Isolation And Screening Of Bacillus Thuringiensis From South China Sea And Preliminary Studies On Chitinase-producing Conditions And Identification Of A Chitin-degrading Strain

Bacillus thuringiensis(Bt) is one of the most widely used insecticidal microbe. Insecticidal crystal proteins(delta-endotoxins) from Bt,which are toxic to pests from many insect orders,are great biocontrol resources against natural pests.As an important kind of insecticidal active substances,chitinase has many valuable biological funtions.Both of them are very valuable in many areas,such as biological control,environmental protection and so on.Therefore,to study on the screening,identification,molecular biology and chitinase production of Bt is of great significance to discover new strains.In this study,a method(NaAc with heating) was developed to isoloate Bt from marine water and sediment samples collected from Luzon,Vietnam upwelling regions in South China Sea.22 Bt strains were obtained from the samples.Under the observation with SEM,it was found that mostly Bt strains had ball-shaped parasporal crystal except that two strains had lozenge-shaped crystal.With a method of Transparent Circle,a comparatively higher chitinase activity Bt strain called B-19 was screened in the medium which chitin is the only carbon source with inorganic salt.In the research,the characters and conditions(eg.tempertature,carbon source,nitrogen source,pH )of B-19 producing chitinase were studied.With a series of research,the optimal chitinase -producing contitions were as the following:cultured in the medium containing 2%colloid chitin and 1.0%yeast extract powder for 36 hours(160r/min).The optimal pH and temperature was 7.0 and 30℃,respectively.Mg²⁺,Ca²⁺,Li⁺ and Ni²⁺ evidently increased its chitinase activity while K⁺ and Zn⁺ decreased its activity..Under the above optimal fermentation conditions,the activity of chitinase from B-19 strain could be enhanced by about 200%.The chitinase from B-19 strain was induced by chitin,while inhibited by NAG and glucose.These elementary results would establish the base for the future research and exploitations of chitinase produced by Bt from marine environment.Meanwhile,a chitin-degrading strain B-Y20 was also screened from the sediment samples.Through the physiological & biochemical test and 16S rDNA molecular identification,B-Y20 belonged to Bacillus licheniformis.This research would advance the utilization and exploitation of chitin-degrading marine microorganism especially from deep sea in the future.,and also provide new strain resource.