Construction And Characteristic Analysis Of Recombinant Chitosanase Surface Display Bacteria

Chitooligosaccharides（COSs）are partially hydrolysed and deacetylated products of chitin/chitosan with a low polymerisation degree（DP）.COSs are water soluble and exhibit various biological activities.Therefore,COSs have various potential applications in medicine,health foods,cosmetics,agriculture,and other areas.A variety of techniques have been developed for the production of COSs,including chemical,physical,and enzymatic methods.In terms of milder reaction conditions,non-pollution,strong specificity and concentrated molecular of products,enzymatic method has showed great potential in the production of COSs.However,traditional enzymatic methods have disadvantages such as complicated purification process,high-cost,and tough purification from a COS-chitosanase system.These made it difficult for the industrial application on COSs production.In this study,the fusion gene inaQ-N-csn46A that contained the N-terminal domain encoding gene（inaQ-N）of ice nucleation protein（INP）from Pseudomonas syringae and the chitosanase（named CSN46A）from Bacillus subtilis 168 strain was constructed.Then,the fusion gene was inserted into a temperature-inducible expression vector named p BLMVL2 and transformed into Escherichia coli BL21（DE3）to obtain a recombinant strain（MB101）.Finally,the recombinant strain（MB102）with two tandemly aligned InaQ-N as the anchor unit was further constructed.The induction conditions of the fusion proteins InaQ-N-CSN46A and2InaQ-N-CSN46A were optimized.The results showed the optimal culturing time of the two fusion proteins was OD₆₀₀=0.9.Then,the induction could begin.The optimized induction temperature was 41.5℃and induction time was 4 h.SDS-PAGE analysis showed that the molecular weights of the two recombinant fusion proteins（InaQ-N-CSN46A and2InaQ-N-CSN46A）were 46 k Da and 65 k Da,respectively.The determination results of growth curves of two recombinant strains indicated that the expression of two fusion proteins in the two systems had less effect on the growth of E.coli.Characteristics of surface displayed chitosanase was further studied.The optimal reaction temperature of InaQ-N-CSN46A and 2InaQ-N-CSN46A fusions was 50℃and reaction pH was 5.5.The chitosanase activity was relatively stable at reaction condition of37-42℃and pH 5-6.The Mg²⁺and Ca²⁺had an activation effect on recombinant chitosanases.The Co²⁺,Fe³⁺,Zn²⁺and Cu²⁺had a negative effect on recombinant chitosanases.The specific enzyme activity of 2InaQ-N-CSN46A reached 761.34±0.78 U/g cell dry weight,which was 45.6%higher than that of InaQ-N-CSN46A under an optimal reaction condition.Compared with the reusability of two surface display systems at different temperatures,according to the results,chitosanase activities in two systems retained more than 65%of their initial activities after 5 cycles of reuse at 37℃.Studies on hydrolytic properties of the displayed CSN46A showed that the final products were chitobiose and chitotriose without observable glucosamine at the optimal reaction temperature（50℃）.This tesult indicated that CSN46A was an endo-type chitosanase.When the reaction temperature was increased to 55℃,the degradation of high-molecular-weight chitosan decelerated.This effect resulted in the gradual accumulation of chitotriose.The results suggested that the surface-displayed protein2InaQ-N-CSN46A showed a higher enzymatic hydrolysis efficiency than InaQ-N-CSN46A.These results confirmed the findings that increasing the InaQ-N domain copy numbers exerts a synergistic effect.This approach could significantly improve the activity and stability of surface-displayed fusion proteins.Therefore,2InaQ-N-CSN46A was the superior choice for COS production.In this study,surface-displayed chitosanase could be expressed by increasing the induction temperature and hydrolysing the substrate chitosan directly without complicated enzyme purification steps.It showed high reaction efficiency and high controllability.It could be reused in batch reactions and provided a new platform for the industrial production of COSs via recombinant enzymes.