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。     

野生茯苓鉴定及其木质纤维素降解酶系研究

为揭示茯苓木质纤维素降解酶系及培养方式对其主要酶系的影响，该研究通过对野生茯苓菌株进行培养特性的显微观察，利用3对引物PCR扩增进行系统发育学的鉴定，经定性培养筛选出优势菌株YX1,采用酶标仪测定不同条件下茯苓纤维素酶、半纤维素酶和木质素降解酶的活力大小。结果表明：(1)茯苓有菌丝体、子实体和菌核3种形态特征。(2)PCR分别获得rDNA-ITS序列1 652 bp、核糖体大亚基序列660 bp和翻译延伸因子序列545 bp,提交至NCBI,登录号分别为ON129554、ON129553和ON155840。(3)纤维素酶和半纤维素酶在有松木屑和无松木屑条件下，外切β-葡聚糖酶(CBH)、内切β-葡聚糖酶(EG)、β-葡萄糖苷酶(BGL)最高分泌量分别为16～17 U·mL^-1、32～35 U·mL^-1、36～37 U·mL^-1;木聚糖酶、甘露聚糖酶和α-葡萄糖苷酶最高分泌量分别为28～38 U·mL^-1、280～342 U·mL^-1、9～11 U·mL^-1...     

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。     

草酸青霉纤维素酶基因和木聚糖酶基因的调控基因POX05145的鉴定

草酸青霉能产生完整的纤维素酶和木聚糖酶酶系,其纤维素酶基因的表达主要受转录因子的调控。前期工作中,通过对草酸青霉菌株HP7-1在不同碳源培养基培养条件下转录组的比较分析,获得了调控纤维素酶和木聚糖酶产量的候选调控基因集。本研究以草酸青霉ΔPoxKu70为出发菌株,通过同源重组法,构建并获得了其中一个候选调控基因POX05145的缺失突变株ΔPOX05145。在微结晶纤维素Avicel诱导培养条件下,与出发菌株ΔPoxKu70相比,ΔPOX05145的纤维素酶产量和木聚糖酶产量发生了显著改变。其中,在诱导第2天时,ΔPOX05145对硝基苯-β-D-纤维二糖苷酶产量和木聚糖酶产量分别上升43.4%和164.7%,对硝基苯-β-D-半乳糖吡喃葡萄糖苷酶产量下降92.8%,但是,滤纸酶产量和羧甲基纤维素酶产量没有显著变化。然而,在诱导第4天时,所有纤维素酶产量和木聚糖酶产量上升100.4%~294.0%。实时荧光定量PCR检测表明POX05145在不同的时间不同程度的调控主要的纤维素酶基因和木聚糖酶基因的表达。序列分析表明POX05145含有一个GAL4类锌指结构的DNA结合功能域和一个保守的真菌特有的转录因子结构域(Fungal_TF_MHR)。     

嗜酸真菌Bispora sp.MEY-1酸性糖苷水解酶的产酶优化及在动物饲料中的应用

嗜酸真菌Bisporasp.MEY-1可以分泌多种酸性糖苷水解酶。以不同的农业废弃物和副产品为碳源诱导该菌产酶以提高液体发酵效率并降低生产成本,结果表明0.5%的魔芋粉和9%的麦麸/玉米芯粉/豆粕混合物诱导效果最佳,木聚糖酶、葡聚糖酶、甘露聚糖酶、纤维素酶、淀粉酶、果胶酶、α-半乳糖苷酶、β-半乳糖苷酶、β-葡萄糖苷酶的产量分别为15、50、3.7、5.7、3.2、0.6、3.4、0.1、1.8U·ml-1。菌株MEY-1最适产酶pH为3,温度为30℃。与优良的商业糖苷水解酶生产菌株Trichoderma reesei RUT C30相比,菌株MEY-1分泌的糖苷水解酶偏酸,且在模拟胃肠道条件下保留了更多的木聚糖酶和葡聚糖酶活性,可降低大麦/豆粕饲料的黏度,在动物饲料工业中具有良好的应用前景。     

