Biomimetic Affinity Purification Of Marine Biological Protease MP

Proteases are enzymes that can catalyze the hydrolysis of proteins or peptides.It is closely related to human beings and involves all aspects of life.Protease can be used for the production of detergent,tanning,fur,protein hydrolyzate,wine,soy sauce,and textile,cosmetics and other biological materials extraction.Because of its unique properties such as salt and alkali resistance,marine biological protease has been paid more and more attention by the market.And it's application in food,medicine,detergent and other aspects are broader.At present,the purification of protease is mainly based on ammonium sulfate precipitation,ion chromatography,gel filtration chromatography or combined with several methods.The purity of protease is low and it's a bottleneck to restrict the application of high purity enzyme.In this paper,the metalloproteinase MP produced by marine bacteria YS-80-122 was purified by affinity chromatography with boric acid Sepharose column and benzamidine Sepharose column.The purification conditions were optimized and analysised the purity of the purified product.And the metal chelate affinity purification of metalloproteinase MP was preliminary studied.Purification of metalloproteinase MP by boric acid Sepharose column: The boric acid Sepharose column?25 mm ×16 mm?was used to purify metalloproteinase MP.The optimal purification conditions were obtained by single factor and response surface test.When enzyme concentration was 100 mg/mL,the optimal loading buffer was glycine-sodium hydroxide of pH 8.6 and the sample rate was 1 mL/min,the elution buffer was disodium hydrogen phosphate-citric acid of pH 5.2 and elution rate was 2 mL/min,the elution buffer added to mannitol of 75 mM,the purification efficiency was enhanced 11 times and the purity above 98.8%.Purification of metalloproteinase MP by benzamidine Sepharose column: In order to obtain a more efficient affinity chromatography column for the purification of metalloproteinase MP,screening four kinds of affinity chromatography columns?25mm×7 mm?including arginine Sepharose column,lysine Sepharose column,benzamidine Sepharose column and trypsin Sepharose column.The results showed that the purification effect of metalloproteinase MP by benzamidine Sepharose column was better.The optimal purification conditions were obtained by single factorand response surface test.When enzyme concentration was 30 mg/mL,the buffer system was pH 8 Tris-HCl,the sample rate was 1 m L/min,the adsorption ion concentration was 100 mM,the elution rate was 1.5 m L/min,the elution ion concentration was 0.8 M,the purification efficiency was enhanced 42.5 times and the purity above 99.9%.Screening of metal chelating affinity ligands: The Sepharose 6B as the carrier,using three kinds of commonly chelating agent IDA,NTA,TED,three connecting arms of different length,and five kinds of different metal ions(Zn²⁺,Cu²⁺,Co²⁺,Ni²⁺,Fe³⁺)were combined to 45 kinds of biomimetic affinity.After screening that five kinds of long chain arm affinity materials?0.2 mL?including IDA-Cu²⁺,IDA-Zn²⁺,IDA-Co²⁺,NTA-Cu²⁺ and NTA-Fe³⁺ have purification effect of metalloproteinase MP.The purified sample with IDA-Cu²⁺ long chain arm affinity material was enhanced 19 times and the purity above 94.6%,which can be applied to the biomimetic affinity purification of metalloproteinase MP.