Design Of Degumming Enzymes System Based On The Structure Of Ramie Gum And Efficient Expression And Modification Of Enzymes

Ramie fiber is a high-performance textile raw material,however,natural ramie fiber contains gum complexe composed of pectin and hemicellulose,which affects the spinnability of the fiber.The traditional degumming methods use high-temperature alkaline boiling to remove the gum,which has drawbacks such as high energy consumption and severe wastewater pollution.In contrast,enzymatic degumming uses pectinase and hemicellulase to degrade gum,which is more environmentally friendly.However,the existing enzymatic degumming process has problems such as poor degumming efficiency and high cost of enzyme preparations,making it difficult to industrially apply.Our laboratory screened a strain of Bacillus subtilis 7-3-3 in the early stage.The extracellular crude enzyme produced by this strain is rich in pectate lyase PelA and various hemicellulases,showing degradation effect on ramie gum.However,it can only remove about 50%of the gum,and the residual gum rate cannot meet the textile requirements.Based on the above background,this article mainly conducts research work from two aspects:the construction of efficient degumming enzyme system in ramie and the efficient expression of degumming enzymes.The main research content and results of the paper are as follows:1.Characterization of ramie pectin structure and design of pectin degradation enzyme systemPectin samples were extracted from ramie fiber before and after enzymatic degumming with PelA crude enzyme solution,and the asmples were characterized and analyzed.The results showed that the pectin in ramie fiber was mainly composed of homogalacturonan(HG)and rhamnogalacturonan Ⅰ(RG-Ⅰ),accounting for 63.3%and 36.7%of the total pectin,respectively.The galacturonic acid residue in HG showed a lower methylation rate(about 3.6%).After degumming,the proportion of RG-Ⅰ reached 50%,and the esterification rate of HG also slightly increased.It is speculated that PelA can degrade unesterified HG,but the lack of enzymes that degrade RG-Ⅰ and esterified HG leads to low degumming efficiency.The pectate lyase,which can degrade HG with low degree of esterification,and rhamnogalacturonan lyase RhgW,which can degrade RG-Ⅰ,were overexpressed in B.subtilis,respectively.The enzymatic degumming experiment showed that the addition of PelC and RhgW increased the gum removal rate of ramie fiber by 64%.The sugar composition analysis of the fiber showed that the composite enzyme system removed more pectin,but it still contained much hemicellulose in degummed fiber.2.Characterization of the hemicellulose structure of ramie fiber and design of hemicellulose degrading enzyme systemAfter deumming with pectinases,the fiber was degummed by hemicellulases(β-1,4-xylanase XynA and β-1,4-mannanase GmuG)were used to treat ramie fiber,but the removal of hemicellulose was still very limited.Therefore,hemicellulose samples were extracted from ramie fiber before and after degumming.The structural analysis results indicated that the sample exctracted from raw ramie fiber mainly contains β-1,6 linked arabinogalactan protein,RG-I,xylan and glucomannan,where the side chains of xylan contain 4-O-methylglucuronic acid and α-L-arabifuros.In the degummed fiber,the relative content of xylan and glucomannan significantly increased,and xylan showed a higher degree of substitution.Then,β-1,4-xylanase xynA,glucuronoxylan XynC,α-L-arabinofuranosidase XynD,rhamnogalacturonan lyase RhgW and glucomannanase Man5A were expressed and purified in Escherichia coli or B.subtilis,respectively.The degumming experiment showed that the residual gum rate of ramie fiber decreased to 1.0%after degumming with the above enzymes.The results of FTIR,sugar composition analysis,X-ray diffraction(XRD),and scanning electron microscopy(SEM)analysis indicated that the residual gum on the fiber surface has been effectively removed.The crystallinity index of the degummed ramie fiber reached 73.8%,with a fineness of 5.34 dtex and a breaking strength of 4.56 cN/dtex,meeting the textile standards.3.Construction of efficient expression vector for B.subtilis and expression of ramie degumming enzymeQuantitative analysis was conducted on the proteome data of B.subtilis 7-3-3,and the promoters and signal peptide coding regions of the highly expressed gene were cloned for the construction of expression vector.Using the pectate lyase pelA as the reporter gene,the overexpression effects of different promoters and signal peptides on PelA was investigated.Finally,a promoter P2454 and signal peptide S2454 that can efficiently express PelA were selected.The pectate lyase activity of the overexpression strain P2454S2454PelA reached 1706 U/mL,which was 68%higher than the PelA overexpression strain constructed in the laboratory.The analysis of promoter P2454 indicated that it only exists in a few Bacillus subtilis strains,coding a hypothetical protein,and P2454 is composed of two distinct proteinsσF and σA-factor recognition promoter is concatenated.The effects of different carbon and nitrogen sources on the expression of PelA in engineering strain P2454S2454PelA were investigated.The optimal nitrogen source for the expression of PelA in engineering strains is peptone,followed by corn syrup.Further rational design optimization was carried out on the promoter core sequence,5’-untranslated region(5’-UTR),and N-terminal coding sequence of the expression vector in this strain.The optimized expression strain fermented in bran corn meal medium for 72 h,and the pectin lyase activity reached 2232 U/mL,which was 30.8%higher than that before optimization.In addition,the expression vector constructed using promoter P2454 and signal peptide S2454 also achieved overexpression of various degumming enzymes.4.Engineering of the thermostability of pectate lyase PelA and its application in ramie degummingIn order to improve the thermaostability of the key enzyme PelA in ramie degumming and further enhance its efficiency under degumming conditions,PelA was modified for thermaostability.Firstly,the PelA of B.subtilis 7-3-3 was analyzed by GROMACS for Molecular dynamics simulations,and the flexible region of PelA was analyzed based on root mean square deviation(RMSF).Then,the amino acid residues near the flexible region of PelA were replaced by the amino acid residues from pectin lyase PL47.PelA mutants and their enzymatic properties were studied.The results showed that after replacing the amino acid residues at positions 147-178 of the PelA flexible region,the optimal reaction temperature of the mutant M175 was increased from 55℃ to 65℃ and the half-life at 50℃was increased from 2.3 h to 7.5 h.Wild type PelA and mutant M175 were used for ramie degumming experiments at 50℃ respectively.Under the same enzyme activity conditions,mutant M175 showed better degumming effect.In addition,the optimal pH of the mutant enzyme decreased from 9.6 to 8.5,which is closer to the optimal pH of the other ramie degumming enzymes,providing a good foundation for the combination of different enzymes.