Study On The Characterization Of Multifunctional Amylase Amy2587 Produced By Seaweed-associated Microorganisms From Indonesia

Hot springs on Indonesia's Halmahera Island are mainly formed by volcanoes.There are many large seaweeds living in the waters around the hot springs.A large number of microorganisms are associated to the surface of the seaweed.These microorganisms are important sources of discovering potential new bioactive substances and enzymes,which have attracted more and more attention of researchers.In this study,three species of seaweed samples collected from Indonesian hot springs were selected as the research objects.Amylase sequences with high activity and good stability were screened by macrogenomic analysis which will be expressed and characterized for industrial application.Firstly,the genomic DNA of three seaweed-associated microorganisms collected from Indonesia was extracted and sequenced.A total of 148464 gene sequences were obtained from three samples.The results of biodiversity annotation showed that the dominant microbia associated to the surface of these macroalgae were Vibrio,Klebsiella,Photobacterium and Acinetobacter.The results of functional annotation information showed that the main gene of microbial carbohydrate-active enzymes?CAZymes?associated to macroalgae samples were GT?Glycosyl Transferase?gene,GH?glycoside hydrolase?gene and CBM?carbohydrate binding module?gene.Through further screening and analysis,116 amylase-coding genes were obtained,which belong to GH13,GH57,GH15,GH66,CBM20,CBM25,CBM26,CBM35 and CBM48 families.Secondly,amy2587,one of the amylase genes containing GH66 family motifs,was selected for gene expression.The length of amy2587 is 1785 bp,coding 587 amino acids,theoretical molecular weight of 67.46 kDa.The recombinant plasmid pET-30a-amy2587was constructed by double digestion and ligation of the target gene and expression vector pET-30a.The recombinant plasmid was transformed into the competent cell E.coli BL21?DE3?.The engineering bacteria containing recombinant plasmids were induced to culture at 16?for 16 h at IPTG concentration of 0.5 mM.The cells were broken and the supernatant was purified by Ni-NTA chromatography column.SDS-PAGE electrophoresis showed that a single target band was obtained with a molecular weight of70 kDa at 80 mM of imidazole eluent concentration,which was consistent with the theoretical molecular weight.The results confirmed that the target gene was correctly expressed in competent cell E.coli BL21?DE3?.Then,the enzymatic properties of recombinant amylase Amy2587 were studied.The results showed that the recombinant amylase Amy2587 not only had the ability to degrade starch,but also could degrade agar,carrageenan,alginate and sodium carboxymethyl cellulose.Among them,the ability to degrade starch was the strongest,and its enzyme activity reached 63.38 U/mL,indicating that recombinant Amy2587 was a multifunctional amylase.Amy2587 at has the optimum temperature of 50?with different substrates and has good thermal stability.The amylase activity of Amy2587 can be maintained about85%for 1-4 h at 50?With the increase of holding time,and the amylase activity decreases gradually with the increase of incubation time,the residual amylase activity of Amy2587 is still more than 60%even after 24 h.The optimum pH was 10.0 for different activity.The relative enzyme activity of amylase and agarase was between 6.0 and 10.0remained at 77%,this indicated that Amy2587 had strong alkali tolerance.Mn²⁺,Fe²⁺,K⁺,and Na⁺metal ions promoted the different activities of Amy2587,but all the activities of Amy2587 were almost lost under the action of Cu²⁺.The K_m values of Amy2587 to starch,agar,carrageenan,alginate and sodium carboxymethyl cellulose were 4.06,10.10,12.25,11.54 and 14.91 mg/mL,respectively,indicating that Amy2587 had the best affinity with starch substrates.Finally,the multifunctional mechanism of Amy2587 is preliminarily studied.Three motifs in the Amy2587 sequence were deleted by gene knockout,and the mutant sequence were expressed and studied.The results showed that the activity of mutants decreased to some extent after knocking out the corresponding motif,which indicated that these functional domains had certain effects on the multiple substrates activity of Amy2587,but could not decide it's multifunctional characteristics.In addition,the effects of fatty acids and fatty acid methyl esters?methyl linoleate,methyl oleate,stearic acid,oleic acid,palmitic acid,linoleic acid and linolenic acid?on the multiple substrates activity of Amy2587 were also studied.The results showed that linoleic acid could obviously inhibit the multiple substrates activity of Amy2587.Oleic acid inhibited the activities of amylase,alginase and sodium carboxymethyl cellulose of Amy2587,but had little effect on the activities of agarase and carrageenase.The multifunctional mechanism of Amy2587 still needs further study.