紫外、~(60)Co复合诱变选育烟色烟管菌漆酶高产突变株

对烟色烟管菌(Bjerkandera fumosa 5.0172)的孢子悬浮液进行紫外诱变筛选得到一株诱变株ZW-7,其产漆酶的活力是原始菌株的1.33倍,继代培养5代后,未见酶活下降。将诱变株ZW-7的孢子悬浮液用60Co-γ射线进行辐射诱变筛选得到一株诱变株Co-11,其产漆酶的活力是ZW-7的1.18倍,为380.5 U/L,较原始菌株的250.6 U/L提高了约58.1%,继代培养5代后,酶活稳定。     

栓孔菌属漆酶高产菌株的初步筛选及其产酶条件的优化

利用显色反应对栓孔菌属(Trametes)进行了漆酶高产菌株的筛选,并对目标菌株的产酶条件进行了优化,在添加愈创木酚的固体培养基中,通过显色反应初步筛选出漆酶高产菌株东方栓孔菌Trametes orientalis Cui 6300;进一步通过单因子分析、正交试验和ABTS法确定了菌株Cui 6300的最适产酶条件:麦芽糖15 g/L,蛋白胨3 g/L,pH 4.8,Cu2+2.0 mmol/L,培养温度28°C,接种饼直径1.5 cm,此时酶活最高可达19.923 U/mL;同时探索了Cu2+浓度及添加时间对其菌丝生物量和漆酶活力的影响。研究表明,Cu2+最适添加浓度为2.0 mmol/L,添加时间为接种后第3天。     

一株产漆酶菌株的筛选鉴定和发酵条件的研究

以愈创木酚为底物,采用平板筛选法筛选得到一株产漆酶菌株WS1-2,形态学特性表明该茵属于绿色木霉.对产酶条件的初步研究结果表明,WS1-2菌株的产酶高峰期出现在接种培养后的第4d.与蔗糖、乳糖、半乳糖和可溶性淀粉相比,以葡萄糖为碳源时,发酵上清液的漆酶活力明显要高,最大值达230U/L.以NH4Cl为氮源,最有利于WS1-2漆酶的产生,漆酶活力最高可达到234U/L.0.01mmol/L的愈创木酚和ABTS对WS1-2产漆酶有明显的诱导作用,3～5mg/L的Tween-80可以明显提高WS1-2的产酶水平.     

茶树内生真菌CSN-4产漆酶培养基及培养条件研究

从茶树内生真菌筛选产漆酶的菌株,分析不同营养因素和培养条件对菌株漆酶酶活力的影响。采用6种显色底物的平板初筛和酶活测定的复筛方法,从15株茶树内生真菌菌株中筛选获得1株产漆酶酶活较高的菌株CSN 4。单因素分析结果显示,液态发酵条件下菌株CSN-4适宜的主要培养基成分是麸皮和蛋白胨;菌株CSN-4分别在麸皮30 g/L、蛋白胨2.5 g/L、CuSO₄·5H₂O 0.015 g/L和茶水6 g/L时发酵产漆酶酶活最高。发酵条件试验结果表明,菌株CSN-4分别在接种量为6个菌饼（直径6 mm）、装液量60 mL/250 mL、pH 4.8、摇床转速120 r/min,培养温度为28 ℃时产漆酶酶活较高。在培养基中添加麸皮和茶水对菌株CSN-4产漆酶有明显的促进作用。经过培养基成分及培养条件优化后,菌株CSN 4产漆酶酶活显著升高,达到2 417 U/L。     

碳源和氮源对彩绒革盖菌液体发酵合成漆酶的影响

研究了碳源、氮源对彩绒革盖菌液体发酵合成漆酶的影响。结果表明,在所选的几种碳、氮源中,麸皮为试验菌株合成漆酶的较好碳源;酵母浸膏、酒石酸铵、蛋白胨均是比较理想的氮源。不同的碳氮比对彩绒革盖菌漆酶的产量有着显著的影响,高碳高氮是其生产漆酶的最佳条件。在适宜的培养条件下该菌株能合成高活力的漆酶,其酶活力可达298 U/mL以上,产酶周期为6~7 d。     

