The Research Of Accessing Thermostable Xylanase By Structure Displacement In Glucanase

Xylanase has broad application prospects in paper making,food and feed.But the thermostability of natural xylanase is bad,and it is the main limitation to its application,so how to improve the thermostability of xylanase is a hotspot of current research.GH12 glucanase Cel12B(17)from Thermotoga maritima is a thermostable enzyme with high Topt(100?),and it can also hydrolyze xylan.Actually,17 is similar to GH11 xylanase on 3D structure.But 17 has no conservative "Thumb" structure of GH11 enzyme,meanwhile has a large random coil which may generate the difference of substrate specificity between GH11 and GH12.Xylanase(23)from Aspergillus niger has been taken early,which belongs to GH11.In order to access thermostable xylanase,Thumb structure from 23 and the large coil of 17 was considered to be introduced into17 and removed from17,respectively.This paper studies and get the following conclusion:(1)Improve the expression quantity of protein by codon optimization.The expression quantity of 17 in Escherichia coli BL21(DE3)was low,which may be caused by secondary structure m RNA of translation starting position.Therefore,the mutants in translation starting position were designed.The expression quantity of optimized enzyme 17 OPT increased by 6.37 times,however the activity of fermentation broth only increased by 4 times,compared to 17.The gray-scale of protein electrophoresis showed that a large amount of enzyme form inclusion body protein after optimizing codon.(2)High density auto-inducing medium increased enzyme expressing.The expression quantity by conventional methods of LB+IPTG was low.This research used auto-inducing medium ZYM-5052 to express protein at low temperature.Ultimately,the new method increased the bacteria density by 2.21 times,and activity by 7.33 times.The soluble protein increased from 50.85% to 50.85%.(3)This study built three mutant enzymes: the structure introduction of Thumb(17OPT-Thu),removal of the main structure(17OPT-R)and boh of above(17OPT-Thu-R).But 17OPT-R and 17OPT-Thu-R form inclusion body totally.The impact of Thumb introduced into 17.The enzymes were characterized byCMC(Carboxymethyl cellulose-sodium)and BWX(Beech wood xylan)(1)17OPT and17OPT-Thu had the same p Hopt and Topt.p Hopt(CMC)was 4.6,p Hopt(BWX)was 5.8,Topt was 100?.These results showed that Thumb structure did not change the optimum reaction conditions.Thermal stability test showed that the activity of17OPT-Thu was still above 95% after incubating 3h at 100?,remaining the thermostable characteristic of the wild-type enzyme.(2)Pure enzyme kinetics parameters determination: after introducing the structure of Thumb,Km(CMC)increased from 5.6 mg/ml to 7.1 mg/ml,Km(BWX)increased from 1.3 mg/ml and 2.2mg/ml,Kcat(CMC)falled to 174.3s-1 from 494.3 s-1,Kcat(BWX)falled to 2.8s-1 from4.3s-1.The introduction of the Thumb structure did not achieve the desired effect,and the affinity of enzyme to two kinds of substrate and enzyme catalytic ability of enzyme were both declined.(3)HPLC product testing experimental results showed that to CMC the product did not change,but to BWX 17OPT-Thu products more xylobiose,xylotriose and xylotetraose which revealed that Thumb structure can work to the substrate.(4)Finally,the molecular docking analysis between enzyme and xylotetraose and cellotetrose finded that the introduction of the Thumb structure changed the enzyme and substrate interactions,intuitively showed that the Thumb structure involves in the enzyme catalytic process on the substrate.For cellotetrose subsrate,the introduction of Thumb did not favor the enzyme catalytic activity.The amino acids that form hydrophobic interaction did not have big changes berore and after introducing Thumb,for xylotetraose substrate.But Glu131,Met142,Tyr187,Glu235,Glu239 in17OPT-Thu form five hydrogen bonds with xylotetraose,enhanced the combination between the enzyme and substrate.These diferences explained the resaults of Enzyme kinetics and HPLC experiment.