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Guodong Liu,
Lei Zhang,
Yuqi Qin,
Gen Zou,
Zhonghai Li,
Xing Yan,
Xiaomin Wei,
Mei Chen,
Ling Chen,
Kai Zheng, [......],
Yaohua Zhong,
Weifeng Liu,
Huajun Zheng,
Shengyue Wang,
Chengshu Wang,
Luying Xun,
Guo-Ping Zhao,
Tianhong Wang,
Zhihua Zhou, Yinbo Qu
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ABSTRACT: Long-term strain improvements through repeated mutagenesis and screening have generated a hyper-producer of cellulases and hemicellulases from Penicillium decumbens 114 which was isolated 30 years ago. Here, the genome of the hyper-producer P. decumbens JU-A10-T was sequenced and compared with that of the wild-type strain 114-2. Further, the transcriptomes and secretomes were compared between the strains. Selective hyper-production of cellulases and hemicellulases but not all the secreted proteins was observed in the mutant, making it a more specific producer of lignocellulolytic enzymes. Functional analysis identified that changes in several transcriptional regulatory elements played crucial roles in the cellulase hyper-producing characteristics of the mutant. Additionally, the mutant showed enhanced supply of amino acids and decreased synthesis of secondary metabolites compared with the wild-type. The results clearly point out that we can target gene regulators and promoters with minimal alterations of the genetic content but maximal effects in genetic engineering.
Scientific Reports 03/2013; 3:1569.
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ABSTRACT: In this study, the functions of β-glucosidases in regulation of the lignocellulolytic enzymes production in Penicillium decumbens 114-2 were investigated. The major extracellular β-glucosidase gene bgl1 and the major intracellular β-glucosidase gene bgl2 were deleted in P. decumbens 114-2 respectively. In Δbgl2, the production of extracellular lignocellulolytic enzymes (including endoglucanases, cellobiohydrolases and xylanases) on insoluble cellulose was significantly promoted, while in Δbgl1 there was no any difference compared with that of 114-2. The enhancement of the production of lignocellulolytic enzymes in Δbgl2 was likely attributed to the accumulation of intracellular cellobiose. Induction experiment in Δbgl1Δbgl2 showed that cellobiose was an inducer of lignocellulolytic enzymes expression in P. decumbens 114-2, and the induction was unrelated to the formation, if any, of gentiobiose or sophorose from cellobiose.
Bioresource technology 03/2013; 137C:33-40. · 4.25 Impact Factor
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ABSTRACT: The current high cost of lignocellulolytic enzymes is a major bottleneck in the economic bioconversion of lignocellulosic biomass to fuels and chemicals. Fungal lignocellulolytic enzyme systems are secreted at high levels, making them the most promising starting points for further development of highly efficient lignocellulolytic enzyme systems. In this paper, recent advances in improvement of fungal lignocellulolytic enzyme systems are reviewed, with an emphasis on the achievements made using genomic approaches. A general strategy for lignocellulolytic enzyme system development is proposed, including the improvement of the hydrolysis efficiencies and productivities of current enzyme systems. The applications of genomic, transcriptomic and proteomic analysis methods in examining the composition of native enzyme systems, discovery of novel enzymes and synergistic proteins from natural sources, and understanding of regulatory mechanisms for lignocellulolytic enzyme biosynthesis are summarized. By combining systems biology and synthetic biology tools, engineered fungal strains are expected to produce high levels of optimized lignocellulolytic enzyme systems.
Biotechnology advances 03/2013; · 8.25 Impact Factor
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ABSTRACT: Cellulases from fungi typically contain one catalytic domain with or without a cellulose-binding domain. We characterized an endo-acting cellulase PdCel5C from industrial cellulase-producing fungus Penicillium decumbens with distinctive domain composition. In addition to a cellulose-binding domain and a catalytic domain, PdCel5C contains two immunoglobulin (Ig)-like domains near the C-terminal end. Truncated mutation experiment reveals that the two Ig-like domains are important for the hydrolytic activity of PdCel5C. Moreover, PdCel5C releases cello-oligosaccharides from cellulosic substrates, which is different from that of most characterized cellulases in the same glycoside hydrolase family 5. To the best of our knowledge, this is the first report on the characterization of an Ig-like domain-containing cellulase in fungi. The results expand our understanding on the diversity of cellulases in fungi, and suggest possible shared catalytic mechanisms between bacterial and fungal cellulases.
