嗜碱芽孢杆菌C-125木糖苷酶基因的表达与酶特征鉴定

嗜碱芽孢杆菌(Bacillus halodurans)C-125菌株的基因组中,一个编码木糖苷酶的基因(BH1068)被克隆并在大肠杆菌中获得高效表达。通过全面分析纯化蛋白,确证了它的木糖苷酶功能。该酶在pH4～9的范围内保持稳定,最适pH值为中性,有较宽的最适温度(35°C～45°C),且能在45°C范围内保持稳定。这些特性使得该酶可在较为宽广的条件下对木聚糖进行酶促降解。该酶对人工合成底物对硝基苯-β-木糖苷(p-nitrophenyl-β-xylose,pNPX)的比活力为174mU/mg蛋白质,且木糖对其反馈抑制较弱(抑制常数Ki为300mmol/L)。结果显示该酶是活性较高且较耐木糖抑制的细菌源木糖苷酶。该酶与商品化的木聚糖酶一起水解山毛举木聚糖(Beechwood xylan)时显示了增效作用,且水解率可获40%。该酶最适pH为中性,对木糖耐受等特性与大多数来源于真菌、最适pH为酸性、对木糖敏感的木糖苷酶将有较好的互补。结果表明该酶在木聚糖或含木聚糖多糖的单糖化过程可能发挥重要作用。     

产耐热木聚糖酶细菌的分离鉴定及酶易错PCR致突变条件优化

从福建省永泰县温泉采集样品中筛选到1株产耐热木聚糖酶嗜热菌株TC-W7,并获得该木聚糖酶基因。在此基础上,采用易错PCR技术在木聚糖酶基因中引入突变,研究Mg2＋浓度、Mn2＋浓度、dTTP/dCTP浓度等条件对突变率的影响。通过形态特征、生理生化试验及16S rRNA序列相似性比对分析,初步鉴定菌株TC-W7为土壤芽胞杆菌（Geobacillus）,菌株TC-W7在最适温度75℃和 pH 8.2条件下,其木聚糖酶活力为215.83 U/mL,Triton X-100和DDT能显著增强该酶的活性。在 Mg2＋浓度为20μmol/L,Mn2＋浓度为0.80μmol/L,dTTP/dCTP浓度为0.30 mmol/L的致突变条件下,碱基突变率为0.98%。 Geobacillus sp. TC-W7产木聚糖酶具有较好的耐热和耐碱等工业应用特性,对该酶易错PCR致突变条件优化结果,可用于后续木聚糖酶的耐热定向进化。     

嗜碱细菌Cellulomonas bogoriensis 69B4T木聚糖酶的表达纯化及性质研究

【目的】对嗜碱细菌Cellulomonas bogoriensis 69B4~T产碱性木聚糖酶进行研究,克隆来源于该菌株的木聚糖酶基因,并对其进行异源表达、纯化及酶学性质的表征,为后续研究碱性木聚糖酶的耐碱机制及应用奠定基础。【方法】采用单因素分析法对菌株产碱性木聚糖酶情况进行研究;通过基因组分析,锚定5个内切木聚糖酶基因,利用同源扩增的方法进行克隆,并在大肠杆菌中重组表达,利用亲和层析对重组酶进行纯化,以木聚糖为底物表征木聚糖酶的酶学性质。【结果】来源于C. bogoriensis 69B4~T的5种木聚糖酶Xyn370、Xyn393、Xyn425、Xyn466和Xyn486均在大肠杆菌内实现了异源表达,并经亲和层析获得纯酶组分,其最适反应温度分别为60、50、40、40、60°C,在50°C范围内保温2h,残余酶活均在90%以上;最适反应p H分别为7.0、8.0、8.0、8.0、9.0,在p H5.0–9.0时具有较好的稳定性;5种重组木聚糖酶对部分金属离子和高浓度盐表现出较好的耐受性,对榉木木聚糖的酶活性最高,均为内切型木聚糖酶。【结论】本研究表达纯化的5种重组木聚糖酶具有耐盐碱的优良特性,且对温度、某些金属离子和化学试剂耐受,为研究木聚糖酶的耐碱机制及工业应用提供了酶源。     

里氏木霉GXC木聚糖酶的研究*

研究了里氏木霉GXC产木聚糖酶的条件和酶学性质。结果表明,适宜产酶碳源为乳糖、甘露糖、棉子糖、木聚糖和麸皮,氮源为牛肉膏和酵母膏;产酶的最适初始pH为4.0,30℃培养60h。对以麸皮为碳源的培养液进行纯化的酶特性研究表明,木聚糖酶的最适反应温度为50℃,pH为5.5,该酶在pH5.0(7.0和40℃以下相对稳定。Fe3+和Mn2+对木聚糖酶有较大的促进作用,Cu～2+、Fe～2+和Ca～2+ 具有抑制作用。     

