Influence of glucose feeding on the ligninolytic enzyme production of the white-rot fungus Phanerochaete chrysosporium

The present work studied the influence of glucose feeding on the ligninolytic enzyme production of Phanerochaete chrysosporium in a nitrogen-limited (C/N ratio is 56/8.8 mmol/L) medium. Several sets of shaking flask experiments were conducted. The results showed that 2 g/L glucose feeding on the first day of the culture (24 h after the inoculation) simulated both fungal biomass growth and enzyme production. The manganese peroxidase (MnP) activity was 2.5 times greater than that produced in cultures without glucose feeding. Furthermore, the glucose feeding mode in fed-batch culture was also investigated. Compared to cultures with glucose feeding every 48 h, cultures with glucose feeding of 1.5 g/L (final concentration) every 24 h produced more enzymes. The peak and total yield of MnP activity were 2.7 and 3 times greater compared to the contrast culture, respectively, and the enzyme was kept stable for 4 days with an activity of over 200 U/L. Translated from Acta Scientise Circumstantiae, 2007, 27(3): 363–368 [译自: 环境科学学报]     

白腐真菌批式发酵动力学模型研究

为了进一步认识白腐真菌发酵产酶机理并指导相关发酵工艺设计,在一系列试验研究的基础上,建立了描述黄孢原毛平革菌批式发酵过程的动力学模型,并采用自由悬浮和固定化2种培养条件下的试验数据分别回归了模型各参数.模拟计算结果表明,生物量、葡萄糖、氨氮、木质素酶浓度的的计算值与实测值的偏差在15%以内.对比2种培养条件下的模型参数发现,二者得到的最大生物量浓度均为1.78 g/L;固定化培养菌体的比增长速率值(0.668 3 d-1)大于自由悬浮培养值(0.514 4d-1);自由悬浮培养中绝大部分葡萄糖消耗于非生长菌体代谢,固定化培养中则部分葡萄糖用于菌体生长,且氨氮消耗更快;菌体的产酶过程与生长无关;自由悬浮培养条件下,菌丝小球可合成MnP(231 U/L),其MnP合成能力为115.8 U·(g·d)-1,且高峰后依然可以保持26.1 U·(g·d)-1,适合采用补料-分批发酵的模式提高MnP的产量及其发酵效率;固定化培养条件下,菌体可同时合成MnP(410 U/L)和LiP(721 U/L),但其LiP和MnP合成能力在酶活高峰之后明显下降,分别由248.9 U·(g·d)-1和80.1 U·(g·d)-1下降到0 U·(g·d)-1和6.04 U·(g·d)-1,故而在考虑补料-分批发酵模式时,补料时机应选择在培养中期的LiP与MnP峰值之前.     

木质素过氧化物酶和锰过氧化物酶对染料脱色的性能比较

通过控制培养基中Mn2+的浓度获得只含有木质素过氧化物酶(LiP)或锰过氧化物酶(MnP)的单一酶培养物,用于比较LiP和MnP对染料降解脱色的条件和能力的差异.结果表明,LiP对橙Ⅰ降解脱色的最佳条件为h2O₂0.15 mmol/L,pH 3.0,温度40 ℃;MnP为h2O₂0.15 mmol/L,pH 3.5,温度40 ℃.最佳条件下,考察LiP和MnP作用下染料浓度和染料类型对脱色的影响,发现LiP作用下脱色率明显高于MnP作用下的脱色率,表明LiP可作用的染料浓度比MnP高,可作用的染料范围比MnP大.由此认为LiP具有比MnP更强的脱色能力.      