Enzyme and microbial technology. 03/2013; 52(3):190-5.
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ABSTRACT: The activity, stability and conformation of laccase were first investigated in an aqueous solution of ionic liquids 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([Bmim]TfO), 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonate ([Bmpyr]TfO) or tetramethylammonium trifluoromethanesulfonate ([TMA]TfO). Compared with control system, high level of [Bmim]TfO or [Bmpyr]TfO destabilizes laccase while [TMA]TfO stabilizes laccase. These effects are more pronounced with the extension of the incubation time. The activity variations are well correlated with the changes of the conformation of laccase evidenced by fluorescence and circular dichroism spectra under specified conditions. The effects of the three ionic liquids on laccase are associated with the chaotropicity of the cations in Hofmeister series. For laccase, [TMA]TfO is not a good activating agent but it greatly enhances the stability of laccase in addition to maintaining the catalytic efficiency of laccase, showing its great potential in real application.
International journal of biological macromolecules 02/2013; 56C:62-68. · 2.37 Impact Factor
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ABSTRACT: Choline acetate is an ionic liquid composed of a kosmotropic anion and a chaotropic cation. According to Hofmeister series, a kosmotropic anion and/or a chaotropic cation could stabilize an enzyme, thereby facilitating the retention of the catalytic activity of the enzyme. In this work, we first report the influence of choline acetate on the activity and stability of lipase in AOT/water/isooctane reverse micelles. The indicator reaction is the lipase-catalyzed hydrolysis of 4-nitrophenyl butyrate. The results show that a low level of choline acetate does not affect the microstructure of the AOT reverse micelles, but the ionic liquid can improve the catalytic efficiency of lipase. Fluorescence spectra show that a high level of choline acetate has an impact on the conformation of lipase, so the activation is mainly due to the influence of choline acetate on the nucleophilicity of water. Infrared spectra demonstrate that choline acetate can form stronger hydrogen bonds with water surrounding lipase, and therefore enhance the nucleophilicity of the water, which makes it easier to attack the acyl enzyme intermediate, thereby increasing the activity of the lipase-catalyzed hydrolysis of the ester. A study on the stability of lipase in AOT reverse micelles indicates that the ionic liquid is able to maintain the activity of lipase to a certain extent. The effect of choline acetate is consistent with that predicted based on Hofmeister series.
Colloids and surfaces. B, Biointerfaces 01/2013; 105C:81-86. · 2.60 Impact Factor
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ABSTRACT: Enzymatic degumming, as an alternative to chemical processing, has attracted wide attention. However, to date, little information about other enzyme components with effective degumming except pectinase has been reported, and there is no report about the effect of bleaching agent (H2O2) on enzymatic degumming and combining enzymatic degumming and H2O2 bleaching process. In this study, we found that the crude enzyme of wild-type Bacillus sp. Y1 had a powerful and fast degumming ability. Its PGL activity was the highest at pH 9.6-10.0 and 60°C and stable at pH 7-10.5 and 30-50°C, having a wide scope of pH and temperature. Its PGL also had a high H2O2 tolerance, and the gum loss and brightness of fibers could be significantly improved when H2O2 was added into it for degumming. The synergistic action was also found between it and H2O2 on the degumming and bleaching of ramie fibers. All showed that it was very suitable for a joint process of enzymatic degumming and H2O2 bleaching. It also contained more proteins compared with a control pectinase, and its high protease content was further substantiated as a factor for effective degumming. Protease and pectinase also had a synergistic action on degumming.
BioMed research international. 01/2013; 2013:212315.
