Bacillus pumilus WL-11木聚糖酶A的纯化、鉴定及其底物降解方式

对一株Bacilluspumilus WL_11木聚糖酶的纯化、酶学性质及其底物降解模式进行了研究。经过硫酸铵盐析、CM_Sephadex及SephadexG_75层析分离纯化,获得一种纯化的WL_11木聚糖酶A ,其分子量为26.0kD ,pI值9.5 ,以燕麦木聚糖为底物时的表观Km 值为16.6mg mL ,Vmax值为12.63μmol (min·mg)。木聚糖酶A的pH稳定范围为6 0至10 4 ,最适作用pH范围则在7.2至8.0之间,是耐碱性木聚糖酶;最适作用温度为45℃～55℃,在37℃、45℃以下时该酶热稳定性均较好;50℃保温时,该酶活力的半衰期大约为2h ,在超过50℃的环境下,该酶的热稳定较差,55℃和60℃时的酶活半衰期分别为35min和15min。WL_11木聚糖酶A对来源于燕麦、桦木和榉木的可溶性木聚糖的酶解结果发现,木聚糖酶A对几种不同来源的木聚糖的降解过程并不一致。采用HPLC法分析上述底物的降解产物生成过程发现木聚糖酶A为内切型木聚糖酶,不同底物的降解产物中都无单糖的积累,且三糖的积累量都较高;与禾本科的燕麦木聚糖底物降解不同的是,木聚糖酶A对硬木木聚糖降解形成的五糖的继续降解能力较强。采用TLC法分析了WL-11粗木聚糖酶降解燕麦木聚糖的过程,结果表明燕麦木聚糖能够被WL-11粗木聚糖酶降解生成系列木寡糖,未检出木糖,这说明WL-11主要合成内切型木聚糖酶A,同时发酵液中不含木糖苷酶,适合用来酶法制备低聚木糖。     

Bacillus pumilus WL-11木聚糖酶A的纯化、鉴定及其底物降解方式

对一株BacilluspumilusWL_11木聚糖酶的纯化、酶学性质及其底物降解模式进行了研究。经过硫酸铵盐析、CM_Sephadex及SephadexG_75层析分离纯化,获得一种纯化的WL_11木聚糖酶A ,其分子量为2 6 0kD ,pI值9 5 ,以燕麦木聚糖为底物时的表观Km 值为16 6mg mL ,Vmax值为12 6 3μmol (min·mg)。木聚糖酶A的pH稳定范围为6 0至10 4 ,最适作用pH范围则在7 2至8 0之间,是耐碱性木聚糖酶;最适作用温度为4 5℃～5 5℃,在37℃、4 5℃以下时该酶热稳定性均较好;5 0℃保温时,该酶活力的半衰期大约为2h ,在超过5 0℃的环境下,该酶的热稳定较差,5 5℃和6 0℃时的酶活半衰期分别为35min和15min。WL_11木聚糖酶A对来源于燕麦、桦木和榉木的可溶性木聚糖的酶解结果发现,木聚糖酶A对几种不同来源的木聚糖的降解过程并不一致。采用HPLC法分析上述底物的降解产物生成过程发现木聚糖酶A为内切型木聚糖酶,不同底物的降解产物中都无单糖的积累,且三糖的积累量都较高;与禾本科的燕麦木聚糖底物降解不同的是,木聚糖酶A对硬木木聚糖降解形成的五糖的继续降解能力较强。采用TLC法分析了WL_11粗木聚糖酶降解燕麦木聚糖的过程,结果表明燕麦木聚糖能够被WL_11粗木聚糖酶降解生成系列木寡糖,未检出木糖,这说明WL_11主要合成内切型木聚     

中度嗜盐菌产木聚糖酶发酵条件的研究

中度嗜盐菌在盐碱环境下生长繁殖,其产生的木聚糖酶也同样具有在盐碱环境下发挥作用的特性。本文对一株中度嗜盐菌的产木聚糖酶活性进行了初步研究。研究包括氮源、液体种子接种量、培养温度、pH值、培养时间等因素对该菌株产木聚糖酶能力的影响。结果表明,最佳培养氮源为蛋白胨;最佳产生木聚糖酶的发酵条件是液体种子接种量为6%,温度为35℃,pH值7,培养时间为4 d。     