白腐真菌F2的生长及产木质素降解酶特性的研究

考察了从我国生物资源中筛分出的白腐真菌F2的生长特性,采用多因素试验法研究了F2菌产木质素降解酶的特性;用统计学方法对试验数据进行了误差分析,并分析了多种影响因素对F2菌产木质素降解酶的影响.实验结果表明:F2菌在PDA培养基平板上和Tien＆Kirk培养基中都生长良好且生长速率较高;在自由悬浮振荡培养(不通氧气)条件下,F2菌产LiP的最高酶活可达60 5U·L-1,MnP的最高酶活可达263 4U·L-1,其产酶能力超过了黄孢原毛平革菌在自由悬浮振荡培养(不另外通入氧气)条件下的产酶能力.本研究还发现向培养基中添加的Cu2+浓度超过1μmol·L-1时会抑制F2菌产LiP,以往的文献未报道过Cu2+的这种抑制作用.     

白腐真菌培养条件对其分泌木质素降解酶的影响

应用摇瓶试验研究了不同白腐真菌培养条件对其产酶的影响.通过控制不同转速、投加载体等方式考察了白腐真菌的产酶情况.结果表明,在悬浮培养方式下,转速为160r/min时获得的锰过氧化物酶最高(481.6U/L),其酶活是静置、120r/min条件下培养的65倍和43倍;投加载体后,载体固定化培养获得的木质素过氧化物酶酶活是悬浮培养的71倍,载体投加量对其产酶影响很大,只有当载体处于非浸没状态时才有利于酶活产生,否则酶活极低.因此,选择载体固定化—非浸没—振荡培养方式培养白腐真菌可获得更高的产酶量和酶活.     

白腐真菌木质素降解酶的反应器发酵

为了获得更高的白腐真菌木质素降解酶的产量和相应的控制策略,应用5L搅拌罐生物反应器对氮限制下(C/N=56/2.2)P.chrysosporium产木质素降解酶进行了放大研究.结果表明,在分批发酵试验中,锰过氧化物酶(MnP)和漆酶(Lac)分别在培养第6d和第7d达到峰值,其酶活随时间的变化规律与摇瓶试验基本相同;而采用氮限制液体培养基进行补料没有获得更高的酶活,因此,可以得出采用发酵液体培养基作为补料液不利于白腐真菌持续产酶.另外,在分批发酵和分批补料发酵过程中,均发现体系pH值变化与白腐真菌生长和次生代谢产酶具有较好的相关性,当白腐真菌进入次生代谢阶段开始产酶时,体系pH值开始下降,随着发酵后期酶活的降低,pH值下降的幅度也逐渐变小,当体系酶活接近为0.0U/L时,pH值基本不再变化.因此,在实际发酵过程中,可以依据体系pH的变化间接了解白腐真菌的生长和产酶情况,而分批补料策略还有待于进一步研究.     

Cooperation between ligninolytic enzymes produced by superior mixed flora

Since the ability to degrade lignin with one kind of white-rot fungi or bacteria was very limited, superior mixed flora‘s ability to degrade lignin was investigated by an orthogonal experiment in this paper. The results showed that superior mixed flora reinforced the ability to degrade lignin, the degradation rates of both sample 9 and 10 were beyond 80% on the day 9. The cooperation between lignin peroxidase(LiP), Mn-dependent peroxidase(MnP) and laccase (Lac) for lignin degradation was also studied. By examining the activities of three enzymes produced by superior mixed flora, it was found that Lac was a key enzyme in the process of biological degradation of lignin but Lip was not; the enzyme activity ratios of Lac/MnP and Lac/LiP were significantly correlative with the degradation rate of lignin at the 0.01 level; and the ratio of MnP/LiP was an important factor affecting the degradation rate of lignin.     

Cometabolic degradation of veratryl alcohol and biphenyl by white rot fungus under nitrogen nutrition-rich condition

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嗜碱细菌复合碳源条件下对麦草木质素的降解

在碱性液体培养条件下(pH≈10.5), 研究复合碳源共代谢最佳综合条件下嗜碱性木质素降解细菌6号菌株产酶、降解能力及菌株的生长状况.结果显示,虫漆酶(Laccase)在培养的第4天酶活达到最高值2915.37U/L、锰依赖过氧化物酶(MnP)在培养的第8天酶活达到最高值1152.88U/L,培养10d麦草中木质素降解49.84%.同时通过扫描电镜分析探讨了6号菌株降解木质素的微观过程,证明了6号菌株优先降解木质素的特性和降解方式.     