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Guodong Liu,
Lei Zhang,
Xiaomin Wei,
Gen Zou,
Yuqi Qin,
Liang Ma,
Jie Li,
Huajun Zheng,
Shengyue Wang,
Chengshu Wang,
Luying Xun,
Guo-Ping Zhao,
Zhihua Zhou, Yinbo Qu
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ABSTRACT: Many Penicillium species could produce extracellular enzyme systems with good lignocellulose hydrolysis performance. However, these species and their enzyme systems are still poorly understood and explored due to the lacking of genetic information. Here, we present the genomic and secretomic analyses of Penicillium decumbens that has been used in industrial production of lignocellulolytic enzymes in China for more than fifteen years. Comparative genomics analysis with the phylogenetically most similar species Penicillium chrysogenum revealed that P. decumbens has evolved with more genes involved in plant cell wall degradation, but fewer genes in cellular metabolism and regulation. Compared with the widely used cellulase producer Trichoderma reesei, P. decumbens has a lignocellulolytic enzyme system with more diverse components, particularly for cellulose binding domain-containing proteins and hemicellulases. Further, proteomic analysis of secretomes revealed that P. decumbens produced significantly more lignocellulolytic enzymes in the medium with cellulose-wheat bran as the carbon source than with glucose. The results expand our knowledge on the genetic information of lignocellulolytic enzyme systems in Penicillium species, and will facilitate rational strain improvement for the production of highly efficient enzyme systems used in lignocellulose utilization from Penicillium species.
PLoS ONE 01/2013; 8(2):e55185. · 4.09 Impact Factor
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ABSTRACT: This study evaluates the effects of some pretreatment processes to improve the enzymatic hydrolysis of oil palm empty fruit bunch (EFB) for ethanol production. The experimental results show that the bisulfite pretreatment was practical for EFB pretreatment. Moreover, the optimum pretreatment conditions of the bisulfite pretreatment (180°C, 30min, 8% NaHSO(3), 1% H(2)SO(4)) were identified. In the experiments, a biorefinery process of EFB was proposed to produce ethanol, xylose products, and lignosulfonates.
Bioresource technology 11/2012; · 4.25 Impact Factor
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ABSTRACT: It is imperative to establish a simple, efficient, and practical method to investigate the Hofmeister effect of ionic liquids (ILs) on the behavior of proteins (enzymes). In this study, the effects of the cations and anions of different ILs in aqueous media on the structural stability of horseradish peroxidase (HRP), a model oxidoreductase, were systematically investigated using electrochemical methods. It is found that without ILs no direct electron transfer current signals of HRP appear at bare glassy carbon electrode (GCE) in phosphate buffer (pH 7.0) even after incubation and accumulation at a negative potential. In the presence of ILs, however, a current signal occurs at GCE, depending on the structure of the IL and its concentration. A linear relationship between the peak currents and the scan rates demonstrates that the direct electron transfer is a surface-confined thin-layer electrochemical process. The redox signal at GCE is from the heme of HRP. An IL has a perturbing effect on the HRP structure. The anodic peak current of HRP at GCE, the catalytic activity of HRP, and the secondary structure of HRP are well correlated. Different cations or anions at varied concentrations have different effects on the structural stability of HRP, resulting in different current signals at GCE. Thus, the anodic peak current of HRP at GCE can be used as an indicator to quantitatively characterize the effect of ILs on the structural stability of HRP. The present Hofmeister series for cations and anions is in good agreement with that reported elsewhere. To our knowledge, this is a first attempt to establish a simple and practical electrochemical method to correlate Hofmeister effects with characteristics of ions and solvents. The present investigation not only deepens our understanding of the complex electrochemical behavior of proteins in ILs media but also offers a practical guidance to designing "green" and biocompatible ILs for protein (enzyme) separation, purification, and enzymatic catalysis and conversion.
The Journal of Physical Chemistry B 08/2012; 116(36):11075-80. · 3.70 Impact Factor
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Guangqi Zhou,
Jing Lü,
Zhonghai Li,
Jingjing Li,
Mingyu Wang, Yinbo Qu,
Lin Xiao,
Shulin Qin,
Haitao Zhao,
Ruirui Xia,
Xu Fang
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ABSTRACT: Penicillium decumbens T. is an important filamentous fungus for the production of cellulases to effectively degrade lignocellulose for second generation biofuel production. In order to enhance the capability of Penicillium decumbens to produce cellulases, we constructed a creB (a deubiquitinating enzyme encoding gene) deletion cassette, and generated a creB knockout strain with homologous double crossover recombination. This mutation resulted in a detectable decrease of carbon catabolite repression (CCR) effect. The filter paper activity, endoglucanase activity, xylanase activity and exoglucanase activity of the deltacreB strain increased by 1.8, 1.71, 2.06 and 2.04 fold, respectively, when comparing with the parent strain Ku-39. A 2.68 fold increase of extracellular protein concentration was also observed. These results suggest that the deletion of creB results in CCR derepression. These data also suggest that CREB influences cellulase production of Penicillium decumbens. In generation, this study provides information that can be helpful for constructing cellulase hyper-producing strain.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 08/2012; 28(8):959-72.