嗜热真菌耐热木聚糖酶的产酶条件和酶谱分析*

嗜热真菌Thermomyces lanuginosus CBS288.54-M18耐热木聚糖酶的产酶条件和酶谱分析结果表明：玉米芯水不溶木聚糖相对于其它来源木聚糖为最佳碳源,而酵母提取物和蛋白胨作为复合氮源时效果最好。培养基最适初始pH值为7．0,最适培养温度为50℃。在最适条件下发酵所产木聚糖酶活力最高达1.834u／mL。另外,SDS-PAGE和酶谱分析(变性和非变性状态下)结果都表明该菌只产生一种分子量约为26kD的G/11族木聚糖酶。     

具有高木二糖形成活力的木聚糖酶生产菌株的筛选与产酶培养基的优化

从土样中筛选出一株产木聚糖酶的青霉,该青霉所产木降糖酶具有很高的木二糖形成活力,经鉴定为顶青霉,其木聚糖酶的合成与分泌受木聚糖等木糖苷类物质的诱导,麸皮对其木聚糖酶的合成也有促进作用,优化产酶液体培养主要成分的配比为：麸皮：玉米芯木聚糖：玉米芯粉;蛋白胨（或尿素）＝1：1：1：0．6（0．4）,摇瓶96h达到最大酶活,最高木聚糖酶活达到289．3U／ml,该菌所产木聚糖酶的最适作用条件为45－50度,PH4．4,在PH4．4－8．0范围内稳定。     

嗜极性木聚糖酶

木聚糖酶内切水解木聚糖主链的1,4-β-D-糖苷键,木聚糖是植物细胞壁中一种主要的多糖。自然界中木聚糖是多种糖类的复合体,这就使得木聚糖酶呈现多态性和多域性,由此需将繁多的木聚糖酶进行归类。木聚糖酶的催化反应属于双置换机制。在已研究的真菌或细菌性木聚糖酶中,大多数在温和的条件下表现出最佳活性,但有很多在极端环境下生长的生物体,为了适应极端环境而产生嗜极性的酶,其中嗜酸的、嗜碱的、嗜热的木聚糖酶,现在已有广泛的研究。对嗜极性木聚糖酶的研究进展作了论述。     

一株培菌白蚁后肠木聚糖降解细菌的分离与鉴定

【目的】从培菌白蚁——黄翅大白蚁肠道微生物菌群中分离能降解木聚糖的细菌。【方法】以木聚糖为唯一碳源,利用刚果红染色,根据透明圈大小进行筛选。通过显微形态,革兰氏染色及16S r RNA基因序列分析进行菌株鉴定。二硝基水杨酸(DNS)法测定细菌生长过程中木聚糖酶酶活变化,比较酶活与菌株生长状况的关系。【结果】从黄翅大白蚁肠道中筛选到一株具有较高木聚糖降解活性的革兰氏阳性菌Mb1,16S r RNA基因序列分析表明为类芽孢杆菌属细菌,命名为Paenibacillus sp.Mb1。该菌培养72 h后菌体浓度达到最高,木聚糖酶酶活主要存在于培养液上清中,酶活在对数期增长快,在培养96 h时达到最高值,之后趋于稳定。【结论】从黄翅大白蚁肠道中分离出一株具有较高木聚糖酶活的类芽孢杆菌,可作为产细菌木聚糖酶的潜在优良菌株。     

培养基初始pH值对木聚糖酶合成的影响

本文探讨了培养基三种初始ｐＨ值对木聚糖酶合成的影响。试验结果表明,培养基初始ｐＨ值对木聚糖酶的合成有重大影响,低ｐＨ有利于提高木聚糖酶活力。在碳源为７ｇ／Ｌ条件下,初始ｐＨ值为４．０时合成的木聚糖酶活力高达３２．８７ＩＵ／ｍｌ,酶产率和得率分别为６５７４．０ＩＵ／Ｌ·ｄ和４６９５．７ＩＵ／ｇ木聚糖。酶活力和酶产率是初始ＰＨ５．０的１．７倍,分别是初始ＰＨ６．０的３．８倍和２．３倍。进一步研究表明,     

