2种野外采集白腐真菌分离及其产漆酶液体培养基优化

[目的]优化2种野外采集白腐真菌菌丝产漆酶液体培养基。[方法]对2种采集的白腐真菌轮纹韧革菌(Stereum ostrea)和朱红密孔菌(Pycnoporus cinnabarinus)通过分离纯化得到白腐真菌菌丝,应用ABTS法测定其产漆酶活性,研究碳源、氮源、金属阳离子、p H、温度、转速对白腐真菌产漆酶的影响。[结果]轮纹韧革菌最佳培养基是葡萄糖20.00 g、硝酸钾5.00 g、NaH_2PO_45.00 g、Cu SO41.00 g、初始p H 6、温度25℃、转速170 r/min,在第11天其产漆酶活性达到最大,为1.557 U/m L,是PDA液体培养基产漆酶活性的2.7倍。朱红密孔菌最佳培养基是麦芽糖25.00 g、酵母膏5.00 g、NaH_2PO_41.00 g、KCl 2.00 g、初始p H 7、温度30℃、转速200 r/min,在第9天其产漆酶活性达到最大,为1.478 U/m L,是PDA液体培养基产漆酶活性的2.3倍。[结论]筛选出2种野外白腐真菌产漆酶最佳培养基,为漆酶的进一步研究提供了参考。     

特异腐质霉来源漆酶基因的克隆及其在毕赤酵母中的表达

漆酶是一种含铜的多酚氧化酶,在有机农药残留和木质素的降解方面具有潜在的工业价值。从特异腐质霉Y1(Humicola insolens Y1)中分离到了漆酶基因Lac1,其全长1 803 bp,编码600个氨基酸,与NCBI蛋白数据库比对结果表明,与来源于Chaetomium globosum CBS 148.51(XP_001228806)的多酚氧化酶序列相似性最高为71%,表明是一个新的漆酶基因。将其克隆入毕赤酵母表达载体pPIC9r中,通过PCR和SDS-PAGE检测证实基因已经整合入酵母基因组中且能分泌表达。在3 L发酵罐中,重组Lac1蛋白表达量达到3.79mg/mL发酵液,酶活性达到1.3 U/mL发酵液。酶学性质分析结果显示,漆酶的最适作用温度为65℃,最适反应pH为4.5,且在pH 6～11的范围内酶的相对活力均在70%以上。     

白腐菌TP21液体培养产漆酶条件的研究

研究了碳源、氮源、各种理化因素以及培养条件对白腐菌TP21漆酶分泌的影响.结果表明:木糖和多糖作碳源、酵母膏作氮源有利于漆酶的分泌,pH在4.0～6.0的范围内对漆酶的分泌影响不大,培养温度、接种量、通气量对漆酶的分泌有较大影响,培养基中添加诱导物对漆酶的产生有促进作用,特别是玉米秸秆价格低廉,是较好的选择.     

Improvement of dispersing property of sodium sulfite-formal-dehydeanthraquinone pulping effluent by treatment withCoriolus versicolor

A strain of the fungusCoriolus versicolor was inoculated periodically into potato dextrose agar (PDA) slants containing the effluent to enhance the natural ability to grow in the effluent. The acclimated strain grown in the 50% effluent-containing PDA slant and the original strain were employed to treat the effluent. The acclimated strain could grow in a higher concentration of the effluent than the original unacclimated one. Both the original and acclimated strains improved the dispersing ability of the effluent, especially the acclimated strain because of its higher laccase secretion. The dispersing ability of the SFP effluent was improved to a level comparable to a commercial lignosulfonate product because it was strongly polymerized by the fungus. During the fungal treatment, more than 50% of the sugars were removed from the effluent, thereby increasing the purity of the SFP lignin product.This work was presented at the 48th annual meeting of the Japan Wood Research Society, April 3–5, 1998, Shizuoka, Japa     

