Isolation,Purification,Properties And Modification Of Groups Of The Beta-amylase From 'Yushu17'

Beta-amylase?BAM,EC 3.2.1.2?is a starch hydrolase widely found in plants and microorganisms.It is an exoenzyme from the perspective of hydrolysis,Acts on the?-1,4 glycosidic from the non-reducing end of the starch until the 2 glucose residues in the?-1,6 glycosidic then stop the reacting,continuous hydrolysis produces maltose,Then ransformation of substrates by Walden,the final product is beta maltose and beta-limit dextrin.In organisms,BAM exists mostly in the form of a single subunit,whereas sweet potato-derived BAM is a tetramer composed of four identical subunits.BAM is used in medicine,brewing,textile and food processing industries,it can be used as a saccharifying agent for the production of beverages and beer in food fermentation.Therefore,domestic and foreign researchers have conducted in-depth research on BAM from different sources.China is the largest producer of sweet potato in the world,Due to the diversification of sweet potato varieties,the promotion area has increased.The consumption of sweet potato is dominated by fresh food and processing,In the process of producing sweet potato starch,a large amount of waste water is generated,and waste water is rich in BAM.If the BAM in the waste water can be recycled,it can be turned into a treasure.An important prerequisite for the study and utilization of high starch sweet potato varieties is to explore the nature of BAM.The existing research results show that the efficiency of recycling BAM is not high,and the recovery efficiency needs to be improved.This study uses the roots of'Yushu17'as the experimental material which was promoted by the Chongqing Sweet Potato Engineering Technology Research Center.The recovery rate of BAM is high purified from the Sweet Potato,efficient recycling of starch by-products can produce huge economic benefits.At the same time,its enzymatic properties and functional groups were studied,and selected BAM from different species on NCBI for bioinformatics analysis.This study lays a solid theoretical basis and experimental basis for the full use of BAM in the starch by-products of sweet potato.The experimental results are as follows:1.The sweet potato beta-amylase gene consists of 7 exons.The length of the gene is 4772 bp.The open reading frame is 1500 bp in length and encodes 499 amino acids.The active site is in the conserved domain.The active site 1 is amino acid 95¹03,active site 2 is amino acid 184¹94.Phylogenetic analysis showed that the beta-amylase genes of sweet potato,peanut,Trifolium repens and soybean were clustered together,corn,rice,barley and wheat were clustered together.Among them,cassava and potatoes are clustered into one.It shows that the close relationship between sweet potato with soybean,Trifolium repens and peanut beta-amylase genes,weaker genetic relationship with maize,rice,barley and wheat beta-amylase genes.The phylogenetic relationship among B.megaterium and the above 12 species is relatively far.2.The fresh'Yushu17'root was peeled and stored at-20°C.The response surface ptimization method showed that the optimum extraction condition was pH 6.7,the material-liquid ratio was 1:10?m/V?.The electrophoretic pure BAM was obtained by homogenization,ethanolfractionation,DEAE-Sepharoseionexchange chromatography and Superdex-200 gel filtration chromatography.The results showed that the specific activity of the enzyme was 333.15 U/mg,the recovery rate was 66.6%and the purification multiple was 9.93 times.3.The molecular weight of'Yushu17'BAM subunit is about 55.12 kD,and the total enzyme molecular weight is about 223.54 kD.It is speculated that this enzyme is a tetramer composed of four identical subunits;the optimal temperature and pH are50°C and 6.2⁶.4,respectively.The enzyme has good stability under the condition of55°C and pH 6⁸,the K_m value of the enzyme to soluble starch measured under optimal reaction conditions was 1.36 g/mL.Urea has double effects on the BAM of'Yushu17',low concentration urea promotes enzyme activity,but it present a weak inhibitory effect at high concentration.Both Fe³⁺and Co²⁺have a promotion effect on the enzyme,Pb²⁺,Zn²⁺,Ag⁺,oxalic acid and SDS have strong inhibitory effect on the enzyme;Cu²⁺,K⁺and Ca²⁺also have a certain inhibitory effect on it.But the difference of the relative enzyme activity was not obvious with the concentration of each ion.Methanol in the organic substance has the greatest inhibitory effect on the enzyme.4.The chemical modification of the BAM side-chain groups with specific chemical modifiers showed that the tryptophan oxime group and cysteine thiol group are the essential groups in the active center of the enzyme and the two group directly participate in the construction of the enzyme activity center.The methionine thioether group?glutamic acid/aspartic acid carboxyl group and arginine thiol group are not the active groups of the enzyme,but they may have a role in maintaining the spatial conformation of the enzyme.Histidine thiol,serine hydroxy,lysine?-NH2,tyrosine phenolic hydroxy and disulfide bonds are not involved in the activity of the enzyme,they are not an essential group of the'Yushu17'BAM active center.