粗毛栓菌诱变菌株SAH-12漆酶的分离纯化及酶学性质研究

粗毛栓菌Trametes gallica诱变菌株SAH-12是通过紫外诱变选育所得的漆酶高产菌株,Active-PAGE分析表明SAH-12在高氮低碳无机盐培养液(LM3)中至少分泌3种漆酶同工酶(Lac1、Lac2、Lac3)。采用硫酸铵盐析、透析和Sephadex-G75分子筛层析从其培养液中分离纯化得到电泳纯的Lac1,纯化倍数6.54,酶活性回收59.7%。Lac1经SDS-PAGE验证为一条带,其表观分子量为61.5kDa。Lac1为一种糖蛋白,含糖量11.6%,等电点pI4.40,催化氧化底物ABTS的最适反应温度为60℃,最适pH为2.6,Km值为25μmol/L。Lac1在40℃(pH4.0)以下和pH1.5～5.0(28℃)范围内稳定。金属离子Fe2+、Ag+、Hg2+和Cr3+与抑制剂DTT、SDS、EDTA和DMSO对Lac1有抑制作用,其中Fe2+和DTT完全抑制酶活,而Cu2+对酶有明显激活作用,Mn2+、Zn2+对酶活影响不大。Lac1不仅可使一些合成染料明显脱色,而且对苹果汁多酚祛除也有较好效果。40℃用该酶(1U/mL)处理苹果汁5h,其多酚含量可降低40%。     

粗毛栓菌诱变菌株SAH-12漆酶的分离纯化及酶学性质研究

粗毛栓菌Trametes gallica诱变菌株SAH-12是通过紫外诱变选育所得的漆酶高产菌株,Active-PAGE分析表明SAH-12在高氮低碳无机盐培养液（LM3）中至少分泌3种漆酶同工酶（Lac1、Lac2、Lac3）。采用硫酸铵盐析、透析和Sephadex-G75分子筛层析从其培养液中分离纯化得到电泳纯的Lac1,纯化倍数6.54,酶活性回收59.7%。Lac1经SDS-PAGE验证为一条带,其表观分子量为61.5kDa。Lac1为一种糖蛋白,含糖量11.6%,等电点pI4.40,催化氧化底物ABTS的最适反应温度为60℃,最适pH为2.6,Km值为25μmol/L。Lac1在40℃（pH4.0）以下和pH1.5～5.0（28℃）范围内稳定。金属离子Fe2＋、Ag＋、Hg2＋和Cr3＋与抑制剂DTT、SDS、EDTA和DMSO对Lac1有抑制作用,其中Fe2＋和DTT完全抑制酶活,而Cu2＋对酶有明显激活作用,Mn2＋、Zn2＋对酶活影响不大。Lac1不仅可使一些合成染料明显脱色,而且对苹果汁多酚祛除也有较好效果。40℃用该酶（1U/mL）处理苹果汁5h,其多酚含量可降低40%。     

链霉菌产木聚糖酶条件和酶学性质以及重离子诱变对产酶的影响研究

以一株由青藏高原牦牛粪中分离出的链霉菌为出发菌株,对其培养特性、产酶条件和酶学性质进行初步研究.通过重离子诱变,筛选出遗传稳定的高产菌株.结果表明,该菌以玉米芯和麸皮(1∶1)为碳源能高效地诱导木聚糖酶的胞外分泌,其最适培养基和培养条件氮源为酵母膏、初始pH7、培养温度为25℃,在此条件下,第4天酶活力达到峰值3480.25 U/mL.说明该酶能够利用农业废弃物高效生产木聚糖酶.该菌株所产木聚糖酶的最适反应温度为15℃、pH4,属低温酸性木聚糖酶.经重离子诱变后,筛选出一株高产菌株SZ10-7,其酶活力可达5 338.42 U/mL.     

