利用毛壳霉固体发酵木聚糖酶

毛壳属真菌大多数具有较强的纤维素降解能力地,其中球毛菌所产生的木聚糖酶活性较强.木聚糖酶通过水解木糖分子间的β-1,4-糖苷键,将木聚糖水解成低聚木糖及少量木糖和阿拉伯糖.     

β-木糖苷酶及其在纤维素乙醇生产中的应用研究进展

将木质纤维素类生物质生物转化生产液体燃料,如纤维素乙醇和大宗化学品,对缓解当前人类社会面临的能源和资源危机以及保护环境具有重要意义。半纤维素是木质纤维素类生物质的主要组成成分之一,它的生物降解转化对实现木质纤维素生物炼制意义重大。由于半纤维素糖种类的多样性和半纤维素结构的复杂性,需要一个复杂的半纤维素酶系才能完成对半纤维素的有效降解。除了木聚糖酶等以外,β-木糖苷酶也是半纤维素酶系的主要组分。在半纤维素降解过程中,β-木糖苷酶将木聚糖酶的水解产物木寡糖和木二糖水解为木糖,不仅在木聚糖的彻底降解过程中起着重要作用,而且可以缓解木寡糖对木聚糖酶和纤维素酶的抑制作用。该文综述了目前在β-木糖苷酶方面的研究进展,包括β-木糖苷酶的分类、酶学性质、酶结构及其催化机制、基因的克隆与表达等,并对β-木糖苷酶在纤维素乙醇生产中的应用情况进行了简述。     

用氢氧化钾和过氧化氢从甘蔗渣中提取木聚糖的条件优化及甘蔗渣木聚糖酶解产低聚木糖的分析

甘蔗渣含有约40%纤维素和20%半纤维素,是生物炼制生产高附加值生化产品的良好原料。本研究通过响应面分析实验优化了氢氧化钾和过氧化氢处理甘蔗渣从中提取木聚糖的工艺参数:氢氧化钾浓度、过氧化氢浓度和处理温度,以甘蔗渣木聚糖提取率为考察指标,获得处理甘蔗渣的最优条件:10.0%KOH,2.36%H_2O_2,50℃,处理时间6 h,甘蔗渣木聚糖提取率达到78%。用重组内切木聚糖酶水解提取的甘蔗渣木聚糖生产低聚木糖,主要包括木二糖和木三糖,在底物浓度36 g/L、反应温度36℃、p H 5.0、木聚糖酶添加量800 U/g的条件下,水解反应24 h,木聚糖产生低聚木糖的转化率为43%。     

微生物木聚糖降解酶系统

木聚糖类半纤维素是产量仅次于纤维素的植物多糖 ,其结构要比纤维素复杂得多 ,完全降解木聚糖 ,实现植物残体的生物转化需要多种水解酶 (即木聚糖降解酶系统 )的协同作用。木聚糖酶在食品、饲料、纺织、能源工业 ,特别是在纸浆和造纸工业中有着广阔的应用前景 ,如人们将极端嗜热和嗜碱菌的木聚糖酶基因克隆到现有工程菌中生产工业用酶 ,用于纸浆的生物漂白和饲料加工。但是木聚糖资源的开发利用要求完整的酶系统。人们通过对具有木聚糖降解酶系统微生物的研究 ,运用基因工程技术将其构建成发酵工程菌 ,直接利用半纤维素生产单细胞蛋白 ;或者…     

微生物木聚糖降解酶研究进展及应用前景

木聚糖是植物半纤维素的主要成分,它是除纤维素外,自然界中最为丰富的多糖。木聚糖的基本结构单元是由β－１．４或β－１．３糖苷键连接的多聚木糖链,在Ｄ－木糖的第二位氧上连接有Ｄ－葡萄糖醛酸或４－Ｏ－甲基葡萄糖醛酸或在第三位氧上连接有Ｌ－阿拉伯呋喃糖。有些木聚糖还在第二或第三位氧上发生乙酰化。不同来源的木聚糖在结构上有一定差异。木聚糖酶是一类木聚糖降解酶系（表１）,对降解自然界大量存在的半纤维素起着重要作用。它们不但可以降解木聚糖生成木糖,而且能以农作物残渣中的半纤维素为原料生产经济价值较高的产品。由…     

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。     

木聚糖酶异源表达的研究进展

木聚糖(xylan)在自然界中的含量极其丰富,在农作物和农林剩余物中大量存在。随着能源资源问题的日益凸显,对木聚糖的应用和研究越来越受到重视。木聚糖酶(xylanase)是可以将木聚糖降解为低聚木糖和木糖的一类水解酶,近年来,为了实现木聚糖酶的高产、高酶活表达,科研工作者做了大量的研究工作,就木聚糖酶异源表达(heterologous expression)的研究进展进行综述。     

