Solid-state Fermentation for Enhanced Production of Laccase using Indigenously Isolated <Emphasis Type="Italic">Ganoderma</Emphasis> sp.

Laccase production by solid-state fermentation (SSF) using an indigenously isolated white rot basidiomycete Ganoderma sp. was studied. Among the various agricultural wastes tested, wheat bran was found to be the best substrate for laccase production. Solid-state fermentation parameters such as optimum substrate, initial moisture content, and inoculum size were optimized using the one-factor-at-a-time method. A maximum laccase yield of 2,400 U/g dry substrate (U/gds) was obtained using wheat bran as substrate with 70% initial moisture content at 25°C and the seven agar plugs as the inoculum. Further enhancement in laccase production was achieved by supplementing the solid-state medium with additional carbon and nitrogen source such as starch and yeast extract. This medium was optimized by response surface methodology, and a fourfold increase in laccase activity (10,050 U/g dry substrate) was achieved. Thus, the indigenous isolate seems to be a potential laccase producer using SSF. The process also promises economic utilization and value addition of agro-residues.     

酶促反应抑制动力学的微量热法研究──氟离子对漆树漆酶催化氧化邻苯二胺的抑制

提出一个适合各类可逆性抑制的热动力学方程,并根据各量之间的关系提出了可逆竞争性抑制、非竞争性抑制、反竞争性抑制的热动力学判据以及计算表观米氏常数K_m,app和抑制常数K_i等酶促反应生化常数的热动力学公式,并实际应用于NaF对漆树漆酶催化氧化邻苯二胺抑制的微量热法测定。实验初步得出此抑制为非竞争性抑制,求出其K_m,app、K_i值。     

固定漆酶聚苯胺-CoC2O4纳米复合物修饰电极的直接电化学

采用循环伏安法、微分脉冲伏安法、交流阻抗谱以及计时电流法等电化学方法,结合红外光谱、紫外-可见分光光度法、原子力显微镜、透射电子显微镜以及原子吸收光谱等辅助手段,表征了固定漆酶的聚苯胺-草酸钴纳米复合物的化学组成、结构和形貌,测试了纳米复合物固酶前后的导电性能的变化,研究了纳米复合物修饰电极上固定漆酶的直接电化学行为,评估了该电极的催化氧还原效能以及作为电化学传感器检测氧分子的性能。实验结果表明该电极在不含电子介体的溶液中以酶活性中心T2作为首要电子受体,将得到电子传递给化学吸附的氧气使其被电还原,其表观电子迁移速率为0.017 s^-1,且具有良好的催化氧还原性能（氧还原起始电位：460 mV vs NHE,转化氧分子为水的表观速率常数为2.6×10^-4 s^-1）,酶电催化氧还原为水分子步骤为反应的速控步。该电极作为电化学传感器对氧具有极低检测限（0.20 μmol·L^-1）,宽线性响应范围（0.4~7.5 μmol·L^-1）以及对底物高亲和力（K_M=122.4 μmol·L^-1）等优势。     

Injury-Triggered Blueing Reactions of Psilocybe “Magic” Mushrooms

Upon injury, psychotropic psilocybin-producing mushrooms instantly develop an intense blue color, the chemical basis and mode of formation of which has remained elusive. We report two enzymes from Psilocybe cubensis that carry out a two-step cascade to prepare psilocybin for oxidative oligomerization that leads to blue products. The phosphatase PsiP removes the 4-O-phosphate group to yield psilocin, while PsiL oxidizes its 4-hydroxy group. The PsiL reaction was monitored by in situ ¹³C NMR spectroscopy, which indicated that oxidative coupling of psilocyl residues occurs primarily via C-5. MS and IR spectroscopy indicated the formation of a heterogeneous mixture of preferentially psilocyl 3- to 13-mers and suggest multiple oligomerization routes, depending on oxidative power and substrate concentration. The results also imply that phosphate ester of psilocybin serves a reversible protective function.     