荷叶离褶伞高产漆酶菌株选育及其漆酶酶学特性

应用原生质体紫外诱变,对一株产漆酶荷叶离褶伞(Lyophyllum decastes)菌株紫外诱变,用愈创木酚马铃薯葡萄糖固体平板变色法初筛,然后用ABTS[2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐]测定漆酶活性进行复筛,获得1株漆酶高产诱变菌株HY1022-01;对HY1022-01所产漆酶粗酶液酶学特性进行研究,结果表明,HY1022-01所产漆酶最大酶活力比出发菌株提高30.32%,最适条件下酶活达991.67 U/mL,且产酶稳定;HY1022-1所产漆酶最适作用温度为35℃,在30~35℃较为稳定;最适反应pH值为3.0,pH在2.2~3.8较为稳定,ABTS的漆酶动力学常数为72.622μM。     

黄孢原毛平革菌产漆酶优化培养及其对刚果红的脱色降解

以白腐真菌模式菌株黄孢原毛平革菌Phanerochaete chrysosporium为研究对象,探讨培养条件、重金属和芳香族化合物对产漆酶的影响,并进一步研究漆酶对刚果红的降解效果。结果表明,P. chrysosporium产漆酶最适培养条件：葡萄糖为碳源,蛋白胨为氮源,碳氮比为90。培养8d后,漆酶酶活为911.1U/L;Mn²⁺严格地控制着P.chrysosporium产漆酶,而Cu²⁺对其影响不大,在添加4.0mmol/L Mn²⁺时,漆酶酶活为2 001.7U/L;芳香族化合物中藜芦醇（veratryl alcohol,VA）、4-香豆酸、香草醛和香草酸对菌体产漆酶能力均有促进作用,最高可提升至1 459.1U/L,而咖啡酸对菌体产漆酶稍有抑制。100U/L漆酶粗酶液可降解40mg/L刚果红,降解率为24.0%;而当介体物质VA存在时,该降解效率可提升至87.7%。     

黄孢原毛平革菌突变株抗碳氮营养阻遏产漆酶碳氮生理调控机理

【目的】通过比较不同碳氮营养及其消耗对产漆酶的影响,了解白腐菌模式种黄孢原毛平革菌解除营养阻遏产漆酶代谢的生理生态特性,揭示白腐菌合成漆酶的碳氮生理调控机理。【方法】分别利用限碳限氮(CL-NL)、限碳富氮(CL-NS)、富碳限氮(CS-NL)与富碳富氮(CS-NS)4种条件培养黄孢原毛平革菌野生型(WT)与突变株,比较两者产漆酶动力学、菌体生长、葡萄糖与氨氮消耗差异及其相关性来揭示解除营养阻遏产漆酶调控生理特性,明确C、N营养对产漆酶的生理调控途径。【结果】突变菌株除消耗速率比野生型略慢外,两者氨消耗趋势一致,但对葡萄糖的消耗比野生型快且氨氮浓度对葡萄糖的消耗影响不大。在CL-NL、CL-NS、CS-NL、CS-NS 4种培养条件下,野生型分别在培养后期的第11、14、19和19天的次生代谢时期产生0.107、0.029、12.84和18.05U/L漆酶,启动漆酶合成及酶峰值出现的时间与基质中葡萄糖耗尽或接近耗尽的时刻,或同氨氮消耗至最低值的时刻相对应;与WT产漆酶特性不同,突变株产漆酶伴随整个培养过程且均有两个产酶高峰,分别在培养的第8、7、12天和12天出现298.83、343.14、271.22、251.49U/L漆酶第一个产酶高峰,在培养的第12、13、19和19天产生257.69、298.78、213.81、216.93U/L漆酶的第二个产酶高峰。碳氮营养对产酶的影响显示:两菌株只要初始碳源浓度相同(限碳或富碳),各自产酶动力学趋势基本一致;相反,即使初始氮源浓度相同但其产酶动力学趋势却不同,说明碳源对黄孢原毛平革菌产漆酶的影响比氮源更为重要。【结论】野生型黄孢原毛平革菌产漆酶受碳或氮饥饿调控,碳、氮各自独立发挥作用且在不同的营养条件下由不同营养素所调控,如在限碳条件下产漆酶主要由葡萄糖饥饿启动,而在富碳条件下则由氨氮饥饿所激发,以碳或氮菌体负荷表示是否达到启动酶合成的调控阀值比单纯碳或氮浓度更为合理。突变菌株漆酶合成的启动不受碳、氮营养所阻遏,可能涉及一个全局调控的改变,解除了漆酶合成的营养阻碍。     