微生物木聚糖酶的研究进展及其在食品领域的应用

木聚糖是半纤维素的主要组成成分,也是自然界第二丰富的可再生资源。木聚糖的结构稳定、组成复杂,很难在自然条件下自我降解,只有通过多种酶组成的木聚糖酶系的协同作用才可以更好地水解木聚糖或含有木聚糖的底物。木聚糖酶系主要由微生物产生,不同来源的木聚糖酶的性质存在较大差异。介绍了木聚糖水解酶系的组成和作用机理,木聚糖酶的分类和酶学性质,并对木聚糖酶在食品领域的应用进行了综述。     

厌氧菌群SVY42产酶条件分析及产木聚糖酶菌株的分离鉴定

以美国内华达州大盆地温泉采集样品为材料,富集获得纤维素及半纤维素高效稳定降解厌氧菌群SVY42,以巨菌草、甘蔗渣、废菇筒、羧甲基纤维素钠、滤纸、木聚糖为碳源,分析菌群SVY42产内切葡聚糖酶(CMC酶)、β-葡萄糖苷酶和木聚糖酶的情况。在此基础上,以木聚糖为底物筛选高产木聚糖酶的菌株。菌群SVY42在以巨菌草作为碳源时的β-葡萄糖苷酶活最高为0.23 U/mL,以木聚糖作为碳源时CMC酶活和木聚糖酶活均为最高,分别为0.31 U/mL和0.35 U/mL。从菌群SVY42中筛选得到1株高产木聚糖酶厌氧菌株SVY42-1,该菌在最适温度41℃和pH 8.0条件下,其木聚糖酶活力为0.26 U/mL,对其进行16S rDNA序列系统进化分析,SVY42-1与已知菌株的最高同源性仅为93.81%,初步鉴定属于新属。     

Purification and characterization of an alkaline xylanase from alkaliphilic Micrococcus sp AR-135

An xylanase producing alkaliphilic Micrococcus sp was isolated from an alkaline soda lake. Xylose and xylan induced enzyme production but no activity was detected when it was grown using other carbohydrate sources. The level of xylanase production was higher in the presence of xylose than in the presence of xylan. The enzyme was purified to homogeneity and its molecular weight was estimated to be 56 kD on SDS-PAGE. The optimum temperature and pH for xylanase activity were 55°C and 7.5–9.0, respectively. Sixty per cent of the maximum activity was displayed at pH 11. The enzyme was very stable in the pH range of 6.5–10 and up to a temperature of 40°C. Xylanase activity was inhibited by Cu²⁺ and Hg²⁺. Received 03 October 1997/ Accepted in revised form 03 February 1998     

Stimulation of xylanase synthesis in Cryptococcus albidus by cyclic AMP

Cryptococcus albidus secretes a xylanase when induced by xylan or beta-methylxyloside, a non-metabolizable inducer, and production of the enzyme is repressed by xylose. The effect of exogenous cAMP on xylanase production was tested under different growth conditions. The cAMP elicited a 1.5 to 2 fold increase in xylanase production during the induction by xylan and B-methylxyloside but did not relieve the repression observed during growth on xylose. Cyclic AMP also affected the growth rate of the cells and did not modulate the activity of pure xylanase in vitro. A 15-nucleotide sequence located upstream from the xylanase gene could be part of a cAMP regulatory sequence.     