Molecular dynamics simulation studies suggests unconventional roles of non-secretary laccases from enteropathogenic gut bacteria and Cryptococcus neoformans serotype D

Laccase in Cryptococcus neoformans is covalently linked to the carbohydrate moiety of the cell wall, which allows it to get access to the different substrates for catalyzing their oxidation and therefore plays a vital role in the virulence. The laccase gene (3.0 kb) from C. neoformans serotype D was amplified, cloned and sequenced for protein modeling, docking and simulation studies. The three dimensional homology models of laccase protein from C. neoformans and other pathogenic gut bacteria were docked with selected biomolecules like prostaglandins (PG), membrane phospholipids, neurotransmitters (serotonin) using GOLD software. The GOLDscore values of laccase from C. neoformans docked with prostaglandinH₂ (59.76), prostaglandinG₂ (59.45), prostaglandinE₂ (60.99), phosphatidylinositol (54.95), phosphatidylcholine (46.26), phosphatidylserine (55.26), arachidonic acid (53.08) and serotonin (46.22) were similar to the laccase from enteropathogenic bacteria but showed a better binding affinity as compared to that of the non-pathogenic bacteria (e.g. Bacillus safensis, Bacillus pumilus and Bacillus subtilis). The RMSD of MD simulation study done for 25 ns using laccase protein from C. neoformans complexed with phosphatidylcholine was found to be highly stable, followed by the laccase-PGE₂ and laccase-serotonin complexes. Furthermore, the binding free energy results were found to support the docking and MD simulation results. The present study implies that few candidate ligands can be intermediate substrate in the catalysis of microbial laccases, which can further play some crucial role in the cell signaling and pathogenesis of enteropathogenic gut micro flora and C. neoformans.     

Comparison of TEMPO and its derivatives as mediators in laccase catalysed oxidation of alcohols

A series of TEMPO (2,2′,6,6′-tetramethylpiperidinyl-1-oxy) derivatives were studied as mediators of laccase (from Trametes versicolor) in the oxidation of benzyl alcohol and 1-phenylethyl alcohol. TEMPO (1), 4-hydroxy-TEMPO (2) and 4-acetylamino-TEMPO (4) turned out to be the most active mediators for laccase. In addition, 4-acetylamino-TEMPO and 4-hydroxy-TEMPO were more active in the oxidation of 1-phenylethanol compared to TEMPO. For these mediators kinetic isotope effects in the range of 2.1-3.2 were observed for α-monodeutero-p-methylbenzyl alcohol oxidation. These values are consistent with a mechanism involving oxoammonium intermediacy. Competition experiments between benzyl alcohol and 1-phenylethanol showed that TEMPO and its derivatives react faster with primary alcohols than with secondary alcohols, also in line with the proposed mechanism.     

壳寡糖化学修饰对云芝漆酶催化活性和酶学性质的影响

利用壳寡糖(COS)对纯化后的云芝漆酶(EC1.10.3.2)进行化学修饰以改善其活性和稳定性.最优化学修饰条件:温度5℃,pH=4.0,100mgNaIO4,20mgCOS.在此条件下得到的修饰酶(COS-TvL)的活性(456.00U/mg)为天然酶(TvL)活性(314.39U/mg)的145.04%.十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDSPAGE)结果表明,TvL的3条肽链相对分子质量为58100,45200和21600,而COS-TvL的3条肽链相对分子质量为62900,53300和39700,说明酶分子修饰成功,氨基修饰率为30.7%.TvL的最适pH值在4.0附近,而COS-TvL的最适pH值在3.5~4.0之间.TvL的最适温度为40℃,而COS-TvL的最适温度为45℃.动力学研究结果显示,TvL的Km=9.929mmol/L,vmax=714.29mmol/(mg·min);而COS-TvL的Km=8.989mmol/L,vmax=1250mmol/(mg·min).通过红外光谱、紫外吸收光谱、紫外差示光谱和荧光发射光谱对TvL和COS-TvL进行了结构表征.     

