Coexpression Of β-Mannanase And Xylanase Gene

Hemicellulase hydrolyse hemicellulose which is a diverse group of enzymes, and the mostimportant hemicellulase are mannanase and xylanase. They have been widely used in feed industry,textile, paper, food and other fields. They are also the key enzymes in ramie degumming. This paperconstructed the β-mannanase and xylanase coexpression system, and made some expression research. Itwill provide materials and methods for mechanism research and improved technology of ramiebiological degumming, and it also will provide reference for enzyme preparation on production andapplication in the future.In the study, constructed a plasmid by polycistronic manner firstly. The β-mannanase and xylanasegene inserted into the vector pET28a one by one, and they linked by Hind III restriction sites,constructing a coexpression plasmid pET28a-man-xyl. In the connection area of Hind III restrictionsites inserted into a5'-UTR sequence of xylanase gene. Secondly, constructed a plasmid by singlecistron manner. The xylanase and β-mannanase gene respectively inserted into the multiple cloning siteMCS-1and MCS-2of vector pACYCDuet-1, constructing a coexpression plasmidpACYCDuet-man-xyl. The two coexpression plasmids were transformed into E.coli BL21(DE3),generating the coexpression strain. After screening by mannanase and xylanase activity detection plate,PCR and double digestion of plasmid, obtaining the coexpression strain of B.pET28a-man-xyl andB.pA-man-xyl.The B.pET28a-man-xyl strain was analyzed by SDS-PAGE, extracellular and intracellularenzyme activity determination. The analysis of SDS-PAGE showed that xylanase and β-mannanasewere expressed extracellularly as individual functional proteins, which was not expression as a fusionprotein. And it also created advantages to keep the activity of protein. The enzymes volume expressedby coexpression strain were significantly higher than the original strain, and the two enzymes werealmost no difference. The analysis of extracellular and intracellular enzyme activity showed that theintracellular enzyme activity was higher than that of extracellular enzymes. The extracellular activity ofβ-mannanase and xylanase were more than intracellular at4h and6h. Thereafter, extracellular enzymeactivity increased with the induction time, but intracellular enzyme activity tended to balance. Afterbeing induced21h, the extracellular activities of β-mannanase and xylanase were713.34U/mL and1455.83U/mL, respectively. They are2.5and11.9times of intracellular, and more than70%of theβ-mannanase and xylanase activity were presented in the extracellular.Fermentation liquor of strain B.pET28a-man-xyl was concentrated by ultrafiltration, SephadexG-100chromatography and dialysis concentrations, which obtained the protein sample ofelectrophoretically pure. The protein weight molecular of purification and prediction were equivalent,and it was consistent with the specific band of strain B.pET28a-man-xyl total protein. Furthermore, itconfined that the two specific band of transformant SDS-PAGE above were β-mannanase and xylanase.Obtaining3.7g hemicellulose from40g ramie rod powder by alkaline extraction, and the rate was9.25%. Calculated by reducing sugar yield, β-mannanase and xylanase used together to hydrolyse the hemicellulose were efficient than alone. After20min later of the hemicellulose hydrolysis by compoundenzyme (β-mannanase and xylanase), more than80%of reducing sugar yield.β-mannanase and xylanase activity of strain B.pA-man-xyl were also higher, and the zenymeactivity mainly concerned on the intracellular. After being induced4h, β-mannanase and xylanaseactivity were1015.12U/mL and1181.00U/mL. Plasmid of strain B.pA-man-xyl was transformed intoDCE-01strain. Molecular biology detection showed that plasmid were successfully transformed intostrains. The expression analysis of transformed strain DCE.pA-man-xyl showed that the plasmidpACYCDuet-man-xyl have expressed in DCE-01strain. The β-mannanase activity was determined. Itshowed that β-mannanase activity has increased, and it is about3times of the original strain DCE-01.The β-mannanase activity of strain DCE.pA-man-xyl was804.52U/mL.