正交实验选择嗜碱细菌降解木质素的最优综合培养条件

在复合碳源的碱性液体培养条件下(pH≈10.5),用正交实验法对影响嗜碱木质素降解细菌6号菌株降解木质素的7种单因素培养条件进行了优化.结果表明,第1碳源用量等单因素在不同程度上对6号菌株产生的木质素降解酶Laccase和MnP以及木质素降解率都有一定促进作用.正交实验表明,其中第1碳源用量和T-80用量是显著性的影响因素,7种单因素的最佳综合水平为:37℃静置培养10d、第1碳源(蔗糖)1g/L、氮源NH₄NO₃1.2g/L、培养基70ml/250ml锥形瓶、初始pH值10.6和第2碳源接种后第4d加入、添加0.3%的表面活性剂T-80.     

Mesoporous silicas synthesis and application for lignin peroxidase immobilization by covalent binding method

Immobilization of enzymes on mesoporous silicas (MS) allows for good reusability. MS with two-dimensional hexagonal pores in diameter up to 14.13 nm were synthesized using Pluronic P123 as template and 1,3,5-triisopropylbenzene as a swelling agent in acetate buffer. The surface of MS was modified by the silanization reagents 3-aminopropyltriethoxysilane. Lignin peroxidase (LiP) was successfully immobilized on the modified MS through covalent binding method by four agents: glutaraldehyde, 1,4- phenylene diisothiocyanate, cyanotic chloride and water-soluble carbodiimide. Results showed that cyanotic chloride provided the best performance for LiP immobilization. The loaded protein concentration was 12.15 mg/g and the immobilized LiP activity was 812.9 U/L. Immobilized LiP had better pH stability. Acid Orange II was used to examine the reusability of immobilized LiP, showing more than 50% of the dye was decolorized at the fifth cycle.     

锰过氧化物酶(MnP)对农药毒死蜱降解的初步研究

以广泛使用的有机磷杀虫剂毒死蜱为研究对象,利用白腐菌P.sordida YK-624菌株所产锰过氧化物酶(MnP)对其进行生物降解的初步研究。首先比较确定了MnP降解毒死蜱所需要的降解体系,即对于毒死蜱的降解,需要在有机酸(丙二酸)存在的缓冲体系中进行,此体系不需要表面活性剂Tween 80,但必须含有Mn2+和葡萄糖氧化酶氧化葡萄糖产生的H2O2。随后对主要影响因素的试验结果表明,MnP降解毒死蜱较好的条件是在pH 4.5的丙二酸缓冲体系中含7.5 mmol/L MnSO4、2.5 mmol/L葡萄糖以及40 U葡萄糖氧化酶,温度30℃。此时,400 U/L的MnP可将30 mg/L的毒死蜱经3 d时间降解77.51%。同时,利用GC-MS对毒死蜱酶降解后的产物进行了初步分析,得到1种可能的酶解产物O,O,O’,O’-四乙基二硫代焦磷酸酯,不同于水解和细菌降解途径,值得进行进一步研究。     

木质素降解酶在不同堆肥基质中的吸附传输特性研究

为了深入探究木质素降解酶在不同堆肥基质中的吸附传输特性,通过批量实验对比了土壤、菜叶、稻草和米糠对木质素降解酶的吸附性能,并进行了动力学分析和等温吸附模型拟合,同时通过柱淋洗实验考察了木质素降解酶在4种堆肥基质中的迁移传输特性.结果表明,堆肥基质对木质素降解酶的吸附与基质种类有关,土壤、菜叶、稻草和米糠对木素过氧化物酶(LiP)和锰过氧化物酶(MnP)的吸附量分别为1.22、 1.27、 1.13、 1.22 U·g-1和5.09、 4.88、 4.43、 3.95 U·g-1.比较LiP和MnP吸附的动力学模型,准二级动力学方程为表征木质素降解酶吸附的最佳模型,其R2值为0.9732～0.9997, Elovich方程拟合较差,准一级动力学方程拟合最差;Langmuir模型对等温吸附数据进行拟合效果最好,而实验数据不适合用Freundlich方程表征.土壤、菜叶、稻草和米糠对LiP和MnP饱和吸附容量分别为1.23、1.30、1.17、1.14 U·g-1和5.70、 5.19、 4.73、 4.14 U·g-1.LiP和MnP在稻草和米糠基质中传输效果较好,可传输到最深层10 mL处,而在土壤和菜叶基质中则被滞留在浅层.     

