Purification, Enzyme Characterization And Hydrolyzing Characterization Of A Thermostable Carrageenase From A Hot Spring Bacterium

This paper aims at screening bacteria strains producing carrageenase with highactivity from hot spring bacterium of Indonesia, optimizing the culture conditions,purifying the enzyme, studying the enzymatic properties, and analyzing thecarrageenan oligosaccharide after degradation by the enzyme. This work effectivelyprovides theoretical and technological support for commercial production ofcarrageenan oligosaccharide and its enzymes.Firstly,94strains of hot spring bacteria were obtained by plate streaking and14ofthem possess the activity of carrageenan degradation. The14strains were re-screenedby plate streaking in combination with Lugol iodine staining. And it was found thatLc50-1exhibited the highest activity of degradation carrageenan, which was3.56U/mL. The strain Lc50-1was identified as Bacillus by morphological observation and16S rDNA sequence analysis.To study the optimal culture medium and fermentation conditions of thermophilicbacterium Bacillus sp. Lc50-1, the response surface methodology was employed. Theranges of factors (temperature, pH, carbon sources, nitrogen sources, metal ions andinoculum) were determined by single test. Then it was identified by initialexperimental design of Plackett-Burman by Design-Expert software that fourimportant factors influencing enzyme activity were temperature, carbon sources,nitrogen sources, and metal ions. Box-Behnken design and response surface analysiswere adopted to further investigate the mutual interaction between the variables andobtain maximum enzyme activity. The optimal fermentation conditions of Bacillus sp.Lc50-1were listed as below:200mL medium in500mL triangular flask, shakingspeed of150r/min, inoculum volume7%, pH7.0and temperature47.5℃. The optimalmedium components were observed as below: peptone0.25%, carrageenan0.3%, KCl21.55mmol/L, NaCl0.3%. The maximum enzyme activity of carrageenase was8.9U/mL. Compared with the activity of carrageenase cultured in fermentationmedium, it increased1.5times under the optimal conditions.The carrageenase of Bacillus sp. Lc50-1was extracted by (NH4)2SO4precipitation and purified by ion-exchange chromatography and Sephacryl S-200HRgel filtration chromatography. The purified carrageenase appeared as a single band onsodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). It wasestimated by SDS-PAGE estimation that the molecular mass was around38kDa.The optimum pH for the carrageenase was9.0. The carrageenase prefers to workin alkaline environment. The optimum temperature for the carrageenase was75℃,and more than20%of the carrageenase activity was retained after incubation at75℃for30min. The activity of carrageenase was lost by the addition of FeCl3, MgCl2, andwas inhibited by the addition of EDTA, SrCl2, MnCl2, KCl, CdCl2. The activity ofcarrageenase was increased by the addition of CoCl2, CaCl2, CuCl2, NaCl, andcarrageenase activities were increased by about1.7-fold and2.2-fold by the addition ofCaCl2, CuCl2, respectively. Analysis of the substrate specificity showed that theenzyme had high activity on λ-carrageenan but no activity on ι-carrageenans,κ-carrageenans, agarose and algin.The hydrolysis characteristics of carrageenase were studied by thin layerchromatography (TLC). It turned out that degradation product was mainly a new typeof neo-λ-carradiaose which was different from those of carrageenases from otherbacterial strains.