白腐菌对培养环境pH的调节及其产漆酶的相关性

研究了不同种属白腐菌多孔菌属C1(Polyporussp.)、侧耳属B2(Pleurotussp.)、香菇属A3(Lentinusedodes)对培养环境pH的调节,及其与菌株产漆酶状况的相关性。结果表明:3株白腐菌均可调节培养液酸化,培养环境的pH可分别由初始的4.5~5.0持续下降至培养结束时3.0~3.5水平,相应C1、B2、A3三者的漆酶合成与分泌在pH值较高(pH 4.5以上)时均表现较弱(酶活分别处于约1 500、200、50 U/mL以下的水平),在pH 3.2~4.5范围内的不同偏酸性条件下则得到较好表达(最高酶活分别达5 000、340、144 U/mL)。统计学分析指出,白腐菌调节环境pH状况与其产漆酶状况之间存在显著或极显著相关性,但不同白腐菌调节培养环境pH的能力之间并不具有显著性差异。     

白腐真菌AH28-2菌株发酵合成漆酶初步研究

从224个野外采集的真菌样品中筛选分离到一株产漆酶活性较高菌株AH28－2,经初步鉴定为白腐真菌,采用单因子相互比较法,研究了该菌株最适发酵产酶条件。应用添加有1g／LKraft木素的液体发酵培养基,接种量5％（V／V）,初始pH8．5,装液量为50％,28℃、150r/min摇瓶振荡培养4－5d,漆酶酶活水平达20184IU／L。     

Aspergillus sp.脂肪酶发酵条件优化及酶学性质的研究

作者为了得到一种热稳定性较好的脂肪酶新酶种,通过研究分离白极端环境的Aspergillus sp．的最佳产酶条件及其所产脂肪酶的酶学性质,得出了该菌产酶的最佳发酵条件为：以1％黄豆饼粉为氮源、0．2％玉米淀粉为碳源,1．5％橄榄油为诱导物,起始pH6．0左右。装量10mL（250mL三角瓶。摇瓶转速180r／min）、发酵时间为96h。在最佳发酵条件下可得最大发酵酶活36U/mL。Aspergillus sp．所产的脂肪酶的酶学性质是：最适pH为9．0,在pH5．0—10．0于20℃下放置24h后,残余酶活仍保持在起始酶活的90％以上;该酶的最适温度为50℃,50℃保温60min后仍保留70％以上的酶活。Aspergillus sp．所产脂肪酶的热稳定性较好。     

白腐真菌AH28-2菌株发酵合成漆酶初步研究*

从 2 2 4个野外采集的真菌样品中筛选分离到一株产漆酶活性较高菌株AH28-2 ,经初步鉴定为白腐真菌。采用单因子相互比较法 ,研究了该菌株最适发酵产酶条件。应用添加有 1g/Lkraft木素的液体发酵培养基 ,接种量 5 %(V/V) ,初始pH8 5 ,装液量为 5 0 %,2 8℃、 1 5 0r/min摇瓶振荡培养 4～ 5d ,漆酶酶活水平达 2 0 1 84IU/L。     

培养方式对Xenorhabdus nematophila生长与抗菌活性的影响

为了明确不同培养方式对Xenorhabdus nematophilaYL001生长和抗菌活性的影响,提高YL001菌株的抗菌活性。采用分批发酵方式研究了摇瓶与发酵罐培养对置nematophila YL001生长和抗菌活性的影响。实验结果表明：在通气量2．5L／min、搅拌转速300r／min条件下,发酵罐的体积氧传递系数KLa明显高于摇瓶,通气及供氧状况好于摇瓶,细胞生长及代谢旺盛,细胞生长量较摇瓶发酵增加了23．9％;但由于pH变化幅度大,抗菌物质的活性单位仅达到摇瓶的发酵水平,为229U／mL。发酵罐pH控制初步研究表明,发酵过程中控制pH为7．5时,细胞生长量和抗菌活性达到23．71g/L和290U／mL,较不控制pH分别增加了21％和25％。通风对X.nematophila YL001生长和抗菌物质的产生有较大的影响,发酵罐培养时通气及供氧状况好于摇瓶,有利于YL001菌株的生长和抗菌物质的产生。     