嗜热拟青霉固体发酵产木聚糖酶的纯化和性质

研究一株新的嗜热拟青霉J18的固体发酵产木聚糖酶的纯化和性质。固体发酵的粗酶液经硫酸铵沉淀、凝胶过滤层析和离子交换层析得到了一种分子量约为26 kDa的电泳纯木聚糖酶,酶活力回收率为33.5%,纯化了5.27倍。该木聚糖酶具有很好的温度和pH稳定性,在pH7.0~pH 9.0下,60℃处理24 h,酶活力能保存80%以上。该酶水解玉米芯木聚糖生成以木二糖、木三糖和木四糖为主的低聚木糖,薄层层析分析表明不含木糖,适合生产低聚木糖。     

木聚糖酶的分子生物学及其应用

木聚糖是一种多聚五碳糖,是植物细胞中的主要半纤维素成分,木聚糖酶是可将木聚糖降解成低聚木糖和木糖的复合酶系,综述了木聚糖酶分子生物学上的研究进展及其在饲料,造纸,食品,能源工业上的应用。     

木聚糖酶分子结构与重要酶学性质关系的研究进展

木聚糖是一种多聚五碳糖 ,是植物细胞中主要的半纤维素成分。木聚糖酶是可将木聚糖降解成低聚木糖和木糖的水解酶 ,它在饲料、造纸、食品、能源工业和环境科学上有着广阔的应用前景。随着分子生物学、结构生物学的发展及蛋白质工程的应用 ,对木聚糖酶结构和功能的研究不断深入。这里重点阐述与酶的活性、热稳定性、作用pH、等电点、底物亲和性及催化效率等重要性质相关的分子结构研究进展 ,讨论了其进一步的研究发展方向。研究木聚糖酶结构与功能的关系 ,对进一步加深木聚糖酶作用机制的了解、指导木聚糖酶的分子改良有重要意义。     

碱性木聚糖酶研究进展*

木聚糖是一种在自然界中含量仅次于纤维素的丰富的可再生资源,木聚糖酶是一类可以将木聚糖水解成单糖和寡糖的酶,利用木聚糖酶将木聚糖分解后的产物被广泛应用于食品、造纸以及纺织等行业。木聚糖酶按其对酸碱环境的耐受能力分为碱性木聚糖酶、中性木聚糖酶和酸性木聚糖酶,其中碱性木聚糖酶适合应用于造纸工业中,尤其在造纸的制浆、促进漂白及废纸脱墨等多种工艺中,可以显著提高纸张质量,有效降低氯气排放量,从而减少对环境的污染。随着生物技术的进步,利用基因工程技术可以对碱性木聚糖酶进行分子改造,以提高其耐碱、耐热能力,扩大其在工业应用中的条件范围。介绍碱性木聚糖酶在分子改造方面的研究进展以及其在造纸漂白和制浆、废纸脱墨中的应用。     

Impact and efficiency of GH10 and GH11 thermostable endoxylanases on wheat bran and alkali-extractable arabinoxylans

The results of a comparative study of two thermostable (1-->4)-beta-xylan endoxylanases using a multi-technical approach indicate that a GH11 xylanase is more useful than a GH10 xylanase for the upgrading of wheat bran into soluble oligosaccharides. Both enzymes liberated complex mixtures of xylooligosaccharides. 13C NMR analysis provided evidence that xylanases cause the co-solubilisation of beta-glucan, which is a result of cell-wall disassembly. The simultaneous use of both xylanases did not result in a synergistic action on wheat bran arabinoxylans, but instead led to the production of a product mixture whose profile resembled that produced by the action of the GH10 xylanase alone. Upon treatment with either xylanase, the diferulic acid levels in residual bran were unaltered, whereas content in ferulic and p-coumaric acids were unequally decreased. With regard to the major differences between the enzymes, the products resulting from the action of the GH10 xylanase were smaller in size than those produced by the GH11 xylanase, indicating a higher proportion of cleavage sites for the GH10 xylanase. The comparison of the kinetic parameters of each xylanase using various alkali-extractable arabinoxylans indicated that the GH10 xylanase was most active on soluble arabinoxylans. In contrast, probably because GH11 xylanase can better penetrate the cell-wall network, this enzyme was more efficient than the GH10 xylanase in the hydrolysis of wheat bran. Indeed the former enzyme displayed a nearly 2-fold higher affinity and a 6.8-fold higher turnover rate in the presence of this important by-product of the milling industry.     