原生质体紫外诱变提高简青霉的木质素降解性能

以简青霉Penicillium simplicissimum (Oudem.) Thom BGA为出发菌株,对其进行原生质体紫外诱变.经过筛选获得2株诱变菌株(J12与J18),它们的木质素降解酶酶活要明显高于出发菌株J.其中诱变菌株J12产生的漆酶和锰过氧化物酶酶活较高,相对于出发菌株J分别提高了145%和47%,达到44.0,50.9 U/g.诱变菌株J18产生的木质素过氧化物酶酶活较高,达到67.1 U/g,相对于出发菌株J提高了40%.且木质素降解率也最高,相对于出发菌株J分别提高了198.1%.经过7次传代, J12和J18的木质素降解酶活性保持稳定,没有降低.     

Polycyclic aromatic hydrocarbon biodegradation and extracellular enzyme secretion in agitated and stationary cultures of Phanerochaete chrysosporium

The extracellular enzyme secretion and biodegradation of polycyclic aromatic hydrocarbons (PAHs) were studied in agitated and shallow stationary liquid cultures of Phanerochaete chrysosporium. Veratryl alcohol and Tween80 were added to cultures as lignin peroxidase (LIP) and manganese peroxidase (MnP) inducer, respectively. Shallow stationary cultures were suitable for the production of enzyme, whereas agitated cultures enhanced overall biodegradation by facilitating interphase mass transfer of PAHs into aqueous phases. The use of a LIP stimulator, veratryl alcohol, did not increase PAH degradation but significantly enhanced LiP activity. In contrast, Tween80 increased both MnP secretion and PAH degradation in shallow stationary cultures. On the other hand, high PAH degradation was observed in agitated cultures in the absence of detectable LIP and MnP activities. The results suggested that extracellular peroxidase activities are not directly related to the PAH degradation, and the increased solubility rather than enzyme activity may be more important in the promotion of PAH degradation.     

木质素过氧化物酶在球型介孔材料上的固定化特性研究

在醋酸-醋酸钠缓冲体系(pH=3.5)中以聚乙二醇-聚丙三醇-聚乙二醇三嵌段共聚物(P123)为模板,正硅酸甲酯(TMOS)为硅源,1,3,5-三异丙基苯(TIPB)为扩孔剂合成了具有规则六方形孔道(11.6nm)的微米级球型颗粒.以合成的介孔材料为载体,采用物理吸附法对木质素过氧化物酶(LiP)进行固定化,研究了初始酶量和固定化时间等因素对固定化效果的影响,以及固定化对LiP酶学性质和稳定性的影响.结果表明,当初始酶量(E)与载体量(MS)比为76.8mg/g,固定化反应12h时,固定化LiP可获得最大蛋白质负载量(8.87mg/g)和最大表观活性(41.45U/mg).与游离酶相比,固定化LiP的最适pH和温度均未有明显变化,但pH稳定性和热稳定性都有了不同程度的提高,在4℃条件下保存7周后固定化LiP活性几乎没有损失,且在重复使用6次后,可保留近30%的活性.     

