阿魏酸酯酶基因工程菌研究进展

阿魏酸酯酶能水解阿魏酸、多聚阿魏酸与木聚糖以及木质素之间形成的酯键,有利于植物细胞壁的降解。介绍阿魏酸酯酶的微生物来源、阿魏酸酯酶基因工程菌构建和阿魏酸酯酶与其他酶相互协同作用等的研究进展。     

木聚糖降解酶系基因代谢调控研究进展

木聚糖是半纤维素的主要组成部分,是一类数量很大的再生生物资源,工业利用前景广阔。木聚糖降解需要多种酶的参与,主要有木聚糖酶、木糖苷酶、α-葡萄糖醛酸酶、乙酰木聚糖酯酶、阿拉伯糖酶、阿魏酸酯酶、p-香豆酸酯酶等。主要综述了木聚糖降解酶系基因代谢调控的研究进展,主要包括转录激活因子XlnR、抑制蛋白CreA、不同诱导物、pH值、HAP-CCAAT复合物等对木聚糖降解酶系基因表达的影响,最后探讨了木聚糖降解酶系基因代谢调控存在的问题,并对今后的研究进行了展望。     

阿魏酸酯酶和纤维素酶在水解汽爆稻草中的协同作用

利用阿魏酸酯酶, 水解天然木质纤维素原料中半纤维素与木质素之间的阿魏酸酯键, 从破坏两者共价键连接的角度, 探索阿魏酸酯酶促进纤维素酶水解汽爆稻草中纤维素的可行性。结果显示, 当阿魏酸酯酶加入量为240 mu/g底物、水解72 h时, 汽爆稻草纤维素的酶解率、不溶性底物失重率较不加阿魏酸酯酶分别增加了32.00%、32.77%; 阿魏酸酯酶(300 mu/g底物)作用120 min后, 纤维素酶对汽爆稻草纤维素的酶解率、不溶性底物失重率分别增加了29.85%、32.48%。通过比较不同酶法处理后的汽爆稻草的可及度和红外光谱图发现, 阿魏酸酯酶能有效地水解原料中的酯键, 提高原料可及度50%以上。由此表明, 阿魏酸酯酶和纤维素酶之间存在较大的协同作用, 添加阿魏酸酯酶能够提高纤维素酶对天然木质纤维素的酶解效率。     

新颖阿魏酸酯酶在黑曲霉中异源表达及其应用

本研究依托桧状青霉的基因组学和蛋白组学数据,发现一种新型的阿魏酸酯酶。利用PCR技术成功扩增得到桧状青霉阿魏酸酯酶的基因,构建真菌表达盒通过原生质体的方法将其转化到黑曲霉中。通过SDS-PAGE检测和酶活力检测验证该阿魏酸酯酶成功地在黑曲霉胞外分泌表达。根据氨基酸序列相似性和底物特异性分析都显示该阿魏酸酯酶属于C型的阿魏酸酯酶,与常见的A型、B型阿魏酸酯酶性质区别较大。将成功表达阿魏酸酯酶的黑曲霉胞外酶液复配里氏木霉酶液后,水解不同生物质材料,纤维素水解效率均有大幅程度地提高,玉米秸秆,麦麸,玉米芯,木薯酒糟的水解效率分别提高68.8%,38.6%,15.6%和20.0%。该研究为新颖阿魏酸酯酶应用以及今后里氏木霉纤维素酶酶系复配提供新的思路。     

一株产阿魏酸酯酶丝状真菌的分离鉴定及其粗酶性质研究

利用平板透明圈法,筛选分离到37株具有阿魏酸酯酶活性的丝状真菌,其中1株编号为HA4087的菌株具有较强的产阿魏酸酯酶能力。经形态学观察、18S rDNA和ITS序列鉴定为互隔交链格孢霉。对该菌株所产阿魏酸酯酶的粗酶性质进行了初步研究,结果表明,在发酵培养基中发酵粗酶活力为86 mU/mL,最适作用温度为55℃,最适pH值为5.5;在55℃保温30 min后酶活力仍为90%以上;在pH4.5-6.5范围内稳定性较好,酶活仍为85%以上。试验获得了产高酶活阿魏酸酯酶的菌株,为阿魏酸酯酶的工业化应用提供前提。     