来源于橄榄绿链霉菌A1的高比活木聚糖酶XYNB的高效表达及性质的比较分析

高效表达高比活木聚糖酶是进一步提高木聚糖酶发酵效价、降低其生产成本的有效途径。将橄榄绿链霉菌(Streptomyces olivaceoviridis) A1的高比活木聚糖酶成熟蛋白编码基因xynB克隆到毕赤酵母表达载体pPIC9中,转化毕赤酵母得到重组酵母,在重组酵母中木聚糖酶基因得到了高效分泌表达,且表达产物具有生物学活性。在3L发酵罐中蛋白表达量约14mg/mL, 酶活性(效价)为1200IU/mL。SDSPAGE分析表明,表达的木聚糖酶XYNBa为糖基化蛋白, 分子量为31kD, 经脱糖基化处理得到21kD 的XYNBb, 与橄榄绿链霉菌A1所产原酶XYNB大小一致。通过对XYNB、XYNBa及XYNBb酶学性质的比较发现：三者在比活性、Vmax及热稳定性方面有较大差异。该酶对不同木聚糖的酶解产物的糖份分析表明：酶解产物的主要成分为木二糖、木三糖和木四糖,占总糖含量的95%以上。     

Production and partial characterization of cellulase free xylanase by Bacillus subtilis C 01 using agriresidues and its application in biobleaching of nonwoody plant pulps

AIMS: To optimize the solid-state cultivation conditions for xylanase production using agriresidues and testing the biobleaching efficiency of xylanase on nonwoody plant fibre materials. METHODS AND RESULTS: An extracellular cellulase free xylanase was produced from Bacillus subtilis C 01 using various inexpensive substrates under solid-state cultivation. High level of xylanase production (135 IU gds(-1)) was observed when grown on wheat bran followed by maize powder (50 IU gds(-1)). The maximum xylanase (136 IU gds(-1)) production was occurred in wheat bran-to-moisture ratio of 1 : 1 at 72 h. The xylanase pretreated pulp samples of banana, silk cotton and cotton showed an increased brightness of 19.6, 11.6 and 7.9%, respectively. CONCLUSIONS: The enzyme-aided biobleaching results indicate that the xylanase has potential application in enhancing the brightness of nonwoody plant fibre pulp. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on biobleaching of banana fibres, silk cotton and cotton pulps using xylanase. The biobleaching results of secondary fibres are promising and can be transferred to paper mills, which utilize nonwoody plant fibres as a raw material for paper production.     

Cloning of a Thermomonospora fusca xylanase gene and its expression in Escherichia coli and Streptomyces lividans.

Thermomonospora fusca chromosomal DNA was partially digested with EcoRI to obtain 4- to 14-kilobase fragments, which were used to construct a library of recombinant phage by ligation with EcoRI arms of lambda gtWES. lambda B. A recombinant phage coding for xylanase activity which contained a 14-kilobase insert was identified. The xylanase gene was localized to a 2.1-kilobase SalI fragment of the EcoRI insert by subcloning onto pBR322 and derivatives of pBR322 that can also replicate in Streptomyces lividans. The xylanase activity produced by S. lividans transformants was 10- to 20-fold higher than that produced by Escherichia coli transformants but only one-fourth the level produced by induced T. fusca. A 30-kilodalton peptide with activity against both Remazol brilliant blue xylan and xylan was produced in S. lividans transformants that carried the 2.1-kilobase SalI fragment of T. fusca DNA and was not produced by control transformants. T. fusca cultures were found to contain a xylanase of a similar size that was induced by growth on xylan or Solka Floc. Antiserum directed against supernatant proteins isolated from a Solka Floc-grown T. fusca culture inhibited the xylanase activity of S. lividans transformants. The cloned T. fusca xylanase gene was expressed at about the same level in S. lividans grown in minimal medium containing either glucose, cellobiose, or xylan. The xylanase bound to and hydrolyzed insoluble xylan. The cloned xylanase appeared to be the same as the major protein in xylan-induced T. fusca culture supernatants, which also contained at least three additional minor proteins with xylanase activity and having apparent molecular masses of 43, 23, and 20 kilodaltons.     

A single domain thermophilic xylanase can bind insoluble xylan: evidence for surface aromatic clusters.