里氏木霉GXC木聚糖酶的研究

研究了里氏木霉GXC产木聚糖酶的条件和酶学性质。结果表明,适宜产酶碳源为乳糖、甘露糖、棉子糖、木聚糖和麸皮,氮源为牛肉膏和酵母膏;产酶的最适初始pH为4.0,30℃培养60h。对以麸皮为碳源的培养液进行纯化的酶特性研究表明,木聚糖酶的最适反应温度为50℃,pH为5.5,该酶在pH5.0(7.0和40℃以下相对稳定。Fe3+和Mn2+对木聚糖酶有较大的促进作用,Cu～2+、Fe～2+和Ca～2+ 具有抑制作用。     

纤维素降解菌Aspergillus sp. YN1的产酶条件及酶学特性

从牛羊粪堆肥中筛选出一株纤维素降解菌Aspergillus sp.YN1,主要研究了液体发酵培养基中碳源、氮源、培养温度、起始pH、通气量以及接种菌龄对菌株YN1的羧甲基纤维素酶活(CMC酶活)及滤纸酶活的影响。研究结果表明,在优化条件下,该菌的CMC酶活、滤纸酶活在培养第3天分别达到0.53U/mL和0.15U/mL。在酶学特性研究中,菌株YN1的CMC酶的最适反应温度为70°C,最适反应pH4.0(酶促反应为30min)。用不同温度处理1h或不同pH处理2h,YN1的CMC酶在30°C?50°C或pH3.0?4.0之间仍可保持80%以上的酶活性,对热及酸表现出较高的稳定性。     

嗜酸青霉酸性木聚糖酶产生特性及部分酶学性质

从煤矿酸性废水中分离到一株产木聚糖酶青霉,通过酸性液体培养研究了菌体生长对pH的响应及木聚糖酶的产生特征,并测定了木聚糖酶的部分应用性质.结果表明:实验菌株嗜酸,菌丝生长最适pH为2.0,孢子萌发生长适宜pH为3.0～4.0;木聚糖诱导菌体在生长稳定期大量产生木聚糖酶,蛋白胨是菌体产酶的适宜氮源;菌株所产木聚糖酶属于酸性木聚糖酶,反应最适pH 3.5、最适温度50 ℃～55 ℃,pH 2.0时酶活达到最高活力的72%,在最适反应条件下保温60 min,残余酶活接近70%,适用于较强酸性的高温加工环境.     

一株产低温β-半乳糖苷酶微杆菌的筛选鉴定、产酶条件及其酶学特性

【背景】低温β-半乳糖苷酶能在低温下仍保持较高的乳糖水解活性,筛选酶学特性适合在牛乳体系中高效水解乳糖的β-半乳糖苷酶生产菌株,是低乳糖牛乳加工产业关注的焦点。【目的】对天山中国一号冰川沉积物中分离的一株产低温β-半乳糖苷酶菌株的产酶条件和酶学特性进行研究。【方法】结合X-Gal平板法初筛和测定粗酶液酶活复筛,获得产低温β-半乳糖苷酶的菌株。通过形态学、生理生化试验及16S rRNA基因测序分析对筛选菌株进行鉴定,单因素摇瓶实验优化菌株的产酶条件,硫酸铵分级沉淀初步纯化β-半乳糖苷酶并对其酶学特性进行分析。【结果】通过形态学、生理生化特征和16S rRNA基因鉴定,确定菌株LW106为微杆菌属(Microbacterium)菌株;该菌株最适产酶温度为25°C,最佳产酶碳源为可溶性淀粉,培养基初始pH为7.0,接种量为3%;对初步纯化的低温β-半乳糖苷酶酶学性质的研究表明,LW106所产β-半乳糖苷酶的最适pH为6.0,最适反应温度为35°C,4°C时酶活为最大酶活的78%,4°C和pH 7.0时的稳定性最好,10 mmol/L的Na+对酶活性基本没有抑制作用,Ca~(2+)对酶活性具有一定的激活作用。【结论】菌株LW106所产低温β-半乳糖苷酶的酶学特性表明该酶在乳品低温加工领域具有进一步研究和应用的价值。     