来自桔青霉的阿魏酸酯酶的分离纯化、理化性质

【目的】从桔青霉的发酵液中分离纯化了胞外阿魏酸酯酶(PcFAE)并进行了酶学性质的研究,初步探讨了PcFAE对麦糟的酶解作用。【方法】利用(NH4)2SO4沉淀、DEAE-Sepharose Fast Flow离子交换层析、Phenyl Sepharose6Fast Flow疏水层析纯化得到电泳纯的阿魏酸酯酶。【结果】从该菌株的发酵液中获得一阿魏酸酯酶,该酶亚基分子量约为31kDa,全酶分子量约为58kDa。其最适pH为6.0,最适温度为45℃-65℃,在pH5.0-6.0及25℃-55℃之间,酶保持了较好的稳定性。Mg2+、Fe2+、Mn2+、Ca2+和Na+对酶活有一定的促进作用,Zn2+对PcFAE酶活有一定的抑制作用,而Cu2+、亮抑肽素、抑肽酶有显著的抑制作用,Hg2+、苯甲基磺酰氟几乎完全抑制了酶活。EDTA对PcFAE活性无明显影响。PcFAE的kcat/Km对香豆酸甲酯、芥子酸甲酯、阿魏酸甲酯、咖啡酸甲酯的值分别为823、416、103、0,PcFAE对MpCA的催化效率最高。PcFAE作用于麦糟,当5U PcFAE/g麦糟时,其阿魏酸的释放量为7.2%。【结论】获得了一阿魏酸酯酶,其理化性质与至今报道的阿魏酸酯酶有所不同,为阿魏酸酯酶的开发提供了重要的实验依据。     

黑曲霉阿魏酸酯酶基因密码子优化及在毕赤酵母中的高效表达

【目的】对黑曲霉(Aspergillus niger)阿魏酸酯酶基因进行克隆和密码子优化,使其在毕赤酵母(Pichia pastoris X-33)中高效表达。【方法】以黑曲霉基因组为模板,经重叠延伸PCR扩增得到阿魏酸酯酶基因(Anfae A),并对Anfae A基因进行毕赤酵母密码子偏好性"随机优化"和"一对一优化",全基因合成后分别与表达载体pPICZαA连接,构建表达载体pPICZαA-Anfae A、pPICZαA-op Anfae A I和pPICZαA-op Anfae A II。经Sac I线性化后电转化至P.pastoris X-33中,筛选阳性转化子。摇瓶发酵4.5 d后,测定并比较重组阿魏酸酯酶(re Anfae A)酶活。【结果】密码子优化前阿魏酸酯酶酶活为6.8±0.1 U/m L,基因"一对一优化"和"随机优化"后的重组酶酶活分别为5.2±0.1 U/m L和39.9±0.1 U/m L,"随机优化"后酶活比优化前提高了近6倍,而"一对一优化"后酶活仅为优化前酶活的76.5%。重组阿魏酸酯酶的最适p H为5.5,且在pH 4.5-7.0稳定性较好;最适反应温度50°C,在45-50°C较稳定。【结论】阿魏酸酯酶基因经密码子"随机优化"后进行重组表达,酶活显著提高,对研究阿魏酸酯酶在毕赤酵母及其它宿主中的高效表达具有一定的借鉴意义,也为大规模工业化应用奠定了基础。     

一组厌氧真菌菌系产阿魏酸酯酶的初步研究

利用玉米秸秆粉为唯一碳源的筛选培养基,从肉牛瘤胃液中筛选构建了一组厌氧真菌菌系,研究了该菌系产阿魏酸酯酶的特征.阿魏酸酯酶的最适pH为8.0,最适温度为40℃,最高酶活力为19.1 mU/mL,在pH 6.0 ～8.0及35～45℃之间,酶活性保持相对稳定.Mg2+对酶活力具有激活作用,Fe2、Cu2+、Fe3+等均抑制酶活力.     