pH控制下绿色木霉（Trichoderma viride）流加发酵生产纤维素酶

绿色木霉（Trichodermaviride）在pH控制发酵条件下,采用流加葡萄糖发酵策略,可显著提高综合滤纸酶活力（FPA）和内切酶（endo—β—1,4-glucanase,EG）、外切酶exo—β-1,4-glucanase,CBH）、纤维二糖酶（cellobiase,CB）酶活。在5L发酵罐中采用pH控制和流加葡萄糖工艺,可提高CB酶含量,改变酶组分之间的比例,使得FPA、EG、CB和CBH酶活分别达到50．0U／mL,210．0U／mL,4．0U／mL和2．5U／mL,比摇瓶发酵分别提高了6．7．4．2、19、2．5倍。     

葡聚糖酶高产菌株的诱变筛选及其摇瓶发酵条件的初步研究

从土壤中分离出的一株产葡聚糖酶酶活31 U／ml的野生菌株,经UV、^60Co、LiCl诱变筛选后得到了产葡聚糖酶高产菌株SB126,经发酵条件优化试验后检测其酶活达到85U／ml,较野生菌株提高了近两倍。葡聚糖酶摇瓶较适发酵条件为：装量30ml(250m1)、转速180r／min、pH7．0、温度30℃、接种量8％、发酵周期5d。     

血红密孔菌高产漆酶菌株的筛选及其对烟梗的生物降解

白腐菌是目前已知的唯一能将木质素彻底降解的微生物,而漆酶在木质素分解过程中起着重要的作用,被广泛应用于农作物秸秆或甘蔗渣等多种类型生物质的生物预处理和生物降解。本研究利用白腐菌产漆酶发酵培养基对30株血红密孔菌Pycnoporus sanguineus菌株进行筛选,得到了多株漆酶高产菌株,并研究了血红密孔菌发酵粗酶液和菌丝对烟梗的生物降解条件。研究结果表明:血红密孔菌及其产生的漆酶表现出了对烟梗木质素较强的生物降解能力。在漆酶浓度为40U/mL、温度30℃、pH4.5的条件下处理24h,烟梗中木质素的降解率可达到50.4%,纤维素和半纤维素的降解率分别为17.5%和17.3%;漆酶浓度为5U/mL、温度30℃、pH4.5的条件下处理48h,木质素降解率可达到65.1%。血红密孔菌菌丝也表现出对烟梗较好的生物降解效果,接种培养7d后烟梗中木质素降解率可达30%以上,21d后木质素的降解率可达79.1%,而纤维素和半纤维素的降解率仅为20%和12%左右。本研究不但为生物质材料的生物预处理和生物降解提供了优质的白腐菌及漆酶资源,还为通过烟梗的生物预处理提高烟草梗丝和卷烟品质提供了重要参数,具有一定的应用前景。     

白腐菌紫外诱变选育高产漆酶突变株及其产酶条件研究

以白腐菌为出发菌株,利用紫外线(UV)进行诱变,筛选高产漆酶突变菌株。通过测定致死率绘制出发菌株的致死曲线,采用PDA-RBBR平板变色法进行初筛,ABTS检测酶活对突变株进行摇瓶复筛。结果表明:利用15 w紫外灯在照射距离为30 cm,照射时间为120 s,致死率为72.1%的条件下进行诱变处理,获得一株高产菌株,其酶活提高79.54%,经过5代传代培养,未见酶活下降,具有较好的遗传稳定性,进一步研究了初始pH值,接种量和培养基装液量等对诱变菌株产酶的影响,结果表明在最佳的培养条件pH值6.0,15%的装液量于28℃下,酶活达214.9 U/L。     

耐冷菌Bacillus cereus SYP-A3-2低温蛋白酶的发酵条件

研究了营养因子及环境条件对中国冰川1号耐冷菌Bacillus cereus SYP-A3-2产低温蛋白酶的影响。结果表明对该菌产酶影响较大的因素分别是温度、酪蛋白浓度和pH,在酪蛋白浓度0．8％、pH7、15℃下摇瓶发酵48h产酶水平可达3,800U／mL。     

