漆酶降解烟梗中木质素及其机理研究

发布时间：2018-08-27 06:40

【摘要】：木质素作为植物骨架的三大主要成分之一,在烟草中的含量在4-8%,这部分木质素是造成烟气中木质气息重,以至于产生强烈的呛感、咳嗽、灼喉等不适的主要原因之一;且热解时会产生儿茶酚、烷基儿茶酚等引起涩口且有促癌活性的物质,影响卷烟品质和安全。因此,在烟草薄片制备过程中的木质素的降解和脱除有利于提升其品质。漆酶能选择性地催化木质素降解,不会产生有毒物质,并且生产通常在低温、常压的温和条件下进行,易于实现工业化。开展漆酶脱除烟梗中木质素以制备高品质的烟草薄片,并利用木质素模型物来探讨漆酶降解烟梗中木质素的机理,对提高烟草薄片的品质提供工艺参数和理论指导具有良好的意义。研究了XSLacc、SUKALacc、YCLacc、DENYKEM PAP-5、DENYKEM INDICLEAN 6G+、GrenLacc 6种不同产地漆酶对烟梗中木质素的降解的影响,结果表明DENYKEM PAP-5对烟梗中木质素降解效果最好。再分别从固液比、反应温度、pH、反应时间、摇床转速等工艺参数对DENYKEM PAP-5酶解工艺条件进行优化,结果表明酶解过程中,固液比越小,酶解效果越好,考虑到酶解率与成本,实验选择的固液比为3.0:100;在漆酶用量1.3%,反应温度为50℃,pH=5.0,反应时间为4h,摇床转速为150 r/min时,烟梗中木质素的降解率为15.92%。研究了香草酸、丁香醛、对羟基苯乙酮等9种天然介体的添加对漆酶降解烟梗中木质素的影响,结果表明,丁香酸对烟梗中木质素的降解有一定的促进作用,降解率为16.64%。优化的酶解烟梗中木质素工艺经工厂中试放大试验后专家评吸,由烟梗制备的烟草薄片的烟气细腻感有提升,木质杂气和枯焦杂气下降比较明显。以异丁香酚和2-溴-4-羟基苯乙酮等物质为原料,在DMF/NaH体系中合成了酚型β-O-4二聚体木质素模型物(Ⅰ),分别研究了反应温度和反应时间对目标产物收率的影响,结果表明反应时间为50℃,反应时间为60 min,模型物(Ⅰ)的收率可以达到75.8%;以4-甲氧基-2-溴苯乙酮和2-甲氧基苯酚为原料,在K2CO3和丙酮体系中合成非酚型β-O-4二聚体模型物(Ⅱ);采用辣根过氧化物酶(HRP)催化异丁香酚合成包含α-O-4和β-5键的香豆满型二聚体木质素模型物(Ⅲ);然后以香豆满型二聚体模型物(Ⅲ)为底物,在过硫酸钾和硫酸亚铁的氧化还原引发体系中催化合成了包含5-5、α-O-4、β-5键的四聚体木质素模型物(Ⅳ),研究了溶剂比、反应时间、反应温度对目标产物收率的的影响,结果表明:丙酮/水为2:1,反应时间为10 min,反应温度为80℃时,模型物(Ⅳ)的收率达到33.8%。分别对合成的模型物进行高分辨质谱(HRMS)、元素分析(EA)、核磁共振光谱(NMR)等表征,确定合成的模型物的结构。以酚型β-O-4二聚体模型物(Ⅰ)、非酚型β-O-4二聚体(Ⅱ)和含β-5、5-5键的酚型四聚体模型物(Ⅳ)作为研究对象,利用优化的酶解条件进行酶解,结果表明:漆酶可以导致木质素酚型模型物(Ⅰ)中β-O-4键的部分断裂,从而导致木质素的降解;而漆酶对于非酚型β-O-4二聚体(Ⅱ)和含β-5、5-5键的酚型四聚体模型物(Ⅳ)中的联接键都不能发生断裂。可以推断漆酶因为断裂酚型木质素中的β-O-4键而导致木质素的部分降解而脱除。
[Abstract]:As one of the three main components of plant skeleton, lignin content in tobacco is 4-8%. This part of lignin is caused by smoke lignin heavy aroma, so that strong choke, cough, burning throat and other discomfort one of the main reasons; and pyrolysis will produce catechins, alkyl catechins and other astringent mouth and carcinogenic substances. Laccase can selectively catalyze the degradation of lignin without producing toxic substances, and the production is usually carried out under mild conditions at low temperature and atmospheric pressure, which is easy to realize industrialization. The lignin was used to prepare high quality tobacco slices and the lignin model was used to study the mechanism of lignin degradation in tobacco stem by laccase. It is of great significance to provide technological parameters and theoretical guidance for improving the quality of tobacco slices. XSLacc, SUKALacc, YCLacc, DENYKEM PAP-5, DENYKEM INDICLEAN 6G+, GrenLacc from six different producing areas were studied. The results showed that DENYKEM PAP-5 had the best effect on lignin degradation in tobacco stem. Then the enzymatic hydrolysis conditions of DENYKEM PAP-5 were optimized from the aspects of solid-liquid ratio, reaction temperature, pH, reaction time and rotational speed of shaker. The results showed that the smaller the solid-liquid