Isolation And Identification Of The Chitinaseproducing Strain And The Characterization Of Its Chitinase

Chitin widely exists in the biological world,and is the second most abundant natural polysaacharide in nature.Degradation products of chitin are widely used in biomedicine,agriculture,food preservation and other industries.Chitin is the main component of Tenebrio molitor L.molts,but a large number of Tenebrio molitor L.molts are discarded in nature,resulting in the waste of natural chitin resources.It is of great significance to screen the Chitinase-Producing Strain with high enzyme activity and to degrade chitin by chitinase for recycling the Tenebrio molitor L.molts.In this study,a Chitinase-Producing Strain named W3 was isolated from the soil of natural landfill of Tenebrio molitor L.molts.Then,the taxonomic status of strain W3,the optimization of enzyme production conditions and enzyme properties were studied in detail.The conclusions are as follows:(1)Three Chitinase-Producing strains were isolated from the soil of natural landfill of Tenebrio molitor L.molts by transparent circle method,and a strain W3 with higher chitinase activity was obtained by Sachales method.The strain W3 was identified as Paenibacillus sp.by 16 S r DNA sequence analysis.(2)The single factor experiments of culture time,liquid volume,medium pH,culture temperature,optimum carbon source and its addition amount were carried out.Box Behnken response surface method was used to further optimize the enzyme production conditions.The results showed that the enzyme activity of strain W3 was the highest,up to 0.182 U/ml,when the culture medium pH was 7.5,the culture temperature was 32 ℃,the carbon source was colloidal chitin,the addition amount was 8 g/L,the liquid volume was 50 ml/150 ml,and the culture time was 99 h.(3)The study of enzymatic properties of crude enzyme showed that the optimum reaction temperature and pH value of crude enzyme were 45 ℃ and 5,respectively;when the temperature was lower than 45 ℃and pH value was 3 ~ 9,the enzyme activity was relatively stable;10 mmol/L Fe3+ inhibited the enzymatic reaction,and 10 mmol/L Cu2+ promoted the enzymatic reaction;the kinetic parameters of enzyme were Km = 2 mg/m L,Vmax = 769.23 μg/m L·h;The crude enzyme also had a certain degradation effect on powder chitin and Tenebrio molitor L.molts;the product of crude enzyme degrading colloidal chitin for 60 min was identified as NAG by TLC analysis.