Inducible Expression,Enzymology Properties And Its Applications Of Chitinase From Recombinant Escherichia Coli

Chitin,as the only abundance basic polysaccharide in nature,was called the sixth essential nutrient after the five elements which is protein,carbohydrate,fat,cellulose and minerals.Chitin oligosaccharide and N-acetamide glucose which are hydrolysis products of chitin,can be widely used in many fields such as pharmaceutical,chemical,food,environment,and their demand are increasing.In this article,firstly,chitinase gene in Lactococus lactis ssp.Lactis IL1403 was linked up with expression vector pETM-11 to construct E.coli BL21（DE3）/pETM-LlChi18A.Secondly,recombinant protein LlChi18A was expressed,isolated and purified.Thirdly,its enzymatic properties and hydrolysis product were reseached.Lastly,this enzyme was used to degrade the waste mycelium to chitin oligosaccharide.Specific research contents and the results obtained are as follows:（1）Induction conditions of Recombinant E.coli BL21（DE3）/pETM-LlChi18A,after optimization to get the best temperature is 25℃,induction time is 12 h and the final IPTG concentration is 0.05 mmol·L^-1.Under the optimized expression condition,the molecular mass of recombinant expressed product is about 54 KDa,and part of the soluble protein.（2）the study of recombinant protein LlChi18A enzyme activity showed that the optimum pH is 3.7 and the optimum temperature is 37℃.When the concentration of 10mmol·L^-1,chitinase LlChi18A activity was strong inhibited with metal ions Zn²⁺,Cu²⁺,Li⁺,Co²⁺and Mn²⁺.At the same time,the Ni²⁺concentration in 5 and 10 mmol·L^-1inhibited the chitinase activity of LlChi18A strongly.However,when 1 mmol/L Na⁺,Li⁺,Mn²⁺,EDTA and 5 mmol/L Ba²⁺,chitinase activity was increased.（3）The optimum temperature of chitinase LlChi18A in the colloidal chitin is 40℃;the optimal pH is 5;Km and Vmax were 2.913mg·mL^-11 and 2.836μmol·min^-1·mg^-1.The optimal temperature is 60℃ in the water-soluble chitosan;the optimal pH is 7;Km and Vmax were 4.04 mg·mL^-11 and 222.2μmol·min^-1·mg^-1.The degradation effect in the water-soluble chitosan is stronger,faster of the degradation rate,better of temperature stability with in the colloidal chitin.Ni²⁺,Mg²⁺,Ca²⁺,Zn²⁺in colloidal chitin have enhanced the chitinase activity.But there is no promoting effect in the water soluble chitosan,and Ni²⁺and Zn²⁺has inhibitory effect.Tween-80 in the colloidal chitin plays a role in promoting,and different concentrations of SDS has inhibitory effect of enzyme activity,and the higher the concentration of the stronger inhibition.（4）Analysis of the degradation products from the different substrates by TLC and HPLC show that the main product of the enzymatic degradation is chitin disaccharide.The enzymatic degradation product from the colloidal chitin is mainly for simple sugar and chitin disaccharide,and degradation products is relatively complicated from the water soluble chitosan,there are mainly for chitin and chitosan oligosaccharides.（5）By two enzymes（chitinase and snailase）chitin in the waste mycelium was directly degradated to chitin oligosaccharide,and the yield of production is 33.5%aftr four days.The chitin oligosaccharide production yield will increase with the increase of reaction time.