嗜热细菌Clostridium thermocellum阿魏酸酯酶基因的克隆、异源表达及酶学特性分析

【目的】阐明嗜热细菌Clostridium thermocellum Xyn Z蛋白的阿魏酸酯酶催化域的酶学特性,为其在生物质能源及其它发酵工业中的应用奠定基础。【方法】分别构建了C.thermocellum Xyn Z的阿魏酸酯酶催化域(FAE)及该阿魏酸酯酶催化域和碳水化合物结合域(FAE-CBM6)编码基因的原核表达载体,并在大肠杆菌菌株BL21(DE3)中异源表达,在此基础上分析比较了温度、pH、底物、金属离子及CBM6结合域对阿魏酸酯酶活性的影响。【结果】重组FAE酶及FAE-CBM6酶发挥催化活性的适宜pH值为5.0-9.0,适宜温度为50-70°C,它们对不同金属离子的响应有差异。【结论】在同一反应条件下,FAE-CBM6酶的酶活均比FAE高,说明CBM6结合域的存在对于阿魏酸酯酶活性有促进作用。     

黑曲霉h408阿魏酸酯酶基因的克隆及在毕赤酵母中的高效表达

【目的】实现在巴斯德毕赤酵母(Pichia pastoris)中高效表达黑曲霉(Aspergillus niger)h408阿魏酸酯酶A基因(AnfaeA),并对重组酶特性进行表征。【方法】采用重叠延伸PCR扩增黑曲霉h408的阿魏酸酯酶A基因。将AnfaeA基因和毕赤酵母表达载体pPIC9K连接,成功构建重组质粒pPIC9K-Anfae,经线性化后电转化P.pastoris GS115,透明圈法筛选活性高的转化子后进行诱导表达。利用紫外吸收法测定温度及pH对重组阿魏酸酯酶活性的影响。【结果】成功从A.niger h408中克隆得到阿魏酸酯酶A的cDNA基因(GenBank:KF911349),并实现了其在P.pastoris GS115中的高效表达。该基因长度为783bp,含有1个开放阅读框架(ORF),编码260个氨基酸,Blast分析显示该基因和GenBank中黑曲霉阿魏酸酯酶序列同源性为99%。翻译的氨基酸序列含有脂酶典型的活性盖子和催化三联体结构。从转化板上获得1株编号为pPIC9K-Anfae5的转化子阿魏酸酯酶活性最高,酶活达24.72 U/mL,比活力为40.84 U/mg,比黑曲霉出发菌株(22.1 mU/mL)提高了1100倍左右。重组阿魏酸酯酶的最适pH为5.0,且在pH 4.0-9.0稳定性较好;最适反应温度50℃,在40-60℃时较稳定。【结论】阿魏酸酯酶在毕赤酵母中的高效分泌表达为其在饲料工业和造纸工业等工业化应用提供了前提,也为后续改进酶学特性的定向进化奠定实验基础。     

Biotechnological applications and potential of fungal feruloyl esterases based on prevalence,classification and biochemical diversity

Feruloyl esterases are part of the enzymatic spectrum employed by fungi and other microorganisms to degrade plant polysaccharides. They release ferulic acid and other aromatic acids from these polymeric structures and have received an increasing interest in industrial applications such as in the food, pulp and paper and bio-fuel industries. This review provides an overview of the current knowledge on fungal feruloyl esterases focussing in particular on the differences in substrate specificity, regulation of their production, prevalence of these enzymes in fungal genomes and industrial applications.     

What can feruloyl esterases do for us?

The role of feruloyl esterases in plant wall development, in gut health, and in the breakdown of plant biomass for the production of bioactive phytochemicals and biofuel is covered in this review. These enzymes have potential roles in stomatal cell function and the phenolic substitutions and cross-linkages between plant cell wall components. As more plant genomes are sequenced, the role of ferulic acid and feruloyl esterases in planta may be better understood. In human and ruminal digestion, these enzymes are important to de-esterify dietary fibre, releasing hydroxycinnamates and derivatives which have been shown to have positive health effects, such as antioxidant, anti-inflammatory and anti-microbial activities. They are also involved in colonic fermentation where their extracellular and intracellular activities in the microbiota improve the breakdown of polysaccharides and increase microbial production of short chain fatty acids. Their specificity can also be employed to synthesize bioactive compounds for cosmetic and health applications. The enzymatic disassembly of cereal straws is greatly enhanced when feruloyl esterase activity is present, although the substrate specificity of the esterase appears to have some bearing on its optimal application. The involvement of feruloyl esterases in the improved enzymatic and microbial saccharification of cereal-derived material demonstrates a high importance for these enzymes in animal feed preparation and bioalcohol production.     

