Studies On The Activity Of Enzymatically Prepared Agar Oligosaccharides

Agar oligosaccharides,with kinds of biological activities,are low molecular weight saccharides that obtained through chemical or biological degradation.Gracilaria lemaneiformis is a class of red algae that is the main source of agar widely planted in China.In order to achieve high-value utilization of Gracilaria lemaneiformis and agar,we isolated and purified the recombinant agarase,its enzymatic properties were studied.The methods for extraction of agar from Gracilaria lemaneiformis was optimized.The enzymatic preparation conditions of agar oligosaccharides from agar using the agarase also studied.The oligosaccharide products were then separated and identified,whose bioactivities were studied.The results were as follows:1.The recombinant agarase was isolated and purified with Hollow fiber column.The agarase,with a molecular weight of 35 kDa,had a good pH and temperature stability,which optimum pH value is 6.5 and optimum temperature is 45?.The specific activity of agarase is 1120.37 U/mg.2.The effects of solvent-to-solid ratio,temperature and extraction time on the yield of agar were investigated using orthogonal test according to single factor experiment.A solvent-to-solid ratio of 28:1(v/w),a temperature of 120? and an extraction time of 90 min were found to be optimal for extracting agar from Gracilaria lemaneiformis.The yield of agar was 32.8%.3.The effects of acid concentration,acid hydrolysis time and substrate concentration on the degree of hydrolysis of agar were investigated using response surface methodology in view of single factor experiment.A hydrochloric acid concentration of 0.29 moL/L,an acid hydrolysis time of 2.69 h and a substrate concentration of 4.76%(w/v)were found to be optimal for degrading agar.The degree of hydrolysis of agar was 96.95%.The effects of pH value,hydrolysis temperature and substrate concentration on the degree of hydrolysis of agar hydrolysed by the agarse were investigated using response surface methodology in view of single factor experiment.A pH value of 6.36,a hydrolysis temperature of 54.47? and a substrate concentration of 0.6%(w/v)were found to be optimal for degrading of agar.The degree of hydrolysis of agar was 89.7%.The products were identified by TLC,HPLC,MS and NMR.The products of acid hydrolysis included agaroligo saccharides with DPs of 2,3,4,5,6,7 and 9.The products of enzymatic hydrolysis included neoagarooligo saccharides with DPs of 2,4,6,8,and 10.The major product was Neoagarotetraose.The agarase was determined to be ?-type.The ?-agarase was determined to be endonuclease because the major product was not changed at different hydrolysis time.4.The product of enzymatic hydrolysis were separated with Sephadex LH-20 column with distilled water as elution liquid.The neoagarooligo saccharides with DPs of 2,4,6 were obtained.5.The results of antioxidant activities showed that NA4 and NA6 have high hydroxyl radical scavenging activity and ABTS radical cation scavenging activity.Howevr the DPPH radical scavenging activity and reducing power were relatively low.6.The product of enzymatic hydrolysis,include mixture,NA4,NA6,more than NA6 have tyrosinase inhibit activity,with IC50 values of 15.58,17.28,16.21,18.28 mg/mL,respectively.The inhibitory activity of NA4 was reversible competitive inhibitor.The inhibition constant was 16 mg/mL.7.ACE activity could be inhibited by NA4,with IC50 values of 31.93 mg/mL.8.The neoagarooligo saccharides had the effect of prebiotic,but the effect was not as good as dextrose solution.