根霉TC1653产纤维素酶提纯及酶学性质研究

对根霉所产纤维素酶酶系进行了分析并研究了部分酶学性质。实验选择超滤和凝胶柱分离相结合的方式提纯纤维素酶,结果显示根霉TC1653纤维素酶系是一个完全酶系,具有一个较为明显的内切葡聚糖酶组分。β-葡萄糖苷酶组分的最适反应温度为70℃,温度高于70℃时,活性迅速下降,但在这种高温下具有最高反应活性的酶很少见,很可能又是一种新的β-葡萄糖苷酶。     

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。     

Coexpression of β-mannanase and xylanase genes in Escherichia coli

[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21h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.     

纤维素酶在生物质转化中的应用研究进展

纤维素酶是木质纤维素转化的重要酶系,主要是由内切β-1,4-葡聚糖酶、外切β-1,4-葡聚糖酶和β-葡萄糖苷酶组成,水解不同位置的糖苷键从而形成葡萄糖。本文总结了纤维素酶近几年来的研究开发与应用研究进展,包含产酶菌和菌种选育、辅助蛋白,介绍了纤维素酶来源和组成、纤维素酶工业化生产的最新研究进展以及纤维素酶在生物质转化方面的应用,纤维素酶在1.5代、2代纤维素燃料乙醇及生物质制气中都有重要的应用。     

东方肉座菌EU7-22木聚糖酶和木糖苷酶基因克隆及生物信息学研究

东方肉座菌EU7-22具有高产半纤维素酶的能力。根据已报道的同属里氏木霉及绿色木霉木聚糖酶,木糖苷酶相关基因序列,设计PCR引物扩增出东方肉座菌内切木聚糖酶(XYNⅠ,XYNⅡ)及β-木糖苷酶(β-BXL)基因。序列经NCBIBlast分析:东方肉座菌xynⅠ基因与里氏木霉xyn1基因(X69573.1)的同源性最高达到91%;xynⅡ基因与绿色木霉xyn2基因(EF079061)同源性最高达到93%;β-bxl基因与里氏木霉β-bxl1基因(Z69257.1)的同源性最高达到94%。生物信息学分析表明内切木聚糖酶Ⅰ和Ⅱ均属于糖基水解酶家族11,N末端前19个氨基酸均为信号肽。内切木聚糖酶Ⅰ分子量为24.13kD,等电点为7.87,含有3个糖基化位点;内切木聚糖酶Ⅱ分子量为24.44kD,等电点为4.86,含有1个糖基化位点;β-木糖苷酶属于糖基水解酶家族3,分子量为87.27kD,等电点为5.49,N末端前20个氨基酸为信号肽,含有8个糖基化位点。利用SWISS-Model对木聚糖酶,木糖苷酶蛋白质三级结构进行了预测和模拟。对木聚糖酶和木糖苷酶基因及其编码蛋白质的生物信息学分析,为进一步研究这些基因的表达与调控、构建高效利用纤维素组份的工程菌株奠定基础。     

Biotechnological production of arbutins (α- and β-arbutins), skin-lightening agents, and their derivatives

Arbutins (α- and β-arbutins) are glycosylated hydroquinones that are commercially used in the cosmetic industry. These compounds have an inhibitory function against tyrosinase, a critical enzyme for generating pigments, which leads to the prevention of melanin formation, resulting in a whitening effect on the skin. Although β-arbutin is found in various plants including bearberry, wheat, and pear, α-arbutin and other arbutin derivatives are synthesized by chemical and enzymatic methods. This article presents a mini-review of recent studies on the production of α-arbutin and other α- and β-arbutin derivatives via enzymatic bioconversion methods. In addition, the structures of α- and β-arbutin derivatives and their biological activities are discussed. The catalytic characteristics of various enzymes used in the biosynthesis of arbutin derivatives are also reviewed.     

