壳聚糖对栓菌属漆酶固定化条件的研究

为了掌握固定化漆酶对苹果酚类化合物生物催化的特异性,以白腐菌漆酶为试材,采用分光光度法手段,首次对壳聚糖固定化的白腐菌漆酶的特性进行了研究。以儿茶素作为标准底物检验催化活性,实验结果如下:用壳聚糖固定化漆酶,单因素实验表明戊二醛(交联剂)的最适浓度5%,交联时间8h,最适pH为5.5,最适酶浓度1U/ml,固定化酶的最适温度为55℃;最适反应条件固定化漆酶的重复使用次数实验表明,经壳聚糖固定化的漆酶重复使用6次,相对酶活仍能保持70%以上。这为苹果加工中漆酶催化混合酚类化合物转化工艺的确定提供了依据。     

利用综合性实验增强微生物学实习效果的体验

以综合性实验"固定化血红密乳菌对染料的脱色实验"为例,探讨了该实验的实验原理和方法,从而引出其对微生物课程实习教学的效果。结果表明:该综合性实验激发了学生的实习兴趣,并取得了较好的教学效果。     

毛栓菌漆酶基因克隆及其酵母表达载体的构建

[目的]研究毛栓菌漆酶基因克隆及其酵母表达载体的构建,为研制高品质酶制剂,实现其工业化生产和规模化应用奠定了基础。[方法]以毛栓菌总RNA为模板,通过RT-PCR克隆去信号肽的漆酶基因;通过BLAST比对,对毛栓菌漆酶基因序列的同源性进行分析;对质粒pPIC9K进行EcoRⅠ和NotⅠ进行酶切,构建其酵母表达载体p9K+Lac。[结果]去信号肽的漆酶基因编码框全长为1 512bp,编码506个氨基酸,分子量为54 KDa;毛栓菌漆酶基因与NCBI中提交的毛栓菌基因序列同源性达到了99%。[结论]通过RT-PCR得到了漆酶基因全长,重组毕赤酵母表达载体p9K+Lac的构建是完全正确的。     

生物酶预处理对秸秆中密度纤维板性能的影响

针对稻草和麦秸原料的特点,分别用木聚糖酶、漆酶／碳源系统(LCS)和脂肪酶对原料进行生物酶预处理用机械磨浆方法制备纤维,以木质纤维板用脲醛树脂作胶黏剂,压制秸秆中密度板,并探讨酶处理对秸秆纤维板性能的影响。结果表明:各种生物酶预处理对稻草和麦秸纤维板的性能都有一定的改善,同软化处理相比,经三种生物酶处理后的长纤维的比例明显增加,而细小纤维的含量则明显降低,IB均有不同程度的改进,其中木聚糖酶对此两项指标的改进效果更好。经木聚糖酶预处理后压制的密度达到0．8 g／cm3以上的纤维板的性能如下:麦草纤维板的IB为0．75 MPa,MOE为3 960 MPa,MOR为37．60 MPa,TS为21．29％。除TS外,均优于GB／T11718-1999标准要求。而稻草纤维板的IB为0．73 MPa,MOE为2 618 MPa,MOR为21．35 MPa,TS为21．19％,其中MOR与TS均离标准要求还有一定的差距。     

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。     

植物漆酶的研究进展

叙述了植物漆酶的来源、结构，并讨论了植物漆酶的酶促反应机理和生物学功能，包括其在木质素的形成、抗病、抗逆以及种皮颜色中的作用。     

漆酶活化工业木质素制备环保型纤维板的工艺参数及产品性能

以木纤维为基本相、漆酶活化工业木质素作为粘接相,通过热压制得了环保型纤维板.同时,通过红外光谱分析了工业木质素酶法催化改性的反应位点,并利用正交试验对主要工艺参数和成板性能的关系进行了研究,分析了各因子对成板性能的影响规律,确定出最优的工艺参数.在试验范围内,最优工艺参数为:活化浓度200U·mL-1,活化时间8 h,...     

枇杷果肉漆酶酶学特性研究

研究枇杷果肉漆酶(EC1.10.3.2)的提取及其理化性质,为探讨漆酶在冷藏枇杷果实木质化败坏中的作用机理提供参考。以‘解放钟’枇杷(Eriobotrya japonica Lind‘l.Jiefangzhong’)果实为试材,采用硫酸铵盐析、透析和DEAE-纤维柱层析等提取漆酶(EC1.10.3.2)研究其理化特性,包括温度、pH、抑制物、金属离子对漆酶活性的影响及其底物特性,测定果实贮藏期间pH变化。结果表明,漆酶的最适温度为45℃,最适pH为4.4,pH为3.2～4.4时酶的稳定性较好。金属离子Cu2+、K+对漆酶有激活作用,而Ag+、Fe2+、Fe3+起抑制作用。EDTA和L-半胱氨酸抑制物对漆酶均有抑制作用,以L-半胱氨酸抑制作用最强。枇杷果肉漆酶以2,2’-连氮-双(3-乙基苯并噻唑-6-磺酸)(简称ABTS)为底物的Km值为0.00512mol/L。     

灰树花的系统发育分析和主要木质素降解酶的测定

对灰树花菌株迁西二号进行了ITS序列扩增与测序(GenBank登录号为GU584099),并对灰树花及相关白腐菌进行了基于ITS序列的系统发育分析,结果表明灰树花与Grifola sordulenta (Mont.) Singer等白腐菌的亲缘关系较近。采用LNAS(低氮天冬酰胺-琥珀酸)培养基添加4种不同底物,对菌株迁西二号在25 ℃恒温下静置培养,得到了不同时间内的培养液,用紫外可见分光光度计分别检测了在470 、420 、310 nm处对于2,6-二甲氧基苯酚 (2,6-DMP)、2,2’-连氮-双(3-乙基苯并噻唑-6-磺酸)(ABTS)、3,4-二甲氧基苯甲醇/藜芦醇(VA)的氧化作用后光密度值的变化情况,作为锰过氧化物酶(MnP)、漆酶(Laccase)和木质素过氧化物酶(LiP)产生和活性大小的依据,从而获得了灰树花木质素降解酶系统的主要酶系及其与培养基的成分和酶作用底物的基本关系。结果表明,灰树花可产生MnP和漆酶,但不产生LiP;对比4种成分不同的培养液酶活力测定结果,未添加底物的培养液MnP最大酶活仅为2.96 U·L^-1,漆酶最大酶活仅为4.49 U·L^-1。添加底物木屑和2,6-DMP后MnP最大酶活为8.06 U·L^-1,漆酶最大酶活为9.85 U·L^-1,表明在培养液内添加酶的底物木屑后能轻微地提高两种酶的分泌量。     

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.