A clone expressing xylanase activity in Escherichia coli has been selected from a genomic plasmid library of the thermophilic Bacillus strain D3. Subcloning from the 9-kb insert located the xylanase activity to a 2.7-kb HindII/BamHI fragment. The DNA sequence of this clone revealed an ORF of 367 codons encoding a single domain type-F or family 10 enzyme, which was designated as XynA. Purification of the enzyme following over-expression in E. coli produced an enzyme of 42 kDa with a temperature optimum of 75 degrees C which can efficiently bind and hydrolyse insoluble xylan. The pH optimum of the enzyme is 6.5, but it is active over a broad pH range. A homology model of the xylanase has been constructed which reveals a series of surface aromatic residues which form hydrophobic clusters. This unusual structural feature is strikingly similar to the situation observed in the structure determined for the type-G xylanase from the Bacillus D3 strain and may constitute a common evolutionary mechanism imposed on different structural frameworks by which these xylanases may bind potential substrates and exhibit thermostability.     

Effect of tunicamycin on xylanase secretion in the yeast Cryptococcus albidus

The yeast Cryptococcus albidus secretes a glycosylated xylanase (48 kDa) in the culture medium in response to beta-methylxyloside as inducer. Addition of tunicamycin to the medium results in the formation of a modified xylanase (40 kDa) which is depleted in carbohydrate content and whose enzymatic activity is 2.5 times less than that of the glycosylated xylanase. The secretion of xylanase was followed under both conditions by pulse-chase experiments. The half-time of secretion of the glycosylated and nonglycosylated forms was 5 and 2 h, respectively. Cell-associated xylanase activity was not detected when the cells were treated with the antibiotic. The absence of cell wall-associated xylanase, after tunicamycin treatment, was confirmed by immunolocalization with anti-xylanase antibodies at the electron microscopic level. The results suggest that the interactions of carbohydrate moiety within the cell wall retarded the secretion of the enzyme to the medium.     

β-甘露聚糖酶和木聚糖酶基因在大肠杆菌中共表达

【目的】β-甘露聚糖酶和木聚糖酶都属于半纤维素酶,它们已经同时运用于工农业生产的许多领域。构建β-甘露聚糖酶和木聚糖酶共表达菌株并进行相关评价。【方法】通过设计一个共同的酶切位点,将菌株Bacillus subtilis BE-91中的β-甘露聚糖酶和木聚糖酶基因串联到表达载体pET28a(+)上,转化大肠杆菌构建了一株能够共表达β-甘露聚糖酶和木聚糖酶的菌株B.pET28a-man-xyl。【结果】菌株诱导21 h后,发酵液中β-甘露聚糖酶和木聚糖酶的酶活分别为713.34 U/mL和1455.83 U/mL,是胞内酶活的11.8倍和2.53倍。【结论】SDS-PAGE分析、水解圈活性检测和胞外酶与胞内酶酶活检测表明:两个酶均以功能蛋白独立分泌到胞外。此外,与β-甘露聚糖酶和木聚糖酶单独酶解半纤维素相比,复合酶的酶解效果更好。菌株的成功构建为复合酶制剂(半纤维素酶制剂)的研究和生产奠定基础。     

Cloning, characterization, and expression of xylanase gene from Bacillus lyticus in Escherichia coli and Bacillus subtilis.

A genomic library of Bacillus lyticus was constructed in lambda GEM 11 vector and screened for the xylanase gene using Congo red plate assay. A 16-kb fragment containing the xylanase gene was obtained which was further subcloned using Mbo I partial digestion in an E. coli pUC 19 vector. A 1.3-kb sub-fragment was obtained which coded for a xylanase gene of Mr 23,650 Da. This fragment was sequenced and the homology was checked with known xylanases. The maximum homology was 97%, which was obtained with an endo xylanase gene from Bacillus species at the DNA level, while the translated sequence showed only one amino acid change from alanine to serine at position number 102. Expression was checked in E. coli, using the native promoter, and an extracellular activity of 5.25 U/mL was obtained. Cloning of the gene was done in Bacillus subtilis using a shuttle vector pHB 201, which resulted in increasing the basal level xylanase activity from 14.02 to 22.01 U/mL.     

Expression of a Clostridium thermocellum xylanase gene in Bacillus subtilis

Summary The native promoter of a xylanase gene isolated from Clostridium thermocellum was replaced with a strong promoter screened from Bacillus subtilis chromosomes. A part of the C-terminal region of the gene which is not related to the xylanase activity was removed. With the modified xylanase gene, B. subtilis was transformed and grown in LB medium. The xylanase gene was expressed well in B. subtilis and extracellular xylanase was produced up to 30 units per ml when the growth reached OD₆₀₀ of 4.8.