Optimization of xylanase production using inexpensive agro-residues by alkalophilic Bacillus subtilis ASH in solid-state fermentation

Alkalophilic Bacillus subtilis ASH produced high levels of xylanase using easily available inexpensive agricultural waste residues such as wheat bran, wheat straw, rice husk, sawdust, gram bran, groundnut and maize bran in solid-state fermentation (SSF). Among these, wheat bran was found to be best substrate. Xylanase production was highest after 72 h of incubation at 37 °C and at a substrate to moisture ratio of 1:2 (w/v). The inoculum level of 15% resulted in maximum production of xylanase. The enzyme production was stimulated by the addition of nutrients such as yeast extract, peptone and beef extract. In contrast, addition of glucose and xylose repressed the production of xylanase. The extent of repression by glucose (10%, w/v) was 81% and it was concentration-dependent. Supplementation of the medium with 4% xylose caused 59% repression. Under optimized conditions, xylanase production in SSF (8,964 U of xylanase/g dry wheat bran) was about twofold greater than in submerged fermentation. Thus, B. subtilis produced a very high level of xylanase in SSF using inexpensive agro-residues, a level which is much higher than that reported by any other bacterial isolate. Furthermore, the enzyme was produced at room temperature and with tap water without the addition of any mineral salt in SSF, leading to a marked decrease in the cost of xylanase production, which enhances its industrial potential.     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.     

Production of xylanase by the ruminal anaerobic fungus Neocallimastix frontalis.

Xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) production was investigated in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released principally into the culture fluid and had pH and temperature optima of 5.5 and 55 degrees C, respectively. In the presence of low concentrations of substrate, the enzyme was stabilized at 50 degrees C. Xylobiose was the principal product of xylanase action, with lesser amounts of longer-chained xylooligosaccharides. No xylose was detected, indicating that xylobiase activity was absent. Activities of xylanase up to 27 U ml-1 (1 U represents 1 micromol of xylose equivalents released min-1) were obtained for cultures grown on xylan (from oat spelt) at 2.5 mg ml-1 in shaken cultures. No growth occurred in unshaken cultures. Xylanase production declined with elevated concentrations of xylan (less than 2.5 mg ml-1), and this was accompanied by an accumulation of xylose and, to a lesser extent, arabinose. Addition of either pentose to cultures grown on low levels of xylan in which neither sugar accumulated suppressed xylanase production, and in growth studies with the paired substrates xylan-xylose, active production of the enzyme occurred during growth on xylan only after xylose had been preferentially utilized. When cellobiose, glucose, and xylose were tested as growth substrates for the production of xylanase (each initially at 2.5 mg ml-1), they were found to be less effective than xylan, and use of xylan from different origins (birch wood or larch wood) as the growth substrate or in the assay system resulted in only marginal differences in enzyme activity. However, elevated production of xylanase occurred during growth on crude hemicellulose (barley straw leaf). The results are discussed in relation to the role of the anaerobic fungi in the ruminal ecosystem, and the possible application of the enzyme in bioconversion processes is also considered.     

Characterization of a bacterial xylanase resistant to repression by glucose and xylose

Summary Bacillus subtilis CD4, when grown in nutrient broth or minimal medium in presence of xylan, produced extracellular xylanase that hydrolyzed xylan optimally at pH 5. The enzyme was induced by xylan, xylose and glucose. Addition of xylose or glucose in xylan containing medium did not affect enzyme production. The structural gene encoding xylanase was cloned and expressed in E. coli. The recombinant enzyme exhibited similar properties like that of native enzyme including resistance to repression by xylose and glucose.     

Thermostability and Substrate Specificity of GH-11 Xylanase from <Emphasis Type="Italic">Thermomyces lanuginosus</Emphasis> VAPS24

The three dimensional structure (3D structure) of GH-11 xylanase from Thermomyces lanuginosus was obtained through homology modeling. To study the enzyme interaction with an end product of enzyme catalysis, the xylanase two sugar molecules xylose and xylobiose has been docked into the active site of GH-11 xylanase through molecular docking. Based on the free binding energy and Inhibition constant, concluded xylose makes more stable complex than xylobiose. Further, the molecular dynamic simulation studies were carried out at different temperature, i.e. 323, 333, 343 and 353 K (i.e. 50, 60, 70 and 80 °C). It has been observed that there was minor structural modification in 3D-structure of xylanase at 323, 333, and 343 K. But the helix and sheets moved out of the initial structure when simulation carried out at during 353 K (80 °C).     

