Screening Of Efficient Chitin Degrading Strain And Application Of Lytic Polysaccharide Monooxygenase

As the second-largest carbohydrate and nitrogenous organic compound,the degradation products of chitin,N-acetamidoglucose,chitooligosaccharides,and their derivatives have various biological activities and are widely used in many fields,which have important market value and research prospects.Biological enzymatic degradation of chitin is characterized by mild conditions,high product purity and,and environmental friendliness,which is the development direction of the modern tributyrin industry.However,there are problems of poor single enzyme degradation activity and low production efficiency,making industrialization difficult.Therefore,it is of great research importance to realize crystalline chitin’s efficient and low-cost enzymatic conversion into high value-added products.A strain of high chitinase producing bacteria was screened from the soil.Based on taxonomic identification,its fermentation enzyme-producing conditions were optimized.The isolation and purification of chitinase,and enzymatic properties were further investigated.Finally,a crystal chitin multi-enzyme degradation system was constructed by expressing lytic polysaccharide monooxygenase to realize the efficient degradation of powdered chitin.（1）Six strains of chitinase producing were obtained from the soil using the transparent circle method with colloidal chitin as the sole carbon source,and further re-screened to obtain chitinase high-producing strains.By morphology,analysis of 16S r DNA gene sequence,and other methods,the strain were identified to belong to the genus Chitinolyticbacter and named Chitiniphilus sp.LY72.（2）The best carbon and nitrogen sources were glucose and peptone from the results of single-factor experiments,followed by Plackett-Burman experiments to determine glucose,chitin,and peptone as the key factors affecting the synthesis of chitinase.Finally,using the steepest climbing experiment combined with response surface analysis,the optimal culture conditions were determined to be 8.24 g/L for glucose,0.1 g/L for chitin,and 6.02 g/L for peptone,at which the maximum enzyme activity was 2.176 U/m L,which was 43%higher than that before optimization.（3）The crude enzyme solution was purified by DEAE-cellulose anion exchange chromatography and Sephadex G-100 molecular sieve chromatography to obtain chitinase with a molecular weight of about 65 k Da.The recovery of chitinase activity reached 19.1%,the purification multiple was 7.67 times,and the specific enzyme activity of the pure enzyme was 5.286 U/mg protein.The optimum reaction temperature is 30℃;the optimum p H is 6.0;it is more stable below 40℃,and the chitinase activity disappears completely at60℃,and the viability still keeps more than 90%after nine weeks of storage.Chitinase is not a metalloenzyme,and Mg²⁺and Mn²⁺have an activating effect;the enzyme has strict substrate specificity and shows the most potent hydrolysis ability for colloidal chitin;the results of substrate analysis show that it has N-Acetylhexosaminidase activities.（4）Cs LPMO heterologous expression strains EBL-22,EBL-28,and EBL-Co were constructed by cloning the lytic polysaccharide monooxygenase（cslpmo）gene using three plasmids of p ET-22b,p ET-28a and p Cold.By analysis of soluble protein,the optimal expression plasmid was p ET-22b,and the expression conditions of EBL-22 were further optimized.After optimization,the soluble protein content of EBL-22 was 928.32 mg/L,and the enzyme activity was 3.6 U/mg protein,which were 32.1%and 45.2%higher than those before optimization,respectively.The isolated and purified Cs LPMO possessed chitin hydrolytic activity and had an excellent synergistic effect on chitin hydrolase.The addition of Cs LPMO could increase the yield of reducing sugars by 1.67 times.