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ABSTRACT: Infrared spectroscopy is a powerful technique for structure characterization. For a protein hosted in a reversed micellar medium, the spectral features of the protein are always interfered by the IR absorption bands of the medium in addition to the congestion in their IR spectra. Fortunately, there is a transparent window in the 2500-2200cm(-1) region. Incorporation of a vibrational probe with IR absorption frequencies in this region into proteins represents a promising strategy for the study of the conformation of a protein in a reverse micelle. In the present work, we incorporated 4-cyanobenzyl group (CN) into bovine serum albumin (BSA) via cysteine alkylation reactions under mild conditions. Circular dichroism spectroscopy showed that the CN modified BSA (CNBSA) could retain its conformation. When CNBSA was hosted in AOT reverse micelle, it was found that the nitrile group on BSA was sensitive to the conformational change of BSA induced by urea as an additive in the reverse micelle. The peak splitting of nitrile group was also observed when the size of AOT reverse micelle and the concentration of an electrolyte were varied. Obviously, the shift of the IR absorption peak and/or peak splitting of nitrile group on BSA are correlated with the change of BSA conformation in AOT reverse micelle. So we conclude that the nitrile infrared probe can be used to study protein conformation in a reverse micelle.
Spectrochimica Acta Part A Molecular and Biomolecular Spectroscopy 07/2012; 97:858-63. · 2.10 Impact Factor
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ABSTRACT: Yeast alcohol dehydrogenase (YADH) showed substantial decrease in its catalytic activity due to the strong electrostatic interaction
between the head groups of sodium bis(2-ethylhexyl) sulfosuccinate (AOT) and YADH in AOT reverse micelles. However, the catalytic
activity of YADH in a nonionic reverse micellar interface (GGDE/TX-100) obtained from a functional nonionic surfactant N-gluconyl
glutamic acid didecyl ester (GGDE) and Triton X-100 (TX-100) was higher than that in AOT reverse micelle under the respective
optimum conditions. A comparison of the kinetic parameters showed that the turnover number kcat in GGDE/TX-100 reverse micelle was 1.4 times as large as that in AOT reverse micelle, but the Michaelis constants in AOT
reverse micelle for ethanol KmB was twice and for coenzyme NAD+ KmA was 5 times higher than their counterparts in GGDE/TX-100 reverse micelle. For the conversion of ethanol, the smaller KmB and larger kcat in GGDE/TX-100 reverse micelle resulted in higher catalytic efficiency kcat/KmB. The stability of YADH in GGDE/TX-100 reverse micelle was also found to be better than that in AOT reverse micelle. They
were mainly attributed to the absence of electric charge on the head groups of GGDE and TX-100 in the GGDE/TX-100 reverse
micelle.
Central European Journal of Chemistry 04/2012; 7(4):787-793. · 1.07 Impact Factor
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ABSTRACT: In this study, four N-glycosylation sites, Asn45, Asn64, Asn270 and Asn384 of Hypocrea jecorina (syn. Trichoderma reesei) Cel7A (family 7 cellobiohydrolase I) were replaced by serines using site-directed mutagenesis. These four mutants and wild
type H. jecorina Cel7A gene were transformed into P. pastoris, and the recombinant enzymes were purified and analyzed. The enzymatic activities of recombinant Cel7A (rCel7A), and mutants
N45S, N270S and N384S were very low while mutant N64S displayed about seven times higher activity than that of rCel7A, and
about 10% of the wild-type Cel7A activity from H. jecorina. The results indicate that N-glycosylation of Asn64 had an effect on the activity of the Cel7A enzyme expressed in P. pastoris, and that glycosylation at this site would be only a subordinate reason for the low activity of the recombinant enzyme.
