Application Of Pectobacterium Carotovorum Sp. HG-49 And ManA Enzyme In Degumming Of Ramie

Boehmeria nivea?L.?Gaudich is a unique natural fiber resources in China.The textile of ramie is excellent in tenacity,breathability and hygroscopicity.The 30%non-fibrous compositions in bast fiber of ramie,which was called gum,was consisted of pectin,hemicellulose,lignin and so on.since pectin is wrapped on the outermost side of the fiber,its removal ratio directly affects the subsequent degumming.Hemicellulose composition is complex,mainly composed of mannans,xylan,mannans accounted for about 70%.These components play a structural role,acting as hemicellulose that bind cellulose.Hemicellulose and cellulose tightly integrate each other,it is difficult to separate these two components.Therefore,improving the removal ratio of pectin and hemicellulose is the emphasis of optimizing the degumming effect.Microbial degumming,with the advantages of high degumming efficiency,low cost,and low pollution,was becoming the main research field in ramie degumming.Pectobacterium carotovorum and its mannanase ManA is used for degumming.After that,it was expected that the pectin and hemicellulose degradation efficiency was improved and then the degumming efficiency was improved.The research results were as follows:Firstly,a degumming strain with capable of efficiently removing pectin was obtained and its degumming conditions was optimized.A degumming strain was screened by degumming ratio as an index,it has been identified as Pectobacterium carotovorum which named Pectobacterium carotovorum sp.HG-49.This bacterium is applied to ramie degumming.The degumming conditions were as follows:the bath ratio was 1:40,the inoculation amount was 5.0%of the system,the age of bacteria was 8 h,the degumming temperature was 35°C,the degumming time was 24 h.Dynamic monitoring of the process showed that during 4–14 h of degumming with HG-49,the cells proliferation is rapid,the increase of enzyme activity is large,and the degradation of gum is accelerated.The relative removal ratios of the components were as follows:pectin?98.12%?,water soluble matter?75.84%?,fat wax?58.86%?,hemicellulose?40.81%?and lignin?16.33%?.The pectin degradation is the most thorough.The results of scanning electron microscopy showed that the degummed ramie fiber was smooth.Secondly,the mannanase gene from HG-49 was cloned and successfully expressed prokaryotic cells.To promote the removal of hemicellulose and lignin,for the main component of hemicellulose,the mannanase gene ManA derived from the Pectobacterium carotovorum sp.HG-49 was cloned and expressed heterologously.The activity of the purified recombinant enzyme was 2845.28±46.21 U/mL.SDS-PAGE showed that the molecular weight was about 73 KDa,which was consistent with the predicted molecular weight.Sequence similarity analysis showed that the recombinant enzyme ManA belongs to the glycoside hydrolase 5 family.Thirdly,The enzymatic properties of recombinant enzyme ManA were studied and proved to be synergistic with HG-49 during ramie degumming.The optimum pH and temperature of the reaction of recombinant enzyme ManA was 7.0,45°C,the optimum storage pH and temperature range of recombinant enzyme ManA was 7.0–8.0,30–45°C.High concentration of Fe²⁺,Mn²⁺,Cu²⁺can activate the enzyme activity,Fe³⁺,Zn²⁺can inhibit the enzyme activity.Mercapto ethanol has an activation effect,SDS,EDTA,ethanol,urea have an inhibitory effect.ManA only recognizes and catalyzes the hydrolysis of mannose-formed polysaccharides and is more likely to specifically hydrolyze the?-1,4-glycosidic bonds formed by glucose and mannose in the main chain.The kinetic constant K_m was 5.71 mg/mL,the V_max was 1.56?mol/?min?mg?.Recombinant enzymes ManA and HG-49 have a synergistic effect in the degumming process,with the increase of ManA activity in the system,the degumming ratio also increased.Compared with HG-49alone,the removal ratio of hemicellulose was improved by 9.98%,the removal ratio of lignin was increased by 2.64%,the total gum removal ratio increased by 5.02%with synergetic degumming.The study above have promoted the application of Pectobacterium carotovorum in ramie degumming industry.