Genome-Wide Identification of Switchgrass Laccases Involved in Lignin Biosynthesis and Heavy-Metal Responses

Plant laccase genes belong to a multigene family, play key roles in lignin polymerization, and participate in the resistance of plants to biotic and abiotic stresses. Switchgrass is an important resource for forage and bioenergy production, yet information about the switchgrass laccase gene family is scarce. Using bioinformatic approaches, a genome-wide analysis of the laccase multigene family in switchgrass was carried out in this study. In total, 49 laccase genes (PvLac1 to PvLac49) were identified; these can be divided into five subclades, and 20 of them were identified as targets of miR397. The tandem and segmental duplication of laccase genes on Chr05 and Chr08 contributed to the expansion of the laccase family. The laccase proteins shared conserved signature sequences but displayed relatively low sequence similarity, indicating the potential functional diversity of switchgrass laccases. Switchgrass laccases exhibited distinct tissue/organ expression patterns, revealing that some laccases might be involved in the lignification process during stem development. All five of the laccase isoforms selected from different subclades responded to heavy metal. The immediate response of lignin-related laccases, as well as the delayed response of low-abundance laccases, to heavy-metal treatment shed light on the multiple roles of laccase isoforms in response to heavy-metal stress.     

Quantitative determination of Botrytis laccase in musts and wines by the syringaldazine test

The syringaldazine assay was adapted for quantitative determination of laccase (EC 1.10.3.2) activity from Botrytis cinerea Pers in musts and new wines. This colorimetric method is fast, specific and sensitive. It makes it possible to assess the level of contamination of crops by Botrytis cinerea. The assay can be performed in the presence of tyrosinase but the phenolic compounds of the grape must first be eliminated because of their inhibition of syringaldazine oxidation by laccase. When measured in botrytised grape, laccase activity is related to the stages of development of the fungus.     

Biovalorization of brewers’ spent grain for the production of laccase and polyphenols

Brewers’ spent grain (BSG) is the main solid by‐product of the brewing process and is typically disposed of as cattle feed. In this study, BSG was evaluated as a substrate for the production of polyphenols and the lignin‐degrading enzyme laccase using fungal solid‐state fermentation by Trametes versicolor. Laccases are finding increasing applications in the food industry and polyphenols have benefits for human health. After 14 days of fermentation with T. versicolor, there was a 3.4‐fold increase in the extraction of total polyphenols compared with untreated BSG. Using BSG as the sole source of carbon and nitrogen, maximum laccase activity was achieved after seven days of treatment with an activity of 560 U/L. Based on these results, BSG is suggested to be a good lignocellulose waste material to produce value‐added products such as the enzyme laccase and polyphenols. Copyright © 2018 The Institute of Brewing & Distilling     

产漆酶真菌筛选及其固态发酵条件的初步研究

目的筛选产漆酶真菌,优化固态发酵培养基组成,研究粗酶的酶学性质.方法平板筛选获得菌株,通过测定漆酶活性优化培养基的组成.结果获得了固态发酵条件下产漆酶活性较高的菌株.优化后培养基的组成:豆粕和木屑比例1:1,木糖0.5%,酒石酸铵2.0%,培养基含水量65%.漆酶最适温度60℃,低于50℃时漆酶较稳定,最适pH 3.5,pH 6.5～9.0之间漆酶稳定性较好.结论得到了产漆酶活性较高的菌株,培养基优化后漆酶活性提高7倍.     

漆酶对蚕丝织物的生物染色

基于漆酶的生物特性,采用漆酶对蚕丝织物进行处理,达到无染料显色的目的,分析了漆酶用量、温度、时间和pH对显色效果的影响,同时对显色后的蚕丝织物进行各种色牢度和显色均匀性测试,并通过红外光谱初步分析了蚕丝织物显色的机理。结果表明,漆酶对蚕丝织物显色的工艺条件为：漆酶用量50%（o.w.f）,温度50℃,时间24h,pH为4。显色蚕丝织物的颜色均匀性、耐皂洗和摩擦牢度都较好,耐酸稳定性较好,而耐晒牢度稍差。红外分析表明漆酶处理使蚕丝纤维中的酪氨酸残基转变为醌类物质而显色。     