Induction of cellulase and hemicellulase activities of Thermoascus aurantiacus by xylan hydrolyzed products

Thermoascus aurantiacus is able to secrete most of the hemicellulolytic and cellulolytic enzymes. To establish the xylanase inducers of T. aurantiacus, the mycelia were first grown on glucose up until the end of the exponential growth phase, followed by washing and re-suspension in a basal medium without a carbon source. Pre-weighed amounts of xylose (final concentration of 3.5 mg/ml), xylobiose (7 mg/ml) and hydrolyzed xylan from sugarcane bagasse (HXSB) which contained xylose, xylobiose and xylotriose (6.8 mg/ml) were evaluated as inducers of xylanase. It was observed that xylose did not suppress enzyme induction of T. aurantiacus when used in low concentrations, regardless of whether it was inoculated with xylobiose. Xylobiose promoted fast enzyme production stopping after 10 h, even at a low consumption rate of the carbon source; therefore xylobiose appears to be the natural inducer of xylanase. In HXSB only a negligible xylanase activity was determined. Xylose present in HXSB was consumed within the first 10 h while xylobiose was partially hydrolyzed at a slow rate. The profile of α-arabinofuranosidase induction was very similar in media induced with xylobiose or HXSB, but induction with xylose showed some positive effects as well. The production profile for the xylanase was accompanied by low levels of cellulolytic activity. In comparison, growth in HXSB resulted in different profiles of both xylanase and cellulase production, excluding the possibility of xylanase acting as endoglucanases.     

The xylanase system of Coniophora cerebella

1. The culture filtrate of the fungus Coniophora cerebella grown on poplar 4-O-methylglucuronoxylan as carbon source and enzyme inducer contained an enzyme system that degraded the polysaccharide to xylose, acidic and neutral oligosaccharides and an enzyme-resistant polymer. Free uronic acid was not produced. 2. Cold ethanol fractionation of the culture filtrate yielded two active fractions, one of which had only xylanase (EC 3.2.1.8) and the other both xylanase and xylosidase (EC 3.2.1.37) activities. Further fractionation on DEAE-cellulose resolved the xylanase and xylosidase activities. 3. The xylanase degraded poplar 4-O-methylglucuronoxylan in an essentially random manner, producing oligosaccharides, but some xylose residues in the vicinity of uronic acid side groups were protected from hydrolysis, preventing a truly random attack. The xylosidase attacked the polysaccharide very slowly, releasing xylose, but the oligosaccharides produced by the action of the xylanase were much more susceptible to hydrolysis by the xylosidase. 4. The products of xylanase action were separated into neutral and acidic fractions. The neutral oligosaccharides were separated by chromatography on charcoal-Celite, and the major products were characterized as xylobiose, xylotriose, xylotetraose and xylopentaose. Some of the acidic sugars were branched, having the uronic acid residue attached to a xylose residue other than the terminal non-reducing one. 5. Gel filtration of various xylanase fractions gave values for the molecular weight of the enzyme from 34000 to 38000.     

Saccharification of xylan by an amyloglucosidase of Termitomyces clypeatus and synergism in the presence of xylanase

Summary An amyloglucosidase from a mycelial culture of the mushroom Termitomyces clypeatus hydrolysed larch wood xylan independently and synergistically with an endo-(14) xylanase of the same fungus. The glucoamylase saccharified xylan predigested with xylanase at a faster rate compared to that of xylanase acting on amylase-digested xylan. However, overall saccharification of xylan in both cases was the same. Only glucose was liberated from xylan by amylase digestion whereas xylose, xylobiose and other oligosaccharides were liberated during xylanase digestion. The synergistic response of enzyme combinations was reflected in the liberation of glucose from xylan, rather than xylose. Glucoamylase and xylanase activities on soluble and insoluble fractions of larch wood xylan with different xylose and glucose contents suggested that synergism in xylanolysis by the presence of glucoamylase was dependent on the activity of the participating xylanase on the xylan preparation. It is suggested that possibly -glucosidic linkages are present in xylan and that amyloglucosidase might be involved in xylanolysis. Correspondence to: S. Sengupta