Characterization,Molecular Modification And Efficient Secretory Expression Of Thermostable Type ? Pullulanase

Pullulanase?EC 3.2.1.41?is a commonly used starch debranching enzyme that selectively hydrolyzes?-1,6 glycosidic bonds in pullulan,amylopectin and limit dextrin.It is a key rate-limiting enzyme for branch hydrolysis and belongs to the?-amylase family.In starch processing,pullulanase is usually used in combination with other amylases such as saccharifying enzyme and?-amylase to increase the rate of starch saccharification and obtain high-purity glucose,maltose and other small molecule syrup.At present,there are two main problems in the research of pullulanase:First,the catalytic efficiency and thermostability of most wild-type pullulanase are low,and it is difficult to perform efficient hydrolysis and resist high temperature during starch processing;Second,the pullulanase production and secretion efficiency of wild-type strains are generally low,which is difficult to meet the large demand for pullulanase in industrial application.In the present study,in order to solve the problems mentioned above,we cloned and expressed the pullulanase Pul_PY derived from a deep-sea hyperthermophilic archaea Pyrococcus yayanosii CH1and improved its catalytic efficiency and thermostability by domain truncation.Through the three-dimensional structure analysis of enzyme molecule and semi-rational design strategy,we improved the catalytic efficiency and thermostability of pullulanase Pul _A derived from Anoxybacillus sp.WB42,and an efficient expression system for the pullulanase production was established in Bacillus subtilis.The main results are listed as follows:?1?Heterologous expression and characterization of pullulanase Pul_PY:Pul _PY was successfully expressed with the assistance of the five protein fusion tags?flag,strep,gst,sumo,and mbp-tag?in E.coli.Sumo-tag Pul_PYY possessed higher pullulanase activity and production than the others.The Pul_PY was purified and characterized.The optimal temperature of Pul_PY was 95?,and it retained50%activity after incubation at 100?for 4 h.The optimal pH of Pul_PY was 6.6,and it retained more than 80%activity after incubation at pH 5.8-8.0 for 4 h.Kinetic analysis showed that the specific activity,K_m,k_cat and k_cat/K_m of Pul_PY were 5.37 U·mg^-1,8.78 mg·mL^-1,12.33 s^-1 and 1.43 mL·mg^-1·s^-11 respectively.Fe²⁺,Co²⁺,DTT and?-mercaptoethanol significantly increased the specific activity of Pul_PY.When adding DTT?1%v/v?,the specific activity of Pul_PY was approximately 3.4-fold higher than the control.Substrate specificity and hydrolysate analysis showed that Pul _PY was a Type ? pullulanase.?2?Construction of the catalytic efficiency and thermostability of Pul_PY:To improve the catalytic efficiency and thermostability of Pul_PY,three truncation mutants?28N,?791C and?28N+?791C were generated based on the NCBI protein conserved domain analysis tool and characterized in detail.The results showed that the optimal temperature of the combined truncated mutant?28N+?791C was 100?,which was 5?higher than that of Pul_PY.After incubation at 100°C,the residual activity of?28N+?791C was higher than that of Pul_PY.The optimum pH of?28N+?791C was 6.4,shifting toward the acidic pH range.After incubation at pH 5.8-8.0 for 4 h,the remaining activity of?28N+?791C was equivalent to that of Pul_PY.Kinetic analysis showed that the specific activity,K_m,k_cat and k_cat/K_m of?28N+?791C were 32.18 U·mg^-1,6.63 mg·mL^-1,54.16 s^-1 and 8.25 mL·mg^-1·s^-1respectively.The substrate bindin capacity and catalytic efficiency of?28N+?791C were 1.3 and5.8 times higher than that of Pul_PY.Circular dichroism?CD?spectrum analysis was used to compare the secondary structures of Pul_PY and?28N+?791C.The result showed that the content of disordered structure in?28N+?791C was reduced by 6.4%and the number of?-helix was increased by 6.2%compared with Pul_PY.The structure of?28N+?791C was more compact than that of Pul_PY.The decrease of the disordered structure content in?28N+?791C was an important reason for its significant improvement in catalytic efficiency and thermostability.?3?Construction of the catalytic efficiency and thermostability of Pul_A:The molecular structure of Pul_A was modeled and analyzed.Based on the strategy of surficial residue replacement and disulfide bond introduction,a small and precise mutation library containing 26 mutants was constructed,and one with improved catalytic efficiency and thermostability(Pul_AC)was successfully selected and characterized.The optimal temperature of Pul_AC was 65?,and it retained more than85%activity after incubation at 67°C for 30 min,which is much higher than the activity maintained by wild-type Pul_A?50%?.The optimal pH of Pul_AC was 6.2,shifting toward acidic conditions compared with the wild type.After incubation at pH 6.6-7.8 for 4 h,Pul_AC retained more than 90%activity,much higher than that of the wild type?60%?.The Tm value of Pul_AC was 80.2°C,which was 2.1°C higher than that of Pul_A.Kinetic analysis showed that the specific activity,K_m,k_cat and k_cat/K_m of Pul_AC were 98.2 U·mg^-1,11.09 mg·mL^-1,134.7 s^-1 and 12.22 mL·mg^-1·s^-11 respectively.The catalytic efficiency of Pul_AC was 1.5 times higher than that of Pul_A.Through comparing the three-dimensional structure differences between Pul_AC and Pul_A,we found that a hydrogen bond was possibly formed between Arg419 and Asp416,and two disulfide bonds were possibly introduced between Cys245 and Cys326,and Cys651 and Cys707 in Pul_AC,which facilitated the transport of the substrate and binding to the??/??₈-barrel catalytic domain and was responsible for the significant improvement in catalytic efficiency and thermostability of Pul_AC.?4?Construction of an efficient expression system of pullulanase:Anoxybacillus sp.WB42mutant pullulanase pul_AC with good thermostability and high activity as a reporter gene,via optimization of the plasmid backbones,promoters and ribosome-binding-site?RBS?sequences,we constructed an efficient expression system of pullulanase in Bacillus subtilis.The recombinant strain B.subtilis WB800/RBS7 produced extracellular pullulanase activity to 154.9 U/mL in a 250 mL shake flask,which was 136.8 times higher than that of the wild type.When the strain was cultured in a 5-L bioreactor,the pullulanase activity was significantly improved to 269.1 U/mL,which was 1.7times higher than that of the shake-flask cultivation.We achieved the highest pullulanase production of Type ? pullulanase to the best of our knowledge.