation and screening, the two mutants, i.e., EGsl and EGs2,had four and five amino acid substitutions, respectively. These substitutions resulted in an increase in melting temperature from Tm= 62.5 °C for the wild-type enzyme to Tm=65.5 °C for the mutant EGsl and 67.5°C for the mutant EGs2.The optimum temperature of the two mutants were also increased by 5 °C .However, the two mutant enzymes had opposite approaches to produce reducing sugar from lichenin with either much higher (28%) for the former or much lower (21.6%) for the latter in comparison with their parental enzyme. The affinities of the two mutants for the lichenin were basically unchanged. The optimum pH were 6.5 for the EGsl and wild-type, and 7.0 for EGs2.The pH stability for all enzymes was good, the activity of parental enzyme and the two mutant enzymes have no loss at pH 6.0-8.0 and pH 6.5-8.0 for 48 hours, respectively. The results demonstrate that directed evolution is an effective approach to improve the thermo stability of a mesophilic enzyme.The measurement of genetic stability for EGsl and EGs2 indicated that the plasmid stability of the two mutants were good and their expression ability of 6-glucanase kept stable after 50 generations, so the genetic stability of EGsl and EGs2 are very satisfactory.The fermentation conditions for EGs2 mutant were optimized by flask shake. The optimum growing conditions for EGs2 mutant were the following: medium volume 40ml, inoculation volume 1.5%, and initial pH value 6.5-7.0. The bacterium began the stable phase after cultured for 6 hours. The inducing conditions for the bacterium growth and p-glucanase expression were optimized when the IPTG and the lactose were used as inducer, respectively. The results show that both IPTG and lactose can be used as the inducer for the P-glucanase expression;the inducing effect of lactose is little better than that of IPTG;IPTG concentration has little effect on the bacterium growth and the product expression, and the lactose concentration has great effect on the bacterium growth and the product expression;the temperature has great effect on the accumulation of the expressed product in the broth, but has little effect on the bacterium growth;the proper inducing conditions are inducing time 4 hours, inducer concentration O.lmmol/L, inducing temperature 34°C for IPTG, and inducing time 4 hours, inducer concentration lOmmol/L, inducing temperature 34°C for lactose, respectively.The protective agents of P-glucanase were screened through single factor experiments;the orthogonal experiment was used to optimize the best combination of different protective agent. The results indicated that the albumin, glutin and CaCl2-2H2O could enhance the enzyme thermostability;the complex protective agent consisted of 0.8 % albumin, 2% glutin and 1 mmol/L CaCl2.2H2O. The Tm of enzyme was improved by 4°C when P-glucanase contained the complex protective agent. The thermal degradation rate was -0.0398 for enzyme with protective agent and -0.084 for enzyme with no protective agent at 36.2°C.The results indicated that the complex protective agent could effectively enhance the thermo stability and storage stability of p-glucanase.The accelerated storage test was carried out to estimate the shelf life of P-glucanase at different temperatures. The samples were kept in water bath at 28 °C, 36.2 *C, 45 °C, and their activities were determined at different time. The results of statistical treatment of data showed that the activity of p-glucanase decreased by 10% after 1.36d,4.37d,20.09d, 30.7d,67.0d,145.9d,2.21y,4.14y and decreasedby 50% after 8.5d,25.47d, 104.9d, 154.0 d, 316.0 d, 648 d, 3154 d, when they were kept at 45°C, 36,2"C, 28°C, 25°C,20°C,15°C, 4°C and 0°C, respectively. The results indicated that the stability of p-glucanase was competent for the storage requirement, and the accelerated storage test could estimate the shelf life of P-glucanase in short period.