Occurrence,properties, and applications of feruloyl esterases

Feruloyl esterases hydrolyze the ester linkages of ferulic and diferulic acids present in plant cell walls. This interesting group of enzymes also has a potentially broad range of applications in the pharmaceutical and agri-food industries. An overview of the current knowledge of fungal feruloyl esterases focusing on the diverse of substrate specificity and potential applications is presented in this review. Furthermore, biological functions of ferulic acid are discussed.     

一株产阿魏酸酯酶青霉菌株的筛选、鉴定及生长特征

从腐烂的木质纤维中筛选了一株产阿魏酸酯酶的菌株HDFE1,根据其形态特征、rDNAITS1-5.8S-ITS2序列及系统发育分析,鉴定菌株HDFE1为青霉属的橘青霉(Penicillium citrinum Thom)。菌株HDFE1最适生长温度为30°C,最适生长pH为6.0。该菌株在30°C、pH6.0、200r/min培养60h时,阿魏酸酯酶酶活力为最高,达20.75U/L。     

Identification of a type-D feruloyl esterase from<Emphasis Type="Italic"> Neurospora crassa</Emphasis>

Feruloyl esterases constitute an interesting group of enzymes that have the potential for use over a broad range of applications in the agri–food industries. In order to expand the range of available enzymes, we have examined the presence of feruoyl esterase genes present in the genome sequence of the filamentous fungus Neurospora crassa. We have identified an orphan gene (contig 3.544), the translation of which shows sequence identity with known feruloyl esterases. This gene was cloned and the corresponding recombinant protein expressed in Pichia pastoris to confirm that the enzyme (NcFaeD-3.544) exhibits feruloyl esterase activity. Unusually the enzyme was capable of p-coumaric acid release from untreated crude plant cell wall materials. The substrate utilisation preferences of the recombinant enzyme place it in the recently recognised type-D sub-class of feruloyl esterase.     

Exploration of members of Aspergillus sections Nigri, Flavi, and Terrei for feruloyl esterase production

The ability of members of Aspergillus sections Nigri, Flavi, and Terrei to produce feruloyl esterases was studied according to their substrate specificity against synthetic methyl esters of hydroxycinnamic acids. Type A feruloyl esterases (FAEA), induced during growth on cereal-derived products, show a preference for the phenolic moiety of substrates that contain methoxy substitutions, as found in methyl sinapinate, whereas type B feruloyl esterases (FAEB) show a preference for the phenolic moiety of substrates that contain hydroxyl substitutions, as occurs in methyl caffeate. All the strains of Aspergillus section Nigri (e.g., A. niger and A. foetidus) were able to produce feruloyl esterases with activity profiles similar to those reported for FAEA and FAEB of A. niger when grown on oat-spelt xylan and sugar beet pulp, respectively. The two genes encoding these proteins, faeA and faeB, were identified by Southern blot analysis. The strains of Aspergillus sections Flavi (e.g., A. flavus, A. flavo-furcatus, and A. tamarii) and Terrei (e.g., A. terreus) were able to produce type A and type B enzymes. faeA was revealed in genomic DNA of these strains, and FAEA was determined by immunodetection in cultures grown in oat-spelt xylan. In addition, type B enzymes, not related to faeB, were efficiently induced by oat-spelt xylan and exhibited very original activity profiles on sugar beet pulp. This work confirms that the members of the genus Aspergillus are good feruloyl esterase producers.     

Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, selected for the biotransformation of ferulic acid to vanillin, are also able to produce cell wall polysaccharide-degrading enzymes and feruloyl esterases

The filamentous fungal strains Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, previously selected for the bioconversion of ferulic acid to vanillic acid and vanillin respectively, were grown on sugar beet pulp. A large spectrum of polysaccharide-degrading enzymes was produced by A. niger and very few levels of feruloyl esterases were found. In contrast, P. cinnabarinus culture filtrate contained low amount of polysaccharide-degrading enzymes and no feruloyl esterases. In order to enhance feruloyl esterases in A. niger cultures, feruloylated oligosaccharide-rich fractions were prepared from sugar beet pulp or cereal bran and used as carbon sources. Number of polysaccharide-degrading enzymes were induced. Feruloyl esterases were much higher in maize bran-based medium than in sugar beet pulp-based medium, demonstrating the ability of carbon sources originating from maize to induce the synthesis of feruloyl esterases. Thus, A. niger I-1472 could be interesting to release ferulic acid from sugar beet pulp or maize bran.     