Biodegradation of pyrene by Phanerochaete chrysosporium and enzyme activities in soils: Effect of SOM, sterilization and aging

The impacts of soil organic matter （SOM）, aging and sterilization on the production of lignin peroxidase （LIP） and manganese peroxidase （MnP） by Phanerochaete chrysosporium during the biodegradation of pyrene in soils were investigated. The biodegradation of pyrene by P. chrysosporium decreased with increasing SOM content, whereas the maximum activities of LiP and MnP increased, which indicates that SOM outweighed pyrene in controlling enzyme production. Sterilization enhanced the degradation of pyrene due to the elimination of competition from indigenous microbes, whereas aging led to a reduction in the degradation of pyrene primarily through changes in its sorbed forms. Both sterilization and aging could reduce SOM content and alter its structure, which also influenced the bioavailability of pyrene and the enzyme activity. The sterilization and aging processes caused changes in the degradation of pyrene, and the enzyme activities were greater in soils with high SOM contents. MnP was related to the degradation of pyrene to a greater extent, whereas LiP was more related to the decomposition of SOM.     

黄孢原毛平革菌在多种氨氮浓度下木质素降解酶的产生

在氨氮浓度0．0308-0．924g／L，下对黄孢原毛平革菌(Phanerochaete chrysosporium)在空气环境中进行自由悬浮振荡培养，在氨氮浓度0．0308-0．308g／L下可检测到木质素过氧化物酶活性，最高酶活为42．8U/L；在氨氮浓度0．0308g／L和0．154g/L下可检测到漆酶活性，最高酶活为35．0U／L，在氨氮浓度不低于0．154g/L时，葡萄糖消耗速率大致相当，明显高于在氨氮浓度0．0308g／L下的葡萄糖消耗速率；氨氮在葡萄糖耗尽时达到最低值，然后开始增大；木质素过氧化物酶和漆酶的活性峰与葡萄糖和氨氮的最大消耗基本对应。实验结果对木质素降解酶发酵和白腐真菌在环境工程中的直接应用具有启发意义。     

葡萄糖补料对白腐真菌P.chrysosporium产木质素降解酶的影响

为了提高木质素降解酶的产量,采用氮限制培养基(C/N=56.0/8.8),研究了葡萄糖补料对P.chrysosporium产木质素降解酶的影响.结果表明,在培养第1d即接种24h后进行葡萄糖补料,可以刺激菌丝体的生长和产酶,补料浓度达到2g·L-1时,补料体系中的MnP酶活可提高至空白对照样(不进行补料)的2.5倍.同时,对葡萄糖分批补料方式进行的试验结果显示,培养过程中每24h进行1次补料,葡萄糖补料终浓度为1.5g·L-1的补料方式,与每48h的补料方式相比,产酶效果更好.MnP酶活峰值和达到峰值时酶的总产量可提高至空白对照样(不进行补料)的2.7倍和3.0倍,且酶活能够在200U·L-1以上稳定4d.实验结果说明,葡萄糖补料可以有效刺激白腐真菌产酶,且连续低浓度葡萄糖补料可获得较优的产酶效果.     

木质素过氧化物酶降解四环素机理的研究

为探究过氧化物酶对四环素类抗生素的降解机理,以白腐真菌Phanerochaete chrysosporium表达的木质素过氧化物酶(LiP)粗酶为研究对象,研究其产酶条件及其降解四环素(TC)的机理。结果表明:低浓度Mn2+可促进产酶,碳源、氮源限制是产酶的重要条件;降解反应进行10min后,H2O2消耗至阈值0.045mmol/L,LiP对TC的降解停止,其降解率达到82%;TC降解反应趋于平衡时,补加TC和H2O2后,LiP依然可以高效发挥作用,其二次降解率达到65%;LiP作用于不同初始浓度(10mg/L、25mg/L、50mg/L、100mg/L)TC,显示LiP对不同浓度TC均可有效降解,TC降解率分别为62%、80%、82%和90%;降解产物的抑菌性试验反映出降解产物较TC母体的抑菌性明显降低;LC/MS捕获到6种可能的降解产物E-TC、TP 461、E-TP 461、TP 477、TP 475和TP 445,并推测出LiP降解TC可能的降解途径。