A specific concanavalin A-mediated binding of bovine serum α-mannosidase to cultured human skin fibroblasts

A specific elevation of cell-associated α-mannosidase was observed in human skin fibroblasts cultured with concanavalin A for 12–72 hours. There was a latency of several hours before the increase of the enzyme activity occurred. When the cells were washed with α-methylmannoside, α-mannosidase activity was not increased. Other lysosomal enzymes including β-mannosidase showed a slight decrease in activity. It was concluded that the elevation of this enzyme activity was the result of a specific binding to the cell surface mediated by concanavalin A.     

Emerging role of alpha2,6-sialic acid as a negative regulator of galectin binding and function

Galectins are β-galactoside-binding lectins that regulate diverse cell behaviors, including adhesion, migration, proliferation, and apoptosis. Galectins can be expressed both intracellularly and extracellularly, and extracellular galectins mediate their effects by associating with cell-surface oligosaccharides. Despite intensive current interest in galectins, strikingly few studies have focused on a key enzyme that acts to inhibit galectin signaling, namely β-galactoside α2,6-sialyltransferase (ST6Gal-I). ST6Gal-I adds an α2,6-linked sialic acid to the terminal galactose of N-linked glycans, and this modification blocks galectin binding to β-galactosides. This minireview summarizes the evidence suggesting that ST6Gal-I activity serves as an "off switch" for galectin function.     

Inhibitory effects of naphthols on the activity of mushroom tyrosinase

Tyrosinase (EC 1.14.18.1), a copper-containing multifunctional oxidase, was known to be a key enzyme for biosynthesis in fungi, plants and animals. In this work, the inhibition properties α-naphthol and β-naphthol toward the activity of tyrosinase have been evaluated, and the effects of α-naphthol and β-naphthol on monophenolase and diphenolase activity of tyrosinase have been investigated. The results showed that both α-naphthol and β-naphthol could potently inhibit both monophenolase activity and diphenolase activity of mushroom tyrosinase, and that β-naphthol exhibited stronger inhibitory effect against tyrosinase than α-naphthol. For monophenolase activity, β-naphthol could not only lengthen the lag time but also decrease the steady-state activity, while α-naphthol just only decreased the steady-state activity. For diphenolase activity, both α-naphthol and β-naphthol displayed revisible inhibition. Kinetic analyses showed that both α-naphthol and β-naphthol were competetive inhibitors.     

Structural features determining the site specificity of a rat liver cAMP-independent protein kinase.

The Seryl and Threonyl residues affected in α_s1 and in β-caseins by rat liver “casein kinase TS” (a cytosolic cAMP-independent protein kinase) have been identified. All of them, as well as the residues affected by the same enzyme in α_s2-casein are characterized by an acidic group two residues to their C terminus and by being located within predicted β-turns. Several other potential sites of phosphorylation, according to their primary structure, but located outside predicted β-turns, are not significantly labeled by the protein kinase. It seems conceivable therefore that both a definite aminoacid sequence including a critical acidic residue, and the existence of a β-turn are required for the activity of this protein kinase.     

Experimental diabetes treated with trigonelline: effect on key enzymes related to diabetes and hypertension, β-cell and liver function

Type 2 diabetes is quite diverse, including the improvement of insulin sensitivity by dipeptidylpeptidase-4 (DPP-4) inhibitor, α-glucosidase inhibitors, and the protection of β-cells islet. The aim of this study was to search the effect of trigonelline (Trig) on DPP-4, α-glucosidase and angiotensin converting enzyme (ACE) activities as well as β-cells architecture, and starch and glucose tolerance test. In surviving diabetic rats, the supplement of Trig potentially inhibited DPP-4 and α-glucosidase activities in both plasma and small intestine. The pancreas islet and less β-cells damage were observed after the administration of trig to diabetic rats. The increase of GLP-1 in surviving diabetic rats suppressed the increase of blood glucose level and improved results in the oral glucose and starch tolerance test. Trig also normalized key enzyme related to hypertension as ACE and improved the hemoglobin A1c and lipid profiles (plasma triglyceride, HDL-cholesterol, LDL-cholesterol, and total cholesterol), and liver indices toxicity. Therefore, these results revealed that Trig was successful in improving glycemic control, metabolic parameters, and liver function in diabetic rats. It is therefore suggested that Trig may be a potential agent for the treatment of type 2 diabetes.     