桑黄漆酶发酵条件的优化及其分离纯化

本文利用单因素和正交试验分析方法,研究了温度、培养基初始pH值、装液量和摇床转速等条件对漆酶产量的影响。采用盐析、透析、离子交换层析（IEC）、葡聚糖凝胶层析和聚丙烯酰胺凝胶电泳（SDS-PAGE）等技术将发酵得到的漆酶进行分离纯化。结果表明：最佳工艺条件是温度28℃,培养基初pH为5.8,摇床转速180r/min,装液量130mL/500mL。对发酵所得粗漆酶液进行了分离纯化,最终漆酶的回收率为24.4%,纯化倍数为8.3。纯化后的漆酶经SDS-PAGE检测,证实为单一蛋白,并与标准蛋白比较计算得到此漆酶的表观分子量为约为69.3KD。     

羟基酪醇酶促聚合产物的表征及其抗氧化和热稳定性

利用漆酶对羟基酪醇（hydroxytyrosol,HT）进行酶促氧化聚合,运用紫外-可见分光光度计、傅里叶红外光谱、凝胶色谱和液相色谱-质谱联用对聚合产物进行表征。结果表明,形成的聚合物呈现多分散性（D=2.1）,主要生成了三聚体、四聚体、六聚体化合物。同时,以VC和2,6-二叔丁基-4-甲基苯酚（dibutyl hydroxy toluene,BHT）为阳性对照,分别通过清除1,1-二苯基-2-三硝基苯肼自由基和热重-傅里叶变换红外光谱（thermogravimetry-Fourier transform infrared spectroscopy,TG-FTIR）联用的方法对其进行抗氧化性及热稳定性评价。结果表明,HT聚合物较HT和BHT具有更强的抗氧化性,分别约是其2.28?倍和5.7?倍,接近于VC（约是其72%）。基于热重微分曲线峰值温度（315.1?℃）和残余质量所占百分比（66.09%）结果可知,HT聚合物的热稳定性较VC（228.1?℃）、BHT（236.2?℃）和HT（314.9?℃）更强,且FTIR结果表明,HT聚合物及其他受试化合物均是以羟基断裂开始,脱氢形成醌式结构或脱水碳碳双键。这些数据可为HT的深加工利用提供理论和实践基础。     

灵芝漆酶活性的测定及其产漆酶条件的优化

从测定漆酶活性的几种常用方法中,筛选出一种快速、稳定的灵芝漆酶测定方法:在基础培养基中加入0.04%愈创木酚,通过测定显色圈直径检测漆酶的活性;同时利用单因素分析和正交试验确定了灵芝的最适产漆酶条件:麦芽糖3%、酵母浸粉0.25%、pH值6.0、Cu2 0.5mmol/L,培养温度25℃.实验结果为灵芝的菌种选育及其漆酶的工业化生产提供了一定的参考.     

灵芝突变株G1502漆酶的分离纯化及酶学性质研究

对灵芝突变株G1502发酵液中漆酶的分离纯化方法及酶学性质进行了初步研究.通过聚丙烯酰胺凝胶电泳(PAGE)分析发现,G1502在发酵时只分泌一种漆酶.发酵液经透析浓缩、DEAE-纤维素柱层析,比活力提高 24.28倍,酶活力回收率18.66%.以愈创木酚为底物时该酶的动力学常数K m=1.94×10-4mol/L,Vmax=2.28×10-6 mol/L·min;愈创木酚浓度高时对酶活性有抑制作用.K 对酶有激活作用,Cu2 激活作用不大,Fe 2 、Co2 、Ca2 、Na 、Ba2 对酶活力有抑制作用.     

漆酶及其在酿酒工业中的应用研究进展

漆酶是一种含铜的多酚氧化酶,在造纸、生物漂白和食品等领域得到了广泛研究与应用。该文综述了漆酶的来源与分布、结构、性质以及其在酿酒工业的应用研究进展,包括在保持啤酒稳定性、葡萄酒澄清、测定葡萄酒中某些化合物和作为葡萄孢灰霉感染指示剂等方面,并展望了漆酶在酿酒工业的发展前景。