红芝LYL263所产漆酶在溶胶-凝胶体系中的固定化研究

[目的]对红芝LYL263漆酶进行不同溶胶-凝胶体系的固定化研究,以期找到适宜该漆酶的固定化材料。[方法]将海藻酸钠-明胶、海藻酸钠-壳聚糖和海藻酸钠-明胶-壳聚糖3种溶胶-凝胶体系用于红芝LYL263所产漆酶的固定化研究,并初步分析固定化酶的酶学性质。[结果]海藻酸钠-明胶-壳聚糖体系固定后的漆酶酶活分别是海藻酸钠-明胶和海藻酸钠-壳聚糖的2.14倍和2.75倍,该体系最佳材料组成为:海藻酸钠浓度2.0%、明胶浓度1.0%、壳聚糖浓度0.3%、氯化钙浓度36.0%。该试验首次将苯甲酸用于包埋法固定化漆酶,单因素试验结果显示,适宜的苯甲酸浓度能有效提高固定化漆酶酶活;该体系固定化漆酶最佳条件为:苯甲酸浓度2mmol/L、戊二醛浓度0.32%、交联时间50 min、酶浓度10.0%,此时固定化酶活高达635.7 U/g。对固定化漆酶酶学性质的研究表明,固定化酶的热稳定性比游离酶高,冻干的固定化酶活是未冻干固定化酶的1.59倍,但冻干的固定化酶操作稳定性较差。[结论]在酶固定化材料中,各个物质的组成对酶固定化有非常大的影响,该研究对红芝LYL263漆酶的固定化材料选择有重要意义。     

食用菌中漆酶的研究进展(英文)

漆酶是已被广泛研究的用来降解酚类、苯胺等异生物质的酶之一,很多漆酶已经从真菌培养物中被提取。主要讨论从食用菌中获得的漆酶,包括漆酶的催化结构和机制,漆酶基因的克隆和表达以及对食用菌研究的潜在应用。     

Transformation of C-labelled lignin and humic substances in forest soil by the saprobic basidiomycetes Gymnopus erythropus and Hypholoma fasciculare

Litter decomposing basidiomycetous fungi produce ligninolytic oxidases and peroxidases which are involved in the transformation of lignin, as well as humic and fulvic acids. The aim of this work was to evaluate their importance in lignin transformation in forest litter. Two litter decomposing basidiomycete species differing in their abilities to degrade lignin - Hypholoma fasciculare, and Gymnopus erythropus - were cultured on sterile or non-sterile oak litter and their transformation of a ¹⁴C-labelled synthetic lignin (dehydrogenation polymer ¹⁴C-DHP) was compared with that of the indigenous litter microflora. Both in sterile and non-sterile litter, colonisation by basidiomycetes led to higher titres of lignocellulose-degrading enzymes, in particular of laccase and Mn-peroxidase (MnP). The titres of the latter were 6 to 40-fold increased in the presence of basidiomycetes compared to non-sterile litter. During 10 weeks, G. erythropus mineralised over 31% of ¹⁴C-DHP in sterile litter and 23% in non-sterile litter compared to 14% in the non-sterile control. Lignin mineralization by H. fasciculare was comparable to the non-sterile control, 12% in sterile litter and 16% in the non-sterile litter. The largest part of ¹⁴C from ¹⁴C-DHP was transformed into humic compounds during litter treatment with both fungi as well as in the control. In addition to the fast lignin mineralization, microcosms containing G. erythropus also showed a lower final content of unaltered lignin and 23-28% of the lignin was converted into water-soluble compounds with relatively low molecular mass (<5 kDa). Both G. erythropus and H. fasciculare were also able to further mineralise humic compounds. During a 10-week fungal treatment of an artificial ¹⁴C-humic acid (¹⁴C-HA) supplemented to the natural humic material of a forest soil, the fungi mineralised 42% and 19% of the labelled material, respectively, under sterile conditions. The ¹⁴C-HA mineralization by introduced basidiomycetes in microcosms containing non-sterile humic material, however, did not significantly differ from that of a non-sterile control and was around 12%. Altogether the results show that saprobic basidiomycetes can considerably differ in their rates of lignin and humic substance conversion. Furthermore, lignin degradation in forest soil can rather slow down by interspecific competition than it is accelerated by cooperation of different microorganisms occupying specific nutritional niches. Therefore, the overall contribution of saprobic basidiomycetes depends on their particular eco-physiological status and the competitive pressure, and may be often lower than initially expected. Significant lignin transformation including partial mineralization is seemingly not exclusively dependent on exceptional high titres of ligninolytic enzymes but also on so far unknown factors. Higher endocellulase production and subsequent weight loss was found in microcosms where saprobic basidiomycetes were combined with indigenous microbes. Potentially, lignin degradation by the basidiomycetes may have increased cellulose availability to the indigenous microbes.     