World Journal of Microbiology and Biotechnology 04/2012; 26(2):323-328. · 1.53 Impact Factor
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ABSTRACT: Four cellobiohydrolase I (CBHI) glycoforms, namely, CBHI-A, CBHI-B, CBHI-C, and CBHI-D, were purified from the cultured broth of Penicillium decumbens JU-A10. All glycoforms had the same amino acid sequence but displayed different characteristics and biological functions. The effects of the N-glycans of the glycoforms on CBH activity were analyzed using mass spectrum data. Longer N-glycan chains at the Asn-137 of CBHI increased CBH activity. After the N-glycans were removed using site-directed mutagenesis and homologous expression in P. decumbens, the specific CBH activity of the recombinant CBHI without N-glycosylation increased by 65% compared with the wild-type CBHI with the highest specific activity. However, the activity was not stable. Only the N-glycosylation at Asn-137 can improve CBH activity by 40%. rCBHI with N-glycosylation only at Asn-470 exhibited no enzymatic activity. CBH activity was affected whether or not the protein was glycosylated, together with the N-glycosylation site and N-glycan structure. N-Glycosylation not only affects CBH activity but may also bring a new feature to a nonhydrolytic CBHI glycoform (CBHI-A). By supplementing CBHI-A to different commercial cellulase preparations, the glucose yield of lignocellulose hydrolysis increased by >20%. After treatment with a low dose (5 mg/g substrate) of CBHI-A at 50 °C for 7 days, the hydrogen-bond intensity and crystalline degree of cotton fibers decreased by 17 and 34%, respectively. These results may provide new guidelines for cellulase engineering.
Journal of Biological Chemistry 03/2012; 287(19):15906-15. · 4.77 Impact Factor
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ABSTRACT: Second-generation bioethanol made from lignocellulosic biomass is considered one of the most promising biofuels. However, the enzymatic hydrolysis of the cellulose component to liberate glucose for ethanol fermentation is one of the major barriers for the process to be economically competitive because of the recalcitrance of feedstock. In this chapter, the progress on the understanding of the mechanisms of lignocellulose degradation, as well as the identification and optimization of fungal cellulases, cellulolytic strains, and cellulase production is reviewed. The physiologic functions and enzymatic mechanisms of two groups of enzymes involved in lignocellulose degradation, cellulases and hemicellulases, are discussed, and the synergism of the cellulase components during lignocellulose degradation is addressed. Furthermore, the methods for screening filamentous fungal strains capable of degrading lignocellulose are evaluated and the production of cellulases by these fungal strains is discussed. Aside from traditional mutagenesis for improving the secretion level and enzymatic activities of cellulases from filamentous fungal species, genetic engineering of strains and protein engineering on cellulase molecules are also highlighted.
Advances in biochemical engineering/biotechnology 01/2012; 128:1-24. · 1.64 Impact Factor
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ABSTRACT: Reed is a widespread-growing, inexpensive, and readily available lignocellulosic material source in northeast China. The objective of this study is to evaluate the liquid hot water (LHW) pretreatment efficiency of reed based on the enzymatic digestibility and ethanol fermentability of water-insoluble solids (WISs) from reed after the LHW pretreatment. Several variables in the LHW pretreatment and enzymatic hydrolysis process were optimized. The conversion of glucan to glucose and glucose concentrations are considered as response variables in different conditions. The optimum conditions for the LHW pretreatment of reed area temperature of 180°C for 20min and a solid-to-liquid ratio of 1 : 10. These optimum conditions for the LHW pretreatment of reed resulted in a cellulose conversion rate of 82.59% in the subsequent enzymatic hydrolysis at 50°C for 72 h with a cellulase loading of 30 filter paper unit per gram of oven-dried WIS. Increasing the pretreatment temperature resulted in a higher enzymatic digestibility of the WIS from reed. Separate hydrolysis and fermentation of WIS showed that the conversion of glucan to ethanol reached 99.5% of the theoretical yield. The LHW pretreatment of reed is a suitable method to acquire a high recovery of fermentable sugars and high ethanol conversion yield.
