| Ramie fiber is known as the"king of natural fibers"and is the indispensable important raw material in textile industry.Establishing a green and environmentally friendly microbial degumming technology to replace the traditional chemical degumming with heavy pollution and high energy consumption,which is in line with the national goals of"carbon peak"and"carbon neutrality".However,at present,the reported degumming strain still existing problems such as incomplete degumming and low efficiency.A high-efficiency degumming strain P.carotovorum HG-49 was obtained in the preliminary study with a 16-hour gum removal rate as high as 82.16%.However,there is still a gap of about 10%with the national standard of refined ramie fibers.How to further improve the gum removal rate of HG-49and reduce or even replace the use of subsequent alkalis is the key breakthrough goal.This paper systematically studies the dynamic change rules of ramie fibers’microbial degumming process,combined with omics data analysis,revealing the mechanism of degumming and determining the key factors affecting degumming efficiency.Mining key degumming enzymes,expressing key enzymes efficiently to enhance the degumming ability of strains HG-49 and further significantly improve the degumming efficiency.The main results are as follows:（1）Revealing the dynamic change rules of ramie fibers’degumming process by strain HG-49.The dynamic changes of the biomass of strains HG-49,the activities of degumming enzymes,the gum degradation rate,the contents of reducing sugar,and the morphological characteristics of ramie fibers in the degumming process were systematically studied.It was found that 4-12 h was the key period of degumming.HG-49 was at logarithmic growth stage,degumming enzymes were highly expressed,gums were degradated with high speed,large amount of reducing sugar generated.The pectinase activity is significantly higher than that of mannanase and xylanase,the removal rate of pectin is as high as 97.05%,and the residual gum is mainly hemicellulose;The fibers dispersed gradually,the color became more and more whiter,the absorption peak of representative functional group of gum materials decreased and the crystallinity of cellulose increased.Analysis of bacterial community structure in open degumming system showed that the abundance of HG-49 was always in an absolute advantage,miscellaneous bacteria had little influence on degumming.The results in this chapter showed that HG-49 had high pectinase activity,which was the key factor to its high-efficiency degumming ability.However,hemicellulase activity was low,resulting in residual hemicellulose be difficult to remove.（2）Illuminating the degradation and metabolism pathway of ramie gum by strain HG-49.The whole genome analysis of strain HG-49 found that its genome is about 5 Mb in length and 51.75%of GC content,including 4251 genes.25 of degumming enzyme genes were found out,23 pectinase genes included,containing abuntant pectinase systems such as pectate lyase and polygalacturonase;only 2 ofβ-xylosidase were found and indicated that the type and quantity of hemicellulase were insufficient.The transcriptome results of the degumming process showed that 7 of pectate lyases played a key role.Degumming enzymes were secreted to extracellular by typeⅡsecretion system to degrade ramie gum and the generated monosaccharides were transported into HG-49 cells mainly through the ABC pathway and phosphotransferase system.The monosaccharides had a complete metabolic pathway in HG-49 cells,they were metabolized mainly through EMP pathway and TCA cycle,which provided energy for strain HG-49’s growth.The microbial degumming mechanism of“bacteria secreting enzymes,enzymes degradeting gums,gums cultivating bacteria”was analyzed from molecular level.The above results showed that HG-49 had hemicellulase activity,and the hemicellulase removal rate reached 73.14%,however,the key hemicellulase was not found through the omics analysis,and which enzyme is responsible for the degradation of ramie bast hemicellulose remains to be further analyzed and explored.（3）The key hemicellulose of strain HG-49 was found out.Based on genome-wide glycoside hydrolase data,expression level during degumming process by transcriptome analysis and enzymic experiments,an endoglucanase Cel12M was found to own high mannase activity.It belongs to the 12 th family of glycoside hydrolase,with a gene coding region length of 795 bp and a recombinant protein size of 27.3 k Da.The recombinant protein showed high activity to mannan substrates,such as konjac gum,guar gum and locust bean gum,especially to konjac gum with a specific activity as high as 2803.7±185.4 U/mg.Therefore,Cel12M was determined to be a multifunctional endoglucanase with high mannanase activity.What’s more,the enzyme did not show activity on refined ramie fibers and won’t destroy fiber strength.The optimum temperature of Cel12M enzyme is 50℃and the optimum p H is 6.5.Mn2+can promote its activity,and Ag+and SDS have obvious inhibitory effect on its activity.The results of reducing sugar content detection and immunofluorescence show that Cel12M enzyme had a positive degradation effect on the mannan of ramie bast fibers and was the key hemicellulose of strain HG-49.However,the expression level and activity of Cel12M were not high enough in the degumming process,resulting in residual hemicellulose exist.Therefore,the next focus was to improve the expression level of Cel12M in strain HG-49.（4）Constructing engineered strain HG-49 that highly expresses Cel12M,strengthening the degumming ability of HG-49.The encoding gene gm622 of Cel12M was recombined with the modified vector p213 and introduced into the strain HG-49 to construct a constitutive secretory expression engineered strain.Compared with the original strain HG-49,the gm622 gene expression level and mannanase activity of the engineered strain were significantly increased;The hemicellulose removal rate was increased by 9.28%,with a total gum removal rate up to 87.28%.The SEM,ATR-FTIR and XRD analysis of degummed ramie fibers showed that the degumming effect of engineered strain was better than that of original strain.The residual gum was further removed,the fiber surface was smoother,the absorption peak of hemicellulose functional groups was further reduced and the crystallinity of cellulose was significantly improved.The dosage of alkali was reduced by 30%,and the obtained refined ramie fibers reached the first-class national standard.In conclusion,this study systematicly revealed the degumming mechanism of ramie fibers by strain HG-49,and determined the key factor affecting degumming efficiency is that the type and quantity of hemicellulase are insufficient,and the expression level is low,resulting in low activity of hemicellulase and part of hemicellulose remained.A key multifunctional hemicellulose was found from strain HG-49 and a high-efficiency degumming engineerd strain HG-49 was constructed.The removal rate of hemicellulose and total gum were increased significantly,the high-quality refined ramie fibers were obtained and the usage of alkali was reduced greatly at the same time.This study provides theoretical and practical guidance for green manufacturing of high-quality ramie fibers. |
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