Extraction Of Chitin Deacetylase From Rhodothermus Marinus And Its Application In The Preparation Of Deacetylated Chitooligosaccharides

Chitin is the second most common polymer in nature and it has potential value in the fields of medicine,cosmetics,food and agriculture.However,due to the water-insoluble properties of chitin,its application has been greatly restricted.Therefore,scientists have turned their targets to chitin hydrolysates,chitosan and chitosan oligosaccharides.However,the latest research has found that when chitosan has a specific degree of polymerization,degree of acetylation and acetylation mode,its biological activity is stronger than chitosan and chitosan,and it has more research and application value.The existing industrial production mainly uses the acid hydrolysis and oxidative degradation of chitosan to obtain deacetylated chitooligosaccharides.However,the products prepared by these technologies are a very complex mixture,which contains chitooligosaccharides of various molecular weights and various degrees of acetylation.The biological activity of chitooligosaccharides is closely related to the specific molecular weight and degree of acetylation.In biological activity experiments,it is difficult to clarify which chitooligosaccharides molecule(s)exert biological activity.This has become a bottleneck for studying the structure-activity relationship of chitooligosaccharide components and related activity mechanisms.Therefore,in order to further determine the biological activity of chitooligosaccharides in follow-up studies,the preparation of chitooligosaccharides with a single degree of polymerization and degree of acetylation is very necessary.Chitin deacetylase belongs to the carbohydrate esterase 4 family(CE4).It is an enzyme that can remove the specific acetyl group of chitooligosaccharides under mild conditions.This enzyme is used to create shells with specific acetylation patterns on a large scale.Chitooligosaccharides have become an important research direction.The current research on chitin deacetylase is focused on chitooligosaccharides with a higher degree of polymerization,while chitin deacetylases that specifically act on chitooligosaccharides with a low degree of polymerization are less and have low catalytic efficiency.Therefore,it is of great significance to develop a chitin deacetylase with better stability,high activity and specific deacetylation function for chitooligosaccharides.Rhodothermus marinus is a species with a known genome sequence.According to previous studies,it has been found that the carbohydrate hydrolases derived from R.marinus have the advantages of good thermal stability and long half-life,and have high industrial application potential.And there is no report about chitin deacetylase from this source.Mining chitin deacetylase from R.marinus can provide an important tool enzyme for the industrial production of chitooligosaccharides.The specific research content is as follows:1.Study on the cloning,expression and purification,activity determination and active site of recombinant RmCBDA2510The target gene was cloned using Rhodothermus marinus genome as a template,named RmCBDA2510,and successfully expressed in E.coli expression cell BL21(DE3).The purified target protein has a single band,and the size is consistent with the theoretical value.To verify the deacetylation activity of chitin deacetylase,chitobiose was used as a substrate to react for 12 h at pH 8.0 and 37℃.After the reaction,the supernatant of the reaction system was taken for MALDI-TOF-MS detection.According to the mass spectrum,it is preliminarily judged that RmCBDA2510 has single-end deacetylation activity.Then using LC-MS to calculate the conversion rate of the reaction through the peak area comparison results,RmCBDA2510 has a better deacetylation activity for chitobiose.In order to verify the transacylation activity of chitin deacetylase,propionate/butyrate with 5 times the concentration of chitobiose was added to the above reaction system.After the reaction under the same conditions,the supernatant was taken for LC-MS detection.The results show that RmCBDA2510 does not have the function of transacylization,which proves that the catalytic reaction of the chitin deacetylase is a one-way reaction rather than a reversible reaction.Through multiple sequence alignment,it was determined that the conserved site of RmCBDA2510 was aspartic acid at position 41.It was subjected to site-directed mutagenesis to transform it into asparagine.The mutant D41N loses its deacetylation function,which proves that Asp41 is the active site of RmCBDA2510.2.Preparation of oligochitooligosaccharides and deacetylated chitooligosaccharides Chitooligosaccharides are prepared by chemical decomposition method.Dissolve 100 mg of chitin in 3 mL of concentrated hydrochloric acid,stir at 4℃ for 2 h to form soluble colloidal chitin,and place it in a water bath for reaction.According to the results of TLC,the most suitable reaction conditions are reaction at 50℃ for 30 min,when the amount of chitosan oligosaccharides is the most produced.According to the results of MALDI-TOFMS detection,the content of chitooligosaccharides decreases with the increase of the degree of polymerization.Use silica gel column and thin layer chromatography to purify the chitosan oligosaccharide polymer.The silica gel column method can purify the reaction product in a large amount,but the obtained chitosan oligosaccharide is not pure.Thin layer chromatography can purify the reaction product in a small amount.To obtain a singlecomponent pure chitosan oligosaccharide.Using the prepared(GlcNAc)3 as a substrate,the activity of RmCBDA2510 was verified under the same reaction conditions.Take the reaction supernatant for detection and analysis by MALDI-TOF-MS and LC-MS.According to the results of mass spectrometry,RmCBDA2510 also has single-end deacetylation activity for(GlcNAc)3,and the reaction conversion rate is 93.6%,which is slightly lower than chitobiose,and can be used for the preparation of deacetylated chitotriose.