Research Of Thermostable ?-carrageenase Produced By Bacillus Sp. Car19 From Indonesia Hot Spring

In the early days of the earth,it was an extreme environment with high temperature and less oxygen which was similar to the hot spring.After the earth formed,the primitive and abundant life was microorganism.Thus,many scientists recognize that study on the microorganism of the hot spring may be helpful to not only reveal the origin of life on earth but also develop more thermostable enzymes which could be applied to industrial production and science study.The DNA polymerase used in PCR was the most successful example of thermostable enzymes in the application of industrial production and science research.In this study,we screened strains with carrageenanase activity that isolated from Indonesia hot spring and studied the characterization of the carrageenase.The results will provide scientific basis for preparation of carrageenan oligosaccharide with carrageenase and application of carrageenan oligosaccharide.In this paper we isolated 16 strains with carrageenase activity from Indonesia hot spring water samples by using carrageenan as the sole carbon source.The results showed that 11 strains were belonged to the genus of Pseudoalteromonas,2 strains belonged to the genus of Bacillus,the remaining three strains belonged to the genus of Colwellia,Shewanella,and Marinomonas respectively.The strain of Bacillus sp.Car19 with highest carrageenase activity and was selected for fermentation conditions optimization.The resluts indicated 9 facts affect Bacillus sp.Car19 carrageenase production and the three main facts were culture temperature,Cu²⁺ concentration and NaCl concentration.Combined with subordinattion factors the final optimized fermentation condition for the maximal activity discriminated from the regression equation were: culture temperature 52 ?,Cu2 + concentration 6.93 mmol/L,NaCl concentration 37.03 g/L,medium pH 6,1% of inoculation quantity,36 h for incubation time,galactose concentration 0.3g/L,ammonium nitrate concentration 7 g/L,carrageenan concentration 0.5 g/L.The supernatant carrageenanse activity of optimised fermentation was up to 15.82 U/m L,which was 1.5 times before fermentation.The enzyme was separated and purified in order to study the characterization of carrageenase secreted by Bacillus sp.Car19.The result showed that the molecular of carrageenase between 40-50 KDa.The enzyme exhibited maximum activity at pH 7.0 and 60 °C,and exhibited higher thermal stability than the reported carrrageenase.The activity was inhibited by Sr²⁺,Fe²⁺,Cd²⁺ and Fe³⁺,especially Sr²⁺,almost 50% activities depression.But the activity was stimulated by metal ions Cu²⁺,Ca²⁺ and Mg²⁺,especially Cu²⁺,improvement for 80% activities.The substrate specifity of the enzyme showed that it could degrade ?-carrageenan and showed no activity toward ?-carrageenan,?-carrageenan,agar,sodium alginate.Thin layer chromatographic analysis indicated that the production of carrageenase degrade ?-carrageenan to generate disaccharide as final products.we perpared carrageenase oligosaccharides and used it to it to tested on their effects on plant improvement germination and against disease.The result showed that 2% solution of carrageenan oligosaccharides diluted to 100 and 1000 times could promote wheat seed growth 29.48% and 27.17% respectively.The prevent efficiency of the 2% solution of carrageenan oligosaccharides diluted to 100 and 200 times prevent cucumber virus disease,cucumber powdery mildew could reach to 55.27%,45.36% and 48.99%,37.82% respectively.It will be our next work that the gene colne and expression of ?-carrageenase from Bacillus sp.Car19 and the physiological activity mechanism of the carrageenan oligosaccharides.