Effect of carbon,nitrogen sources and inducers on ligninolytic enzyme production by Morchella crassipes

The effect of different carbon, nitrogen sources and inducers on growth and ligninolytic activity by Morel mushroom Morchella crassipes was investigated. The maximum growth was observed in mineral salts broth containing glucose as the carbon source and sodium nitrate as the nitrogen source. Among the inducers, chemical inducers inhibited the growth whereas in natural substrates, growth was not affected much. Manganese peroxidase and lignin peroxidase activity were not detected in the medium with different carbon and nitrogen sources, whereas laccase activity varied depending on carbon source (0.7–3.48 U/ml). Among the inducers, natural inducers resulted in an increase in the enzyme activities. Maximum laccase activity was observed in rice straw (12. 6 U/ml) followed by ABTS (11.6 U/ml); Manganese peroxidase activity was maximum in rice straw (14.32 U/l) wheat straw (12.16 U/l) and phenol red (15 U/l) as the inducers, whereas for Lignin peroxidase activity, rice straw (22 U/l), wheat straw (16 U/l) and veratrylalcohol (20 U/l) served as the best inducers.     

Enhancement of laccase production by Pleurotus ostreatus and its use for the decolorization of anthraquinone dye

The white-rot fungus Pleurotus ostreatus strain 32 is an excellent producer of the industrially important enzyme laccase. Laccase was the only ligninolytic activity detected in the supernatant when the fungus was grown in liquid culture with or without shaking. Growth and laccase production in static cultivation were superior to that in agitated cultivation, and N-limited culture is of benefit to laccase production. When using cellobiose and peptone as carbon and nitrogen source, a higher activity level was obtained. 2,2′-Azino-di-(3-ethylbenzothialozin-6-sulfonic acid) (ABTS) (1 mM) was shown to be the best inducer of laccase production, reaching maximum values of about 400 U/ml. Cu²⁺ (1 mM) also had a positive effect on laccase production, activity being enhanced to 360 U/ml. In addition, anthraquinone dye SN4R can be effectively decolorized by crude laccase (30 U/ml), the rate of which was 66%. The decolorization rate was increased by 90% with ABTS (0.16%) addition as a mediator of laccase.     

Pestalotiopsis mangiferae isolated from cocoa leaves and concomitant tannase and gallic acid production

The enzyme tannase is of great industrial and biotechnological importance for the hydrolysis of vegetable tannins, reducing their undesirable effects and generating products for a wide range of processes. Thus, the search for new microorganisms that permit more stable tannase production is of considerable importance. A strain of P. mangiferae isolated from cocoa leaves was selected and investigated for its capacity to produce tannase enzymes and gallic acid through submerged fermentation. The assessment of the variables affecting tannase production by P. mangiferae showed that tannic acid, ammonium nitrate and temperature were the most significant (8.4 U/mL). The variables were analyzed using Response Surface Methodology - RSM (Box-Behnken design), with the best conditions for tannase production being: 1.9% carbon source, 1% nitrogen source and temperature of 23 °C. Tannase activity doubled (16.9 U/mL) after the optimization process when compared to the initial fermentation. A pH of 7.0 was optimal for the tannase and it presented stability above 80% with pH between 4.0 and 7.0 after 2h of incubation. The optimal temperature was 30 °C and activity remained at above 80% at 40–60 °C after 1 h. Production of gallic acid was achieved with 1% tannic acid (0.9 mg/mL) and P. mangiferae had not used up the gallic acid produced by tannic acid hydrolysis after 144 h of fermentation. A 5% tannic acid concentration was the best for gallic acid production (1.6 mg/mL). These results demonstrate P. mangiferae’s potential for tannase and gallic acid production for biotechnological applications.     

