Determination Of ROS Free Radicals Produced By Laccase-Catalyzing Jute Lignin And Their Effects On Grafting Modification

In recent years,jute and other lignin-rich plant fibers have attracted great attention in the field of composite materials due to their excellent mechanical properties,easy biodegradability,wide raw source,low price and many other advantages.At the same time,,enzymatic biochemical modification of laccase catalysis jute has become a research hotspot according to its poor chemical composition,low performance uniformity and weak interfacial bonding with hydrophobic substrates,which greatly limit the expansion of their applications.At present,researches mainly focus on the optimization of process conditions during the enzymatic modification of laccase and the property study of as-obtained composite materials.There are few researches reporting the free radicals formation,detection,effect factors,and its effect on enzymatic modification.In this work,based on the study of the content,distribution and structure of lignin in jute and other rich lignin fibers,we successfully captured and detected the intermediate products,reactive oxygen species?ROS?free radicals,which were produced by laccase catalyzed oxidation of jute lignin.We also improved the methods and conditions for the detection of ROS free radicals using electron paramagnetic resonance?EPR?spectroscopy.The effects of process parameters on ROS free radicals during the catalysis of jute lignin were also studied and the ROS free radicals produced by laccase catalyzed jute lignin were quantitatively analyzed.Then,the effects of inorganic ions,chelators and mediators on the catalytic performance of laccase were systematically studied based on the initial enzyme activity,thermal stability of laccase catalyzed guaiacol and the ROS free radicals production in the total process of laccase catalyzed oxidation of jute lignin.Finally,the effect of free radical produced by laccase catalysis of lignin on grafting efficiency was studied,and the modified jute was characterized and its properties were determined.The main research contents and conclusions were as follows:1.The determination of lignin content and structure in jute and other lignin-rich fibersThe lignin content in jute and other lignin-rich fibers was determined by Klason method.Among difference lignin-rich fibers,the lignin content in Chinese fir was the highest?about31%?,followed by bamboo?about 24%?.The lignin content in jute and kenaf is about 14%and about 12%,respectively.Marijuana and flax have the lowest lignin content.The molecular weight and dispersion coefficient of milled wood lignin?MWL?of Jute and other lignin-rich fibers were determined by gel permeation chromatography?GPC?.The content information of element C,H,O,N,methoxy group,C₉ unit empirical formula and phenolic hydroxyl group etc.of MWL from lignin-rich fibers were investigated by means of organic element analysis,¹H-NMR analysis and measurement of phenolic hydroxyl.Based on the methoxy content of MWL,the lignin-rich fibers can be arranged from high to low as follows:bamboo,Chinese fir,hemp,jute,kenaf,flax.In addition,through the infrared spectrum analysis,jute and kenaf fiber MWL belong to GS4 type lignin,hemp MWL belongs to GS3 type lignin,flax MWL belongs to GS1 type lignin,bamboo MWL belongs to HGSa type lignin and Chinese fir MWL belongs to G type lignin.The proportions of three basic lignin structural units?guaiacyl:syringyl:hydroxyphenyl?in jute and other lignin-rich fibers of MWL are shown as following:jute?1.00:2.58:0?,kenaf?1.00:3.91:0?,hemp?1.00:1.23:0?,flax?1.00:1.09:0?,bamboo?1.00:1.29:0.61?,Chinese fir?1.00:0.86:0?.Based on histochemical staining and microscope observation of four kinds of bast fibers,they showed non-uniform distribution of lignin in different micro domains.The lignin in jute and kenaf are mostly distributed in the cellular intercellular layers.2.The capture and detection of ROS free radical produced by laccase catalyzed oxidation of juteLaccase can catalyze the oxidation of jute to form ROS free radicals,which will lead to subsequent radical graft polymerization.With spin trapping technique and electron paramagnetic resonance?EPR?spectroscopy technology,we used PBN as the capture agent,and ROS free radicals intermediate from laccase catalyzed oxidation of jute lignin were successfully detected and characterized?g=2.005?.We improved the test conditions for ROS free radicals produced by laccase catalyzed oxidation of jute:The sample tube is a quartz tube with an inner diameter of 2.5 mm,the effective sample height is 3.5 cm,the amount of spin trapping agent PBN is 10 mM?final concentration?and the sample EPR detection time should be controlled within 0 h⁷2 h.On this basis,the process parameters of laccase catalyzed oxidation of jute lignin to produce ROS radicals were optimized by EPR detection technology:pH 3.5,treatment temperature 60?,enzyme dosage 0.54 U/mL,treatment time 1h,copper ion concentration10 mM.Meanwhile,the standard curve of ROS radical and EPR signal intensity was established by using Fenton reaction system:y=0.05458 x+10.02942?R²=0.98917?.The ROS free radicals produced by laccase catalyzed oxidation of jute lignin were also quantitatively analyzed.The number of ROS free radicals is?1.1275±0.1338?