Journal of Biomedicine and Biotechnology 01/2012; 2012:276278. · 2.44 Impact Factor
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ABSTRACT: In the water-in-[Bmim][PF(6)] microemulsion stabilized by both AOT and Triton X-100, the lipase-catalyzed hydrolysis of 4-nitrophenyl butyrate (p-NPB) was investigated to evaluate the catalytic efficiency of lipase in this novel microemulsion. The structural parameters of the microemulsion and the conditions of the enzymatic reaction affect the catalytic activity of lipase, especially the concentration of Tris-HCl buffer. Under optimum conditions, the catalytic activity of lipase in the present microemulsion is much higher than that in H(2)O saturated [Bmim][PF(6)]. When the partitioning of the substrate in the microemulsion is taken into account, the catalytic efficiency of lipase in this novel microemulsion is 14.3 times that in H(2)O saturated [Bmim][PF(6)] due to the significant decrease of the Michaelis constant in the microemulsion. Due to the large interface, high water activity, and probably the activating effect of the imidazolium cation in the water pool, the present microemulsion is demonstrated to be a promising medium for the lipase-catalyzed hydrolytic reaction. To demonstrate an important biocatalytic application in the IL-based microemulsion, the lipase-catalyzed synthesis of the flavoring agent benzyl acetate via transesterification of vinyl acetate with benzyl alcohol was also studied in the medium. Due to the high dispersion of lipase, large interface and removal of the byproduct, a maximum yield of 94% was obtained, indicating that the novel microemulsion is really important and useful.
Colloids and surfaces. B, Biointerfaces 12/2011; 92:360-6. · 2.60 Impact Factor
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ABSTRACT: Penicillium decumbens 114-2 is a fast-growing filamentous fungus which secretes a variety of lignocellulolytic enzymes. Its catabolite-repression-resistant mutant JU-A10 with high secretion capacity of cellulolytic enzymes has been used industrially for biomass hydrolysis. Transcription levels of 6 important lignocellulolytic enzymes genes (cel5A, cel6A, cel7A, cel7B, xyn10A, and xyn11A) from both strains were determined on different carbon sources (glucose, sorbose, lactose, cellobiose, cellulose, and cellulose-wheat bran), by means of a real-time quantitative polymerase chain reaction. For both strains, the 6 genes are coordinately regulated at transcriptional level. Glucose and cellobiose repressed whereas cellulose and cellulose-wheat bran induced expression of 6 genes in both strains. Expression levels of all genes tested in the mutant strain JU-A10 were substantially higher than those in wild-type strain 114-2 on all carbon sources. On glucose repression condition, the mutant JU-A10 appeared obviously derepressed. Lactose was first proved to have an inductive effect on lignocellulolytic enzyme genes expression at lower concentration in Penicillium spp.
Comptes rendus biologies 11/2011; 334(11):806-11. · 1.71 Impact Factor
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ABSTRACT: The direct electrochemistry and bioelectrocatalysis of horseradish peroxidase (HRP) in Nafion films at glassy carbon electrode (GCE) was investigated in three [BF(4)](-)-type room-temperature ionic liquids (ILs) to understand the structural effect of imidazolium cations. The three ILs are 1-ethyl-3-methylimidazolium tetrafluoroborate ([Emim][BF(4)]), 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF(4)]) and 1-hexyl-3-methylimidazolium tetrafluoroborate ([Hmim][BF(4)]). A small amount of water in the three ILs is indispensable for maintaining the electrochemical activity of HRP in Nafion films, and the optimum water contents decrease with the increase of alkyl chain length on imidazole ring. Analysis shows that the optimum water contents are primarily determined by the hydrophilicity of ILs used. In contrast to aqueous medium, ILs media facilitate the direct electron transfer of HRP, and the electrochemical parameters obtained in different ILs are obviously related to the nature of ILs. The direct electron transfer between HRP and GCE is a surface-confined quasi-reversible single electron transfer process. The apparent heterogeneous electron transfer rate constant decreases gradually with the increase of alkyl chain length on imidazole ring, but the changing extent is relatively small. The electrocatalytic reduction current of H(2)O(2) at the present electrode decreases obviously with the increase of alkyl chain length, and the mass transfer of H(2)O(2) via diffusion in ILs should be responsible for the change. In addition, the modified electrode has good stability and reproducibility; the ability to tolerate high levels of F(-) has been greatly enhanced due to the use of Nafion film. When an appropriate mediator is included in the sensing layer, a sensitive nonaqueous biosensor could be fabricated.
Colloids and surfaces. B, Biointerfaces 10/2011; 87(1):61-6. · 2.60 Impact Factor