甜菜果胶酶促交联对蛋白质乳化液稳定性的影响

研究了辣根过氧化物酶（HRP）和漆酶（Laccase）催化甜菜果胶间交联的反应条件及其对蛋白质乳化液稳定特性的影响。紫外〖CD*2〗可见分光光度法和傅里叶变换红外光谱结果均表明,HRP和Laccase在水相中可催化甜菜果胶间形成共价交联。HRP的反应体系为：5mL反应体系中含6mg甜菜果胶,20U HRP,0.5%（质量分数） H2O2,35℃下反应2h;Laccase的反应体系为：5mL反应体系中含6mg甜菜果胶,15U Laccase,40℃下反应24h。激光衍射粒度仪测定结果表明,不同酶反应体系下,蛋白质乳液特性不同。Laccase交联体系引起牛血清白蛋白（BSA）、乳清分离蛋白（WPI）乳状液发生分层现象,随反应时间延长,分层更加明显;而在HRP反应体系下,BSA乳状液的稳定性能明显提高。     

Production of lignocellulose-degrading enzymes and degradation of leaf litter by saprotrophic basidiomycetes isolated from a Quercus petraea forest

Due to the production of lignocellulose-degrading enzymes, saprotrophic basidiomycetes can significantly contribute to the turnover of soil organic matter. The production of lignin- and polysaccharide-degrading enzymes and changes of the chemical composition of litter were studied with three isolates from a Quercus petraea forest. These isolates were capable of fresh litter degradation and were identified as Gymnopus sp., Hypholoma fasciculare and Rhodocollybia butyracea. Within 12 weeks of incubation, H. fasciculare decomposed 23%, R. butyracea 32% and Gymnopus sp. 38% of the substrate dry mass. All fungi produced laccase and Mn-peroxidase (MnP) and none of them produced lignin peroxidase or other Mn-independent peroxidases. There was a clear distinction in the enzyme production pattern between R. butyracea or H. fasciculare compared to Gymnopus sp. The two former species caused the fastest mass loss during the initial phase of litter degradation, accompanied by the temporary production of laccase (and MnP in H. fasciculare) and also high production of hydrolytic enzymes that later decreased. In contrast, Gymnopus sp. showed a stable rate of litter mass loss over the whole incubation period with a later onset of ligninolytic enzyme production and a longer lasting production of both lignin and cellulose-degrading enzymes. The activity of endo-cleaving polysaccharide hydrolases in this fungus was relatively low but it produced the most cellobiose hydrolase. All fungi decreased the C/N ratio of the litter from 24 to 15-19 and Gymnopus sp. also caused a substantial decrease in the lignin content. Analytical pyrolysis mass spectrometry of litter decomposed by this fungus showed changes in the litter composition similar to those caused by white-rot fungi during wood decay. These changes were less pronounced in the case of H. fasciculare and R. butyracea. All fungi also changed the mean masses of humic acid and fulvic acid fractions isolated from degraded litter. The humic acid fraction after degradation by all three fungi contained more lignin and less carbohydrates. Compared to the decomposition by saprotrophic basidiomycetes, litter degradation in situ on the site of fungal isolation resulted in the relative enrichment of lignin and differences in lignin composition revealed by analytical pyrolysis. It can most probably be explained by the participation of non-basidiomycetous fungi and bacteria during natural litter decomposition.