Effects of frontal ganglion removal and starvation on activity and distribution of six gut enzymes in Locusta

A study has been made on the effects of frontal ganglion removal and starvation on the activities and distribution of α- and β-glucosidase, α- and β-galactosidase, trehalase, and ‘trypsin’ in various regions of the alimentary canal of adult locusts. Both treatments resulted in a reduction in the amount of enzyme activity. In addition, the distribution of enzyme activity was changed by comparison with the operated control insects; the foregut of starved and operated animals showing a smaller proportion of the overall gut enzyme activity. The results are discussed in relation to the control of enzyme secretion.     

Studies of glucosidase activities from surface sediments in mangrove swamp

Four transects including sixteen stations were established in the Fugong mangrove (117°54′-117°55′E, 24°22′-24°24′N) of the Jiulong River Estuary, Fujian, China. Besides geochemical characterization and estimation of bacterial abundances, the distribution of α- and β-glucosidase activity was studied to explore the degradation of carbohydrates which can be expected to occur in high quantities in mangrove systems. The distribution pattern of microbial α-glucosidase and β-glucosidase activities was investigated using a fluorogenic model substrate (FMS) technique in order to allow better understanding of in situ enzyme activities, as well as their relation to bacterial biomass, metabolic activity and environmental factors in mangrove sediments. The results showed that the enzyme activities of α-glucosidase (10.83~100.86 µmol g^- 1 h^- 1) and β-glucosidase (39.60~222.75 µmol g^- 1 h^- 1) varied among the different stations, and the enzyme activities of β-glucosidase were higher than those of α-glucosidase at all stations. The extracellular enzyme activities were positively related to organic C, organic matter and bacterial abundance. In addition, the use of the FMS technique to measure extracellular enzyme activities of mangrove sediments could help us to evaluate their catabolic behavior in situ and so lead to a better understanding of the bacterial role in material cycle of mangrove swamp ecosystems.     

The purification and properties of an aryl beta-hexosidase from bovine liver.

An aryl β-hexosidase was purified 800-fold from bovine liver. The purified enzyme hydrolyzed p-nitrophenyl glycosylpyranoside derivatives of β-d-galactose, β-d-glucose, β-d-xylose, β-d-mannose, and α-l-arabinose, but did not hydrolyze several other p-nitrophenyl glycosides. The enzyme also catalyzed hydrolysis of a variety of plant arylglucosides. Disaccharides, polysaccharides, glycolipids, glycoproteins, and glycosaminoglycans containing terminal nonreducing β-d-galactopyranosyl or β-d-glucopyranosyl residues were not hydrolyzed. The pH optima for the several substrates tested ranged from 7.0 to 9.5. The purified enzyme was homogeneous by disc gel electrophoresis and had a molecular weight of 41,000 by Sephadex gel filtration and 46,000 by disc gel electrophoresis performed in the presence of sodium dodecyl sulfate. The enzyme readily transferred glycosyl residues from susceptible β-galactosides or β-glucosides to other sugars; the resulting products were not hydrolyzed by the enzyme. Methyl α-d-glucopyranoside was the most efficient carbohydrate acceptor compound tested. The enzyme exhibited a K_m for p-nitrophenyl β-d-galactopyranoside of 1.78 × 10^?3m and for p-nitrophenyl β-d-glucopyranoside, 2.50 × 10^?3m when incubations were conducted in the presence of 0.15 m methyl α-d-glucopyranoside. Aryl β-hexosidase was found in the cytosol of all mammalian livers tested, but could not be detected in liver of birds, reptiles, or fish; low levels were detected in frog liver. Analysis of bovine extracts indicated that the enzyme occurred in liver, kidney, and intestinal mucosa; it was not detected in testis, spleen, serum, or muscle.