Application Of Pectinase In Fiber-refining And The Separation And Purification Of Pectinase

Pectin is the key materials in bonding the cell walls.Pectin enzyme is one kind of important commercial enzyme which can degrade the pectin,which is mainly concentrate in the middle lamella of cell walls.After the biomass material treated by the pectinase enzyme,the fiber-refining would be easy and efficiently,Besides,the pectinase enzyme is mild and friendly to the environment.In order to improve the existing fiber-refining technics,it researched the influence of pectinase treatment on fiber-refining of biomass materials（including wood,bamboo and industrial hemp stalk）in this experiment by pectinase bought in the markets in this experiment.In order to further improve the application of pectinase in wood processing we focused on that pectinase enzyme producingfrom the endophytic bacteria of industrial hemp seed（Named HS028）and identified the strain as Bacillus mojavensis by method of 16 S rRNA.To optimize the pectinase enzyme production conditions,the fermented liquid,shaking bed temperature,amount of pectin substitutes,the inoculation quantity and Zn²⁺ concentration,etc.were changed.Researched on the mechanism of pectinase production,the separation and purification of the pectinase.To study the influence of pectinase enzyme treatment on the surface structure of biomass material.The main research results as followed:（1）As the biomass materials treated by the pectin enzyme for the fiber-refining.The results shown that the effects of treatment temperature of pectinase on biomass materials were very significant（p<0.05）.The effect of treatment time of pectinase on bamboo fiber-refining was significant（p<0.05）.The effect of treatment concentration of pectinase on wood fiber yield was significant（p<0.05）.（2）Preparation of pectin enzyme by liquid fermentation method and measuring the activity of pectinase by the DNS method.To optimize the pectinase fermentation conditions of HS028 strain,the results shown that the best fluid amount was 60 ml;the best of bed temperature was 37 degrees;Adding different concentration of Zn²⁺ of HS028 strain can inhibition of pectinase activity;The best fermented liquid substrates was pectin.（3）The strain identified results is Bacillus mojavensis by 16 S rRNA method.By sequencing measuring genome-wide of pectinase and comparing lyase genes sequence in the database.Lyase genes corresponding amino acid sequence is deduced by Primer Premier 5 software.This amino acid sequence of pectin lyase was compared in the NCBI databases.Have been known that pectin lyase isoelectric point of HS028 was 9.30,and the molecular weight was 39.30 kDa by pectin lyase gene sequence.（4）Researched separation and purification of pectinase made from HS028 strain,and we can analysis purpose protein bands which will be appear on the map by vertical protein gel electrophoresis.according to the molecular weight was 39.30 kDa.（5）After the biomass materials treated by the pectinase,the morphology of biomass materials showed by the scanning electron microscope.The results shown that pectin lyase can degrade pectic substance among the middle lamella of fiber cells and have the effect on the fiber-refining.In a word,the application of pectinase in fiber-refining was feasible in wood processing,and laid a foundation to improve the biomass material of fiber separation process.