Cloning And Tandem-Expression Of Key Enzymes Genes For Biological Degumming

Biological degumming is a biological process of non-cellulose degradation by using biocatalyst,which has the purpose of getting cellulose fiber to meet subsequent machining requirements,and has the advantages of low energy consumption,low pollution,low cost,etc.Not only can it effectively solve the industry's serious problems of energy saving,emission reduction,and consumption reduction,but also can promote the fiber spinnability and yarn quality to meet the requirements of spinning,which is the direction of industrial development.Currently,bacteria degumming is a primary way among all the bio-degumming methods.Therefore,the quality and effectiveness of the strains'are the key points.In our research,we cloned three key bio-degumming related genes?pectinase gene,xylanase gene,mannanase gene?from DCE-01?Dickeya.dadantii DCE-01?and BE-91?Bacillus.subtilis?,and simulation analysis of these genes'sequence and structure was also done by bio-informatics softwares.Then,all the genes were tandem connected on the MCS region of the expression vector by T4 ligase through different combinations and orders.By using the tool of molecular biology,biochemistry and enzymology,etc.,the expression of all 4 key genes were detected and analysed after transformed into Escherichia coli and the degumming strain DCE-01.The main study results were indicated as follows:?1?According to the sequences of DCE-01 and BE-91 xylanase gene acquired,specific PCR primers were designed and pectinase gene pel419?DCE-01?,xylanase gene xyl?DCE-01?and 91xyl?BE-91?,mannanase gene man?DCE-01?were cloned respectively.The sequencing results in consistency with the original sequences and showed:the full-length of these 4 genes were 1128bp,1137bp,1251bp,642bp and code for 375AA,378 AA,416 AA,213 AA amino acids respectively.Homology of nucleotides between these 4 genes and organisms from other microbial sources are among76%-100%,molecular weights of the protein are from 24.28 to 45.74 kDa,pI are from 5.71 to 8.67.All had signal peptide and the trans-membrane sites are from the 24_PP^th to the 32_PP^nd AA.?2?The genes?pel419,xyl,91xyl and man?were tandem connected in the MCS region of the expression vector through different combinations and different orders and 6 recombinant plasmids for multiple biological degumming gene co-expression were constructed?28PXM,28MPX,28XMP,28PXM?91X?,28MPX?91X?,28XMP?91X??,four recombinant plasmids containing each parental genes?28P,28X,28M,28X?91X??were also acquired.After been transformed into E.coli,all genes were expressed and the conditions were optimized.The optimum substrate is polygalacturonic acid,xylose and konjac flour oat,respectively.The best concentration of IPTG is 1.0mmol/L.The optimum speed for culture shaking is 210rpm.The best induction time is 21 h.Protein bands of pel419,91xyl,man could be observed clearly and hydrolysis circles of xylan and mannan could be detected by plate method.?3?The expression of all key genes related to biological degumming in 6 recombinant strains for multiple gene co-expression and 4 recombinant strains containing each parental genes were detected by DNS method.Details were indicated as follows:Through different connecting orders:the enzyme activity of pectinase,xylanase,mannanase are among 5.57-414.74U/mL?2.05-671.80U/mL?17.52-87601.32U/mL?Closer distance between the gene and the promoter,led to higher enzyme activity,in which,genes expression at the first position is about1.57-52.49 times of the second position and gene expression at the second position is about 1.42-23.40times of the third position.The decreasing magnitude from the 1_PP^st position to the 2_PP^nd position is larger than magnitude from the 2_PP^nd to the 3_PP^rd.The decreasing magnitude also varied widely with the kind of genes,higher enzyme activity,led to larger decreasing magnitude.Through different combinations:although both are xylanase,enzyme activity of 91xyl is about30.52-146.36 times of the activity of xyl in DCE-01;91xyl gene has a significant effect on promoting expression of man gene and pel419 gene.If xyl gene was replaced by 91xyl gene,the enzyme activity of pectinase could have 1.93-5.32 times increase from its original level and enzyme activity of mannanase is 4.07-29.70 times.In addition,the synergy was perfect among pel419 gene,man gene and 91xyl gene.?4?enzymatic properties test of multiple gene engineering strain 28PXM?91X?/B21 and its parental strains showed:extracellular enzyme activity is higher than that of endo-enzyme for pectinase in 28P/BL21 and xylanase in 28X?91X?/BL21,however,it is contrary for mannanase in 28M/BL21 and all 3 key enzyme in 28PXM?91X?/B21.The change tendency of the thermal stability and pH tolerance in 28PXM?91X?/B21 are almost the same as those of parental strains and relative enzyme activity of all key enzymes is more than 80%when temperature is around 30?-35?.pH tolerance of every key enzyme varies from strains to strains in the same pH region.Comparing the pectinase in the parental strains,expression could be enhanced from the original by adding NH⁺_P4P,Zn_PP²⁺,Pb_PP²⁺and EDTA.Expression of xylanase could be enhanced by adding Ca_PP²⁺,NH⁺_P4P,Fe_PP³⁺,Cu_PP²⁺and K⁺_PP,but decreased by adding Zn_PP²⁺and Mn_PP²⁺;Expression of mannanase could be changed from its original activity.K_m value of three key enzymes in 28PXM?91X?was about 0.67,0.53,0.63 times of the corresponding parents strains and V_m is 1.93,1.77,2.89 times respectively.?5?After recombinant plasmid 28PXM?91X?was transformed into DCE-01,we got new type of engineering strain 28PXM?91X?/DCE-01 and found the enzyme activity of pectinase,xylanase and mannanase could be enhanced to 1.8 times,30.1 times and 24.9 times comparing with original strain DCE-01 and the COD value,reducing sugar content,fermentation weightlessness rate of the new strain increased as 114.3%,121%,123.9%of the DCE-01's,which indicate better degumming performance.