| With the decrease of forest wood resources,the recycling of wastepaper has drawn increasingly more attention.However,the stickies generated in the process of wastepaper recycling will flocculate and deposite in the pulp,resulting in production accidents and affecting product output.The biological method can prevent the flocculation of the stickies by enzymatically hydrolyzing the ester bond between the stickies components.Cutinase(EC3.1.1.74)is a multifunctional enzyme that can hydrolyze various polyesters.Relevant studies have shown that some anchor peptides possess the ability to bind polyester.In this paper,the cutinase from Humicola insolens(HiC),was selected to conduct the degradation test on the stickies in pulp,and the enzyme preparation made by high density fermentation was used in the industrial production of recycled paper.Then,the anchor peptides Tachystatin A2(TA2)and OMP25 were fused at the protein terminus of HiC to improve the degradation efficiency of stickies.After that,the enzymatic properties of the fusion protein and the degradation efficiency of the stickies model substrate were determined.The main research results are as follows:(1)Preliminary application of HiC in stickies processing.Firstly,HiC was applied to the degradation experiment of stickies in pulp,and it was found that HiC has a certain degradation effect on stickies in pulp.The high-density fermentation of the engineered strain Pichia pastoris KM71/pPIC9K-HiC was carried out with 40 L fermenter,and the highest enzyme activity was2372 U·m L-1;3000 L fermenter was used for high-density fermentation of the above-mentioned engineered bacteria,the highest enzyme activity was 2213 U·m L-1.The optimization experiment of additive formula confirmed,with 20%glycerol+3‰potassium sorbate+3‰sodium benzoate+10%PEG600,could improve the storage stability of HiC to the greatest extent.In the industrial production of recycled paper,compared with Optimyze?525,which was currently used in the production workshop,HiC was found to be 49.53%lower in stickies content and 33.90%in the amount of stickies in deinked pulp treated,the number of stickies was33.90%lower,the number of paper breaks per day during the industrial production was reduced by 12.50%,the average maintenance time of the machine was reduced by 19.05%,and the weight of sticky deposits was reduced by 30.63%,(2)Molecular construction,recombinant expression and enzymatic characterization of the fusion protein.Previous studies have shown that the anchor peptide TA2 and OMP25 have the ability to bind polyester.The HiC gene was linked to the anchor peptide TA2 and OMP25 genes respectively,and the constructed plasmids pPIC9K-HiC-TA2 and pPIC9K-HiC-OMP25 were transformed into P.pastoris KM71.The maximum enzyme activity of shaker fermentation was21.09 U·m L-1 and 55.22 U·m L-1 respectively.The optimal temperature of the fusion proteins HiC-TA2 and HiC-OMP25 was 80°C,and the half-life period at 30°C and 50°C were 192hours and 168 hours respectively.The optimal pH range was 8.0-8.5,the storage stability was the best at pH 9.0.The maximum enzyme activities of recombinant strains were228.58 U·m L-1 and 631.42 U·m L-1 in 3 L fermenter,which were 10.83 and 11.43 times of those in shaking flask,respectively.(3)Characterization of stickies model substrates degradation by fusion proteins.The degradation experiments were carried out by using PEA and PVAc as stickies model substrates.The results showed that the degradation efficiency of the fusion proteins HiC-TA2 and HiC-OMP25 on PEA was better that of HiC.After HiC-TA2 and HiC-OMP25 treatment,the turbidity change of HiC-TA2 and HiC-OMP25 were 1.46 and 1.48 times higher than that of HiC,the particle size reduction of HiC-TA2 and HiC-OMP25 were 6.78 times and 6.45 times of that of HiC.In the detection of ethanol content of degradation products,HiC-TA2 and HiC-OMP25 were 1.29 times and 1.13 times of that of HiC.When the fusion proteins HiC-TA2 and HiC-OMP25 were used to degrade PVAC,the particle size of the reaction system decreased by505.40 nm and 497.30 nm respectively.In the detection of acetic acid content of degradation products,HiC-OMP25 and HiC-TA2 were 4.86 and 1.91 times of that of HiC respectively.The turbidity variation values of HiC-TA2 and HiC-OMP25 were 5.88 and 3.41 times higher than those of HiC after grinding and degrading the stickies sediments collected from the recycled paper production line. |
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