ratio, the better the enzymatic hydrolysis effect. Considering the enzymatic hydrolysis rate and cost, the solid-liquid ratio was 3.0:100, the degradation rate of lignin in tobacco stem was 15.92% when laccase dosage was 1.3%, reaction temperature was 50%, pH was 5.0, reaction time was 4 h, and the rotational speed of shaker was 150 r/min. The degradation of wood in tobacco stem by laccase of 9 natural mediators, such as vanillic acid, eugenol aldehyde and p-hydroxyphenylacetone, was studied. The results showed that eugenoic acid could promote the degradation of lignin in tobacco stem, and the degradation rate was 16.64%. The optimized enzymatic lignin hydrolysis process was evaluated by experts after pilot scale-up test in a factory. The smoke fineness of tobacco leaf prepared from tobacco stem was improved, and the lignin impurity and charred impurity decreased significantly. Phenolic beta-O-4 dimer lignin model compound (I) was synthesized from isoeugenol and 2-bromo-4-hydroxyacetophenone in DMF/NaH system. The effects of reaction temperature and time on the yield of the target product were studied. The results showed that the yield of the model compound (I) could reach 75. 8%; non-phenolic beta-O-4 dimer model compounds (II) were synthesized from 4-methoxy-2-bromoacetophenone and 2-methoxyphenol in K2CO3 and acetone systems; isoeugenol was catalyzed by horseradish peroxidase (HRP) to synthesize coumarin dimer lignin model compounds (III) containing alpha-O-4 and beta-5 bonds; then coumarin dimer model compounds (III) were prepared by coumarin dimer model compounds (HRP). A tetramer lignin model compound (IV) containing 5-5, a-O-4, and a-5 bond was synthesized by using potassium persulfate and ferrous sulfate as substrates. The effects of solvent ratio, reaction time and reaction temperature on the yield of the target product were studied. The results showed that acetone/water ratio was 2:1, reaction time was 10 min, reaction temperature was 80 C. High resolution mass spectrometry (HRMS), elemental analysis (EA), nuclear magnetic resonance spectroscopy (NMR) were used to characterize the synthesized model compounds, and the structure of the synthesized model compounds was determined. The results showed that laccase could induce the partial breakage of the beta-O-4 bond in the phenolic lignin model compound (I) and lead to the degradation of lignin, while laccase could not induce the bond formation in the non-phenolic beta-O-4 dimer (II) and the phenolic tetramer model compound (IV) containing the beta-5,5-5 bond. Fracture. It can be deduced that laccase is partially degraded by breaking the beta-O-4 bond in phenolic lignin.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q814;O636.2

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