Characterization And Application Of The Key Enzymes For Efficient Ramie Degumming By Pectobacterium Carotovorum

Ramie fiber with the longest fiber and excellent fiber strength among natural plant fibers,has moisture breathable,antibiotic,wear-resistant and other quality performance,is used extensively in high quality products.Ramie fibre is refined from degummed ramie bast,and the gum mainly includes pectin,hemicellulose,lignin.Traditional chemical degumming with high temperature and intense alkali in ramie textile industry causes severe environmental pollution.Alternative,biodegumming could promote fiber quality,and cut down consumption of alkali and coal dramatically,is the focal point and difficulty of the current domestic and abroad research.In our research,a promising strain Pectobacterium carotovorum HG-49 for ramie degumming with gum content reduction by82.2%,removed almost all pectin by 95%,but resulted the high residual hemicellulose by31%as the basis of residual gum.Therefore,a certain amount of alkali boiling and high temperature was needed to remove the residual gum.To improve degumming by P.carotovorum HG-49 and reduce the consumption of alkali and energy,the key enzymes screening and biodegumming effect appraisement were carried out in this study,and then engineering strain for efficient ramie degumming was constructed.The main results were as below:(1)One key enzyme ManB for efficient ramie degumming with P.carotovorum HG-49 was identified and highly expressed by Pichia pastoris.According to heteromannan as the basis of hemicellulose from ramie bast,and the variation of pH values from 7.0 to 9.3 during ramie degumming by P.carotovorum HG-49,one representative mannanase with high specific activity toward galactoglucomannan was engaged for construction of high-yielding strains and promoted ramie biodegumming.The?-mannanase ManB gene(GenBank accession no.KJ806638.1)from codon optimization andsynthesis was introduced into a multi-copy secretion expression vector pAOHr by independent design and then transformed into Pichia pastoris GS115.The highest secretion yield was obtained from a transformant containing six copies of ManB gene.The recombinant?-mannanase ManB was secreted into the medium at a high level of 3.34 g/L having an activity of 1649 IU/mL in 30 L fermenter.ManB showed optimum temperature at 70-75~oC,optimum pH at 7-9,and was quite stable at pH 6-10.The specific activity of ManB was 494.0 IU/mg and 152.8 IU/mg toward LBG and KGM,respectively.The combined application of ManB(400 IU/mL)and P.carotovorum HG-49 for ramie degumming improved gum content reduction by 85.6%.(2)A unique multifunctional glycosidase Cel5M(GenBank accession no.MH544226.1)with high mannanase activity was screened from the released genome of P.carotovorum HG-49 and improved ramie gum content reduction by 88.9%combine with the strain,as well its characterization and function were identified systematically.The Cel5M containing N?catalytic domain(GH5)and C?carbohydrate binding module(CBM3)showed a broad substrate spectrum.The specific activity of Cel5M was 1061.1 IU/mg,647.3 IU/mg,563.4 IU/mg,315.1 IU/mg and 297.1 IU/mg toward KGM,CMC-Na,oat spelt xylan,LBG and guar gum,respectively.Cel5M showed optimum temperature at65~oC,optimum pH at 6.5,and was quite stable at pH 5-9.Cel5M was more stable at50-75~oC and pH 5.0-9.0 toward KGM.Additionally,Specificity activity and sensitivity toward metal ions of 5?catalytic domain from Cel5M can be influenced by its 3?carbohydrate binding module.(3)The effective degumming strain was constructed with homologously over-expression of Cel5M by P.carotovorum HG-49 for increasing the enzyme production and ramie degumming effect.The Cel5M gene was introduced into expression vector pUC18-YJ,and then transformed into the primary P.carotovorum HG-49.The over-expression recombinant strain Cel5M-P.carotovorum HG-49 was screened and identified.During ramie degumming,gum content reduction was improved to 87.7%by the engineering strain Cel5M-P.carotovorum HG-49 higher than 82.2%by primary P.carotovorum HG-49.The engineering strain showed genetic stability and potential ability for ramie degumming industry.In conclusion,this newly discovered mannanase could improve degumming rate of remie bast,especially hemicellulose solubilization with P.carotovorum HG-49.By homologous overexpression of Cel5M in P.carotovorum HG-49,the problem of hemicellulose residue during ramie degumming was effectively solved and then the eco-friendly production and application for ramie biodegumming could be promoted significantily.