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。     

毛栓菌产漆酶条件优化及该酶对合成染料脱色的特性

【目的】提高菌株Trametes hirsuta SYBC-L19漆酶产量,并研究该酶对合成染料脱色的性质。【方法】通过单因素和响应面设计,对产漆酶培养基进行优化。【结果】最优培养基为:玉米粉20.0 g/L、马铃薯淀粉32.4 g/L、酒石酸铵2.9 g/L、吐温80 0.5 g/L、CuSO4.5H2O 2.0 mmol/L、香兰素0.54 mmol/L、NaH2PO4.2H2O 2.0 g/L、MgSO4.7H2O0.5 g/L、MnSO4.H2O 0.1 g/L;最佳培养条件为:培养温度30°C,初始pH 6.0,装液量40 mL/250 mL,接种量8%。【结论】培养8 d酶活达35 U/mL,是优化前的39倍。对漆酶催化合成染料脱色进行了考察,发现该酶在60°C下对偶氮类染料AR1和RB5能迅速脱色,5 min内即可完成。     

Copper induction of lignin-modifying enzymes in the white-rot fungus Trametes trogii

Trametes trogii, a white rot basidiomycete involved in wood decay worldwide, produces several ligninolytic enzymes, laccase being the dominant one, with higher titers than those reported for most other white rot fungi studied up to date. The effect of copper on in vitro production of extracellular ligninolytic activities was studied. CuSO(4)·5H(2)O concentrations from 1.6 μM to 1.5 mM were tested in a synthetic medium with glucose 20 g/L and asparagine 3 g/L. The addition of copper (up to 1 mM) did not affect growth but strongly stimulated ligninolytic enzyme production; faster decolorization of the polymeric dye Poly R-478 was observed as well. Maximal production of manganese peroxidase, laccase, and glyoxal oxidase [1.28 U/mL, 93.8 U/mL (with a specific activity of 720 U/mg protein), and 0.46 U/mL respectively] was attained with 1 mM CuSO(4)·5H(2)O. However, higher copper concentrations inhibited growth and notably decreased manganese peroxidase production, although they did not affect laccase secretion. Laccase activity in the culture filtrate was maximal at 50 C and pH 3.4, and the enzyme was completely stable at pH 4.4 and above, and at 30 C for up to 5 d. Denaturing polyacrylamide gel electrophoresis of extracellular culture fluids showed two laccase activity bands (mol wt 38 and 60 kDa respectively). The pattern of isoenzyme production was not affected by medium composition but differed with culture age.     

Improving the bioremediation of phenolic wastewaters by Trametes versicolor

The successful bioremediation of a phenolic wastewater by Trametes versicolor was found to be dependent on a range of factors including: fungal growth, culture age and activity and enzyme (laccase) production. These aspects were enhanced by the optimisation of the growth medium used and time of addition of the pollutant to the fungal cultures. Different media containing 'high' (20 g/L), 'low' (2 g/L) and 'sufficient' (10 g/L) concentrations of carbon and nitrogen sources were investigated. The medium containing both glucose and peptone at 10 g/L resulted in the highest Growth Related Productivity (the product of specific yield and micro) of laccase (1.46 Units of laccase activity)/gram biomass/day and was used in all further experiments. The use of the guaiacol as an inducer further increased laccase activity 780% without inhibiting growth; similarly the phenolic effluent studied boosted activity almost 5 times. The timing of the addition of the phenolic effluent was found to have important consequences in its removal and at least 8 days of prior growth was required. Under these conditions, 0.125 g phenol/g biomass and 0.231 g o-cresol/g biomass were removed from solution per day.     

桑木层孔菌液体培养条件的研究

对桑木层孔菌(Phellinus mori)液体发酵条件进行了研究,以生物量和胞外多糖为指标,通过L16(45)和L9(34)正交表进行了两次正交试验,筛选出桑木层孔菌最适液体培养条件为:麦芽糖30 g/L,酵母浸粉和蛋白胨15 g/L(质量比2 1),KH2PO4和CaCl25.5 g/L(质量比1 1),初始pH6.0;通过单因素试验筛选出最适装液量为120 mL/250 mL,最适接种量为10%。在此条件下液体发酵培养7 d后,桑木层孔菌生物量达到23.375 g/L,胞外多糖产量达到3.993 g/L。     

Extracellular ligninolytic enzymes by Lentinus polychrous Lév. under solid-state fermentation of potential agro-industrial wastes and their effectiveness in decolorization of synthetic dyes