Three Feruloyl Esterases in Cellulosilyticum ruminicola H1 Act Synergistically To Hydrolyze Esterified Polysaccharides

Feruloyl esterases (Faes) constitute a subclass of carboxyl esterases that specifically hydrolyze the ester linkages between ferulate and polysaccharides in plant cell walls. Until now, the described microbial Faes were mainly from fungi. In this study, we report that Cellulosilyticum ruminicola H1, a previously described fibrolytic rumen bacterium, possesses three different active feruloyl esterases, FaeI, FaeII, and FaeIII. Phylogenetic analysis classified the described bacterial Faes into two types, FaeI and FaeII in type I and FaeIII in type II. Substrate specificity assays indicated that FaeI is more active against the ester bonds in natural hemicelluloses and FaeIII preferentially attacks the ferulate esters with a small moiety, such as methyl groups, while FaeII is active on both types of substrates. Among the three feruloyl esterase genes, faeI was the only one induced significantly by xylose and xylan, while pectin appeared to moderately induce the three genes during the late log phase to stationary phase. Western blot analysis determined that FaeI and FaeIII were secreted and cytoplasmic proteins, respectively, whereas FaeII seemed to be cell associated. The addition of FaeI and FaeII but not FaeIII enhanced the activity of a xylanase on maize cob, suggesting a synergy of the former two with xylanase. Hence, we propose that the three feruloyl esterases work in concert to hydrolyze ferulate esters in natural hemicelluloses.     

Microbial carbohydrate esterases in cold adapted environments

Psychrophiles produce cold-evolved enzymes that display a high catalytic efficiency, associated with a low thermal stability. In recent years, these enzymes have attracted the attention of scientists because of their peculiar properties that render them particularly useful in investigating the relationship existing between enzyme stability and flexibility on one hand, and enzyme activity on the other hand. Among these enzymes, the esterases, and particularly the feruloyl esterases, have potential uses over a broad range of applications in the agro-food industries. In recent years, the number of microbial feruloyl esterase activities has increased in the growing genome databases. Based on substrate utilization data and supported by primary sequence identity, four subclasses of esterase have been characterized so far. Up to the present, ten genomes from psychrophilic bacteria have been completely sequenced and additional fourteen genomes are under investigation. From the bacteria strains whose genome has been completely sequenced, we analyzed the presence of esterase genes, both the putative genes and the determined experimentally genes, and performed a ClustalW analysis for feruloyl esterases. Major details will be presented for the ORF PSHAa1385 from P. haloplanktis TAC125 that recently has been studied in our research group. In addition, the potential biotechnology applications of this class of enzymes will be discussed.     

Identification of two feruloyl esterases in Dickeya dadantii 3937 and induction of the major feruloyl esterase and of pectate lyases by ferulic acid

The plant-pathogenic bacterium Dickeya dadantii (formerly Erwinia chrysanthemi) produces a large array of plant cell wall-degrading enzymes. Using an in situ detection test, we showed that it produces two feruloyl esterases, FaeD and FaeT. These enzymes cleave the ester link between ferulate and the pectic or xylan chains. FaeD and FaeT belong to the carbohydrate esterase family CE10, and they are the first two feruloyl esterases to be identified in this family. Cleavage of synthetic substrates revealed strong activation of FaeD and FaeT by ferulic acid. The gene faeT appeared to be weakly expressed, and its product, FaeT, is a cytoplasmic protein. In contrast, the gene faeD is strongly induced in the presence of ferulic acid, and FaeD is an extracellular protein secreted by the Out system, responsible for pectinase secretion. The product of the adjacent gene faeR is involved in the positive control of faeD in response to ferulic acid. Moreover, ferulic acid acts in synergy with polygalacturonate to induce pectate lyases, the main virulence determinant of soft rot disease. Feruloyl esterases dissociate internal cross-links in the polysaccharide network of the plant cell wall, suppress the polysaccharide esterifications, and liberate ferulic acid, which contributes to the induction of pectate lyases. Together, these effects of feruloyl esterases could facilitate soft rot disease caused by pectinolytic bacteria.