×10¹⁸ spins/g dry jute.3.The effect of inorganic ions,chelators and mediators on catalytic capacity of laccase catalysis of jute lignin and its model compounds and on the free radical in the systemThe effects of inorganic ions on laccase catalyzed oxidation of jute lignin and its model compounds were studied by spectrophotometry and EPR spectroscopy techniques.The results showed that little effect of SO₄^2-,K⁺,Na⁺,Mg²⁺and Ca²⁺was observed on initial activity of laccase and laccase stability;Mn²⁺?at high concentration?,Ag+and NO₃^-have a moderate inhibitory effect on the initial activity of laccase;Cl^-,Fe²⁺and Fe³⁺have a strong inhibitory effect on the initial activity of laccase.The inhibition rate of Fe²⁺?inhibition rate of about98%at 1 mM?was greater than that of Fe³⁺?inhibition rate of about 55%at 1 mM?.Cu²⁺has little effect on the initial activity of laccase,but moderate amount of Cu²⁺is beneficial for the improvement of thermal stability of laccase?when its concentration is 15mM,the enzyme activity could be increased by nearly 24%?,which will promote the formation of ROS free radicals in laccase catalyzed oxidation of jute lignin.On the contrary,long time interaction between Ag⁺ion and laccase will eventually lead to laccase degeneration and inactivation,which will finally affect the catalytic performance of laccase.Fe²⁺and Fe³⁺showed similar great effect on the stability of laccase.Fe²⁺tends to inhibit the catalytic process of laccase by constantly reducing the free radical intermediates.Then the formed Fe³⁺ion will eventually lead to laccase degeneration and inactivation due to its destruction effect on laccase activity structure.Although the chelator EDTMPA exhibited an activation effect on the initial laccase at low concentrations??5 mM?,most chelating agents may have a strong inhibitory effect on the laccase catalytic process according to the effect of EDTA-2Na on ROS free radicals produced by the laccase catalyzed oxidation of jute lignin.And also,the addition of mediators ABTS and DMP is favorable for the production of ROS free radicals during the laccase catalyzed oxidation of jute lignin.In the process of laccase catalyzed oxidation of jute and other lignin-rich fibers,inorganic anions such as Fe²⁺,Fe³⁺,Ag⁺and inorganic cations such as Cl^-?NO₃^-should be avoided.The chelators should also be used with special cautious.In addition,a moderate amount of Cu²⁺or mediator can be added to improve the catalytic activity of laccase.4.The effect of free radical produced by laccase catalyzed oxidation of jute lignin on enzymatic graft modificationThe process conditions of laccase catalyzed graft of jute fabric with a series of gallate monomers were compared with that of the producing of ROS free radical from jute lignin catalyzed by laccase by saponification-back titration.At the same time,the process parameters of enzymatic grafting were optimized.And the influence of the concentration of free radical on the grafting rate was analyzed..On this basis,Fourier transform infrared spectroscopy?FT-IR?and scanning electron microscopy?SEM?were used to characterize and analyze the surface of jute fabrics before and after grafting.Finally,the hydrophobic properties and thermal stability of modified jute fabric were determined.The results showed that the influence of process parameters on the free radicals of lignin catalyzed by laccase was similar to that of enzymatic grafting.And the ROS free radical intermediates have great influence on enzyme-catalyzed graft modification.laccase can catalyze jute lignin and the gallate series monomer?Phenolic compounds with low molecular weight?to form free radicals,and then initiate the hydrophobic modification of jute.Under the condition of buffer pH 3.5,incubation temperature 50 ^oC,laccase dosage 2.5 U/m L,gallate series monomer dosage 10 mM,dosage of exogenous Cu²⁺ions 10 mM and incubation time 4 h,ideal grafting rate can be obtained for jute?octyl gallate?OG?grafting rate was4.04%,dodecyl gallate?DG?grafting rate was 5.85%?.The hydrophobic properties of modified jute were enhanced with the increase of hydrophobic alkyl groups of grafted monomers.Compared with ungrafted jute?water contact angle was 68.32°,the wetting time was 4.7s?,the jute fiber grafted with DG showed excellent hydrophobic properties?water contact angle was 127.8°,the wetting time was more than 30 min?compared with ungrafted jute?water contact angle was 68.32°,wetting time was 4.7 s?.The hydrophobic properties of jute grafted with OG took the second place?water contact angle was 114.12°,wetting time was more than 245s?.The jute grafted with propyl gallate?PG?can only show transitory hydrophobic property.The thermal stability of the grafted jute fabric decreased slightly.