| Carrageenans are gel-forming linear sulfated galactans extracted from the extracellular matrix of red marine algae. They consist of linear chains of galactopyranose residues linked by alternating a-1.3and β-1,4linkages. Based on the number and position of sulfate substitutions, carrageenans are mainly classified into three types:κ-, ι-, and γ-carrageenans. Sulfated oligosaccharides from marine algae have diverse biological and physiological activities, including anticoagulation, anti-inflammation, anti-thrombosis, anti-tumor activity, and viral inactivation, which depend on structural parameters such as carbohydrate structure, molecular mass, degree of sulfate esterification, and the linking position of sulfo groups. Therefore, degraded carrageenans have drawn considerable interest.As a polysaccharide hydrolase,carrageenase degrades carrageenan to mainly even carrageenan oligosaccharides by β-1,4glycosidic bond.Most of reported carrageenase belongs to glycohydrolase family. The structure, function, substrate specificity studies of different types of carrageenase have important theoretical and practical value. Further, the carrageenase can also be used in the research of the relationship between sulfate groups structure and function, and also has its significance in carrageenan oligosaccharides’industrial applications and bacterial metabolism of carrageenan. And for the mild conditions and substrate specificity of the enzymatic degradation, carrageenase can solve the shortcomings of difficult to control, oligosaccharides heterogeneity in the traditional chemical and physical degradation.Our laboratory separated eight carrageenan and agar degrading bacteria from the coastal waters of Dongshan Island, aquaculture seawater and red algae. According to the size of the clear zone on the plate and enzyme activity of fermentation broth, No.KL-A strain was found to have good specificity degradation ι-carrageenan capacity. After the16rRNA sequencing of the strain KL-A, it be attributed to Cellulophaga sp. and named Cellulophaga sp.KL-A. In order to evaluate the potential application of strains, response surface medium(RSM) optimization was used to improve the carrageenase production of the strains.By the early single factor experiments and the fermentation medium components of related marine bacteria, we used RSM to find the most suitable components of the fermentation medium for enzyme production, ι-carrageenan, glucose and CaCl2concentrations were proved to have remarkable effects on carrageenase activity. Optimal medium as follows:1.05g/L i-carrageenan,14.73g/L glucose,1.35g/L CaCl2,10g/L yeast extract,0.15g/L ferric citrate,4g/L MgSO4,0.06g/L SrCl2,0.06g/L K2HPO4. After RSM optimization, the strain KL-A reach the maximum activity of925.2U/g cell dry weight(CDW) at72hours of the growth period, which is20.6times the enzyme activity value in the base medium and9.6times enzyme activity value in optimization of carbon and nitrogen sources. Meanwhile, optimization medium cell dry weight is2.4times the basal medium. This article is the first response surface medium optimization i-carrageenase for Cellulophaga sp., and significantly increased the enzyme production capacity.By genome sequencing, we preliminarily found8ORFs carrageenase or agarase gene-related from Cellulophaga sp. KL-A. Because of the strain KL-A efficient degradation i-carrageenan in preliminary experiment, we cloned and expressed the two i-carrageenase of ORFs sequences, then studied the enzymatic properties and the degradation products. The cgiA2_Ce gene contain1476bp full-length gene sequence, encoding a493aa residues of the protein. Protein molecular size predicted online is53.55kD, with isoelectric point of9.46. The recombinant plasmid pET-32a-CgiA2_Ce was transformed into E.coli BL21(DE3) expression host. The His-tagged recombinase CgiA2_Ce was purified with Nickel-affinity chromatography column.The recombinant ι-carrageenase CgiA2_Ce can specificially degrade ι-carrageenan. The optimum temperature of the enzyme is40℃, the relative enzyme activity between37-43℃maintained above80%, it still retains more than80%of the maximum at a temperature of40℃within lhr. The optimum reaction buffer system was Tris-HCl buffer pH7.5, and the recombinant enzyme showed a relativlet wide pH range, to maintain more than70%of the maximum activity in the pH6.5-8.0range. Among the tested ions, K+and Mg2+slightly promoted the enzyme activity, while Na+and Ca2+significantly promoted (34%-57%). Based on the measurement of the double-reciprocal plots to the Michaelis constant, Km and Vmax values were obtained in0.93mg/mL and573.5U/mg. The smaller Km and larger Vmax of Michaelis constant values indicate that i-carrageenan is a better kinetics substrate to the recombinant i-carrageenase CgiA2_Ce.A rapid reduction of i-carrageenan polysaccharide accompanying an increase of oligosaccharides with various degrees of polymerization(DPs) by TLC and TOF-MS analysis, indicated that CgiA2_Ce was an endo-type hydrolase, and end products of the degradation are mainly disaccharide and tetrasaccharide. Endo-type carrageenase can degraded high molecular weight carrageenan to low molecular weight and low viscosity oligosaccharides.In the future, a single oligosaccharide of low polymerization degree or some oligosaccharides of high polymerization degree are expected by hydrolysis precess control. |