Molecular diversity of tannic acid degrading bacteria isolated from tannery soil

AIMS: The aim of this study was to enrich and isolate bacteria from a tannery soil that were capable of utilizing tannic acid and gallic acid as sole source of carbon aerobically, and to characterize their diversity in order to identify efficient strains that can be used for tannin bioremediation. METHODS AND RESULTS: Bacterial strains were isolated after enrichment in minimal medium with tannic acid or gallic acid as sole carbon source. Polymerase chain reaction (PCR) restricted fragment length polymorphism of 16S rDNA [amplified ribosomal DNA restriction analysis (ARDRA)] and BOX-PCR was used to characterize their diversity. Two strains showing relatively high efficiency in degrading tannic acid and gallic acid were identified on the basis of carbon source utilization pattern (BIOLOG) and 16S rDNA sequence. CONCLUSIONS: Bacterial strains capable of degrading tannic acid and gallic acid could be grouped into six and seven clusters on the basis of ARDRA and BOX-PCR, respectively. On the basis of 16S rDNA sequence, the most efficient isolate degrading tannic acid belonged to Pseudomonas citronellolis, whereas the most efficient gallic acid degrader showed maximum phylogenetic relatedness to P. plecoglossicida. SIGNIFICANCE AND IMPACT OF THE STUDY: Aerobic tannic acid degraders such as the two strains isolated in this study can be used for tannin bioremediation, and in the study of genes involved in the production of tannase, an industrially important enzyme.     

Tannic acid interferes with the commonly used laccase-detection assay based on ABTS as the substrate

Laccase enzymatic activity in biological samples is usually detected spectrophotometrically through its capacity to oxidize several specific aromatic compounds. One of the most commonly used substrates is the compound 2-2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), which becomes green-blue coloured when it is oxidized by laccase. In this work we study the interference of tannic acid with the spectrophotometric assay to detect laccase by using ABTS as the substrate. Our data show that under the normal reaction conditions of this assay, but in the absence of any catalyst, tannic acid is able to carry out the chemical reduction of the oxidized specie of ABTS, thus decreasing the overall detectable laccase-activity values observed when this enzyme is present in the reaction mixture. Therefore, our results represent an important warning concerning a commonly used method for measuring, detecting or screening laccases in biological samples that may content tannic acid or structural-related molecules.     

影响白腐真菌AH28-2菌株漆酶合成的因子和发酵条件

White-rot fungus AH28-2, a newly isolated strain, produced effectively laccase by induction when grown on a synthetic medium. Aromatic compounds of low molecular weight had an inducing influence on laccase production and its isoenzyme compositions. The using of o-toluidine or syringic acid had the best inducing effect. Cu2+ concentration in medium had distinguished effect on laccase production. Enzyme activity was notably increased by Cu2+ and reached the maximum when Cu2+ final concentration was 5 mumol/L. Mn2+ inhibited the synthesis of laccase. Carbon and nitrogen limitation were not beneficial to laccase synthesis, while high nutrient organic medium was beneficial to the growth of cell and the synthesis of laccase. Using cellobiose as the sole carbon source, the highest level enzyme activity reached 82,923. 7 u/L under the condition of optimum fermentation with ABTS as substrate. This enzyme activity was 2.9-fold higher compared to the reported data on international references in recent years.     

Effects of carbon and nitrogen sources on Pleurotus ostreatus ligninolytic enzyme activity

Summary The effect of various carbon and nitrogen sources on laccase, manganese-dependent peroxidase (MnP), and peroxidase production by two strains of Pleurotus ostreatus was investigated. The maximal laccase yield of P. ostreatus 98 and P. ostreatus 108 varied depending upon the carbon source from 5 to 62 U l⁻¹ and from 55 to 390 U l⁻¹, respectively. The highest MnP and peroxidase activities were revealed in medium supplemented by xylan. Laccase, MnP, and peroxidase activities of mushrooms decreased with supplementation of defined medium by inorganic nitrogen sources. Peptone followed by casein hydrolysate appeared to be the best nitrogen sources for laccase accumulation by both fungi. However, their positive effects on enzyme accumulation were due to a higher biomass production. The secretion of MnP and peroxidase by P. ostreatus 108 was stimulated with supplementation of casein hydrolysate to the control medium since the specific MnP and peroxidase activities increased 15-fold and 3.5-fold, respectively.