介孔SiO2/Fe3O4中空磁性微球的漆酶固定化

以介孔SiO2/Fe3O4磁性中空微球作为载体,采用物理吸附法对漆酶进行固定化,考察了时间、温度和pH值对漆酶固定化效果的影响,并对固定漆酶的活性及稳定性进行了研究.结果表明,介孔SiO2/Fe3O4磁性中空微球吸附漆酶分子后,介孔材料的比表面积与孔体积均减小.在3 h时复合微球对漆酶的吸附达到平衡,复合微球中介孔SiO2对漆酶的有效固定量为689 mg/g,大大高于纯介孔材料MCM-41的漆酶固定量(319 mg/g).在pH=3～6的条件下,复合微球中固定漆酶仍保持70%以上的相对酶活.当温度不高于60℃时,固定漆酶的相对酶活仍保持65%以上.固定漆酶的pH稳定性和热稳定性都明显优于游离漆酶,固定漆酶的米氏常数为1.05 mmol/L,与游离漆酶相比,固定漆酶与底物的亲和力有所降低.当2,4-二氯苯酚的浓度为10 mg/L时,固定漆酶对其去除率在6 h时达到81.6%,表现出很好的催化活性.     

C-N coupling of 3-methylcatechol with primary amines using native and recombinant laccases from Trametes versicolor and Pycnoporus cinnabarinus

Five laccase genes from Pycnoporus cinnabarinus and Trametes versicolor encoding for different isoenzymes have been cloned, recombinantly expressed and characterized. Following C-N coupling of primary linear, branched-chained and cyclic amines to 3-methylcatechol was mediated by native and recombinant laccases yielding the corresponding secondary amines. Formation of C5-monoaminated ortho-methylquinones occurred within 1-2 h; prolonged incubation led to the formation of high-molecular mass products. No difference between the use of native or recombinant isoenzymes from P. cinnabarinus or T. versicolor was observed. Optimization of the reaction conditions included variation of amine donor ratios, pH, amount and type of enzyme preparations. The formation of by-products could be suppressed at pH values corresponding to the enzymes optima (pH 4-5). A total of 10 secondary amines were synthesized with product formations of up to 80%. Furthermore, all purified secondary amines were characterized by NMR-, LC-MS- and HRMS-analysis and log P values were determined.     

Comparative Spectroelectrochemical Studies of Lyophilized and Nonlyophilized Laccases from Cerrena unicolor Basidiomycete

The electrochemical, spectroelectrochemical, and kinetic investigations of two preparations of Cerrena unicolor laccase, lyophilized (LLAC) and nonlyophilized frozen enzymes (FLAC), were performed. It was found that the value of the redox potential of the T1 site of C. unicolor laccase is ca. 750 vs. NHE. It was also shown that one of the redox potentials of the T2/T3 cluster of C. unicolor laccase is close to 400 mV, as was previously confirmed for other blue multicopper oxidases, such as trees and fungal laccases, ascorbate oxidase, and bilirubin oxidase. Furthermore, the poor stability of both preparations, but especially of LLAC, in their reduced state was confirmed using mediated and mediatorless spectroelectrochemical studies. DET‐based biocatalytic reduction of O₂ by C. unicolor laccase was only obtained, when FLAC was directly adsorbed on a spectrographic graphite electrode. Moreover, only low values of the steady‐state potentials of gold and graphite electrodes modified by C. unicolor laccase were also found. Heterogeneity of the 3‐D structures of laccase molecules, conformational changes, and partial denaturation of the enzyme, which appeared after enzyme isolation, purification, and especially lyophilization, were found to be the reasons for the low bioelectrocatalytic current, the high K_M‐value towards O₂, and the unusual electrochemical behavior of C. unicolor laccase used in the present study. In spite of the comparable specific activity and long‐term stability of both preparations in homogeneous solution, the stability of immobilized LLAC was found to be inadmissibly low for both fundamental studies and possible electrochemical applications. Indeed, FLAC is a much better source of enzyme than its lyophilized counterpart.