Six agro-industrial wastes were evaluated as a support for ligninolytic enzyme production by the white-rot fungus Lentinus polychrous Lév. under solid-state fermentation. Enzyme production was markedly different according to the substrate used. Rice bran (RB) yielded the highest laccase activity of 1,449 U/L (after 21 days of culture) with specific activity of 4.4 U/g substrate. Rice bran supplemented with rice husk (RH) (2:1 by wt) showed high laccase activity of 1,425 U/L with specific activity of 10.0 U/g substrate (after 17 days of culture). The crude enzyme of the RH-RB culture also contained manganese peroxidase (MnP) and manganese-independent peroxidase (MIP) activities in relative proportions of 1.9:1.4:1 of laccase:MnP:MIP, respectively. Zymogram studies showed the same isoenzyme pattern with these ligninolytic enzymes. The high enzyme production level and low substrate cost of SSF-L. polychrous Lév. suggest that it has potential for industrial applications. Our studies showed that the crude enzyme from this culture exhibited in vitro decolorization of Indigo Carmine. The highest efficiency of dye decolorization was observed under alkaline conditions (pH 9.0) at an initial dye concentration of 10 mg/L. The rather high pH conditions and high efficiency in Indigo Carmine decolorization make the enzyme further interest for the applications in treatment of waste water from the textile industry, which contains synthetic dyes.     

Production of biomass and ligninolytic enzymes by Pleurotus ostreatus in submerged culture.

The production of biomass and ligninolytic enzymes by Pleurotus ostreatus was analysed in synthetic medium with yeast extract and different glucose concentrations (0.5 - 20 g/l), at different pH (3.5-6.5) and incubation temperatures (23-32 degrees C). The best culture condition were: initial glucose concentration of 5 g/l, initial pH between 5.5-6.5 and incubation temperature between 26-29 degrees C. The saturation constant for glucose (Ks) was 1.75 g/l. The biomass concentration reached 8.6 g/l with a glucose addition of 20.0 g/l to the culture medium. The control of pH allowed an increment of 0.5 g/l of biomass concentration. The birreactor produced pellets with a homogeneous distribution of diameter size of 3.4 -/+ 0.2 mm. Approximately, 307 U/l of laccase and 0.41 U/l of manganese peroxidase were obtained in extracellular liquid medium and 0.015 U/g of laccase and 0.809 U/g of manganese peroxidase were obtained in solid substrate. Lignin peroxidase activity was not detected at any condition.     

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。     

1株甲苯降解真菌的筛选鉴定及其降解甲苯的特性

从实验室保存的7株真菌筛选到1株能高效降解甲苯的菌株H1,基于形态特征、ITS序列系统学分析,将H1菌株鉴定为毛栓菌（Trametes hirsuta）。利用正交设计实验方法研究了温度、pH值、甲苯浓度和吐温80浓度对H1菌株降解甲苯的影响,研究得出该菌株降解甲苯的最适条件为30℃、pH 5.0、甲苯浓度300mg/L、吐温80浓度0.05%,在该条件下H1对甲苯的最大降解率为85.3%,降解率比未优化之前有了显著提高。比较了H1菌株在3种培养基产生漆酶的能力,H1在土豆葡萄糖培养基产酶能力最强,在第7天达到酶活高峰16 500 U/L。H1在甲苯为唯一碳源的培养基中,漆酶酶活最低,培养7 d时漆酶酶活为589 U/L。     

Production of carbonyl reductase by Metschnikowia koreensis

A new strain of the yeast Metschnikowia koreensis was grown in shake flasks and a stirred bioreactor for the production of carbonyl reductase. The optimal conditions in the bioreactor for maximizing the biomass specific activity of the enzyme were found to be: a medium composed of glucose (20 g/L), peptone (5 g/L), yeast extract (5 g/L) and zinc sulfate (0.3g/L); the pH controlled at 7; the temperature controlled at 25 °C; an agitation speed of 500 rpm; and an aeration rate of 0.25 vvm. In the bioreactor, a biomass specific enzyme activity of 115.6 U/gDCW was obtained and the maximum biomass concentration was 15.3 gDCW/L. The biomass specific enzyme activity obtained in the optimized bioreactor culture was 11-fold higher than the best result achieved in shake flasks. The bioreactor culture afforded a 2.7-fold higher biomass concentration than could be attained in shake flasks.