土壤中产木聚糖酶菌株的筛选及发酵条件优化

【背景】木聚糖广泛存在于木质纤维类生物质中,是世界上含量最丰富的半纤维素,利用产酶微生物对木质纤维类生物质进行发酵处理是木质纤维类生物质资源化和能源化的有效手段。【目的】通过产木聚糖酶菌株的筛选鉴定、酶学特性分析和发酵条件优化,获得开发多纤维农林废弃物生产新型多元化饲料添加剂的材料。【方法】利用青藏高原土壤筛选产木聚糖酶菌株,通过形态学观察和rDNAITS区域序列分析鉴定菌株XC70种属,对其所产酶的酶学特性及该菌的生长规律和产酶规律进行分析,并利用单因素法和正交试验法优化其发酵条件。【结果】菌株XC70经形态学和分子生物学方法鉴定为草酸青霉(Penicillium oxalicum)。菌株XC70所产木聚糖酶的最适反应条件为:pH 5.0,70°C,温度低于50°C时稳定性较好,具备一定的耐酸性,Na+和K+对木聚糖酶活力具有促进作用(P0.05),在发酵54 h后菌体量和上清液酶活力大小均达到高峰。经过单因素法和正交试验法优化后确定了该菌的最优发酵条件为:蛋白胨7 g/L,玉米秸秆50 g/L,KCl 4 g/L,培养基初始pH 4.0,28°C,摇床转速200r/min,接种量2%。在此发酵条件下,木聚糖酶活力可达到1 489.33U/mL,与优化前相比提高了3倍多。【结论】从青藏高原土壤中筛选获得的菌株XC70具有一定的产木聚糖酶能力,其所产生的酸性木聚糖酶可用于降解多纤维物质开发新型饲料添加剂,具有一定的应用潜力和开发价值。     

一株产生淀粉分解酶犁头霉的分离鉴定及其酶学性质

从保宁麸醋醋曲中定向筛选得到一株产生淀粉分解酶的菌株CQB43,其酶活力为105.2 U/mL,RDA值达到27.9%。通过形态观察和分子生物学鉴定确定该菌株为伞枝犁头霉Absidia corymbifera。对该菌株分泌生淀粉酶酶学性质的研究结果表明,该酶在pH为4.0-5.6的范围相对稳定,最适pH是5.0;在60°C以下的范围内具有较好的热稳定性,最适作用温度为40°C。研究金属离子对其活力影响的结果表明,Co2+对该生淀粉糖化酶有激活作用,Fe3+和Ca2+对该酶有抑制作用。     

Production of high level of cellulase-free and thermostable xylanase by a wild strain of Thermomyces lanuginosus using beechwood xylan

Thermomyces lanuginosus, isolated from self-heated jute stacks in Bangladesh, was studied for production of high level of cellulase-free thermostable xylanase at 50°C using xylan. Optimization of the medium composition was carried out on shake-flask level using Graeco-Latin square technique. This increased xylanase production from 527 nkat ml⁻¹ in the original medium to 9168–9502 nkat ml⁻¹ in the optimized medium under optimized culture conditions e.g. initial medium pH (6.0–6.5), culture temperature (50°C) and time (5–6 d). The lag phase was very much shorter in the laboratory reactor compared to which existed in the shake cultures and 7111 nkat of xylanase activity were obtained per ml of culture filtrate at 60 h of cultivation. With a 15 min reaction time, the optimal pH and temperature for the xylanase activity were at 6.5 and 65°C, respectively. The enzyme was almost stable over a broad range of pH 3–9 at 20°C, with an optimum stability at pH 6.5. After 51 h heating at 50°C the enzyme retained 60%, 100% and 90% activity at pH 5.0, 6.5 and 8.0, respectively. The crude enzyme could hydrolyse xylan effectively and in only 6 h 67.3%, 54.0% and 49.2% saccharifications were achieved for 2%, 5% and 10% substrate levels, respectively. The principal product of hydrolysis was xylobiose together with smaller amounts of xylooligosaccharides (degree of polymerization 3–7) and xylose.     

Xylanases, xylanase families and extremophilic xylanases

Xylanases are hydrolytic enzymes which randomly cleave the beta 1,4 backbone of the complex plant cell wall polysaccharide xylan. Diverse forms of these enzymes exist, displaying varying folds, mechanisms of action, substrate specificities, hydrolytic activities (yields, rates and products) and physicochemical characteristics. Research has mainly focused on only two of the xylanase containing glycoside hydrolase families, namely families 10 and 11, yet enzymes with xylanase activity belonging to families 5, 7, 8 and 43 have also been identified and studied, albeit to a lesser extent. Driven by industrial demands for enzymes that can operate under process conditions, a number of extremophilic xylanases have been isolated, in particular those from thermophiles, alkaliphiles and acidiphiles, while little attention has been paid to cold-adapted xylanases. Here, the diverse physicochemical and functional characteristics, as well as the folds and mechanisms of action of all six xylanase containing families will be discussed. The adaptation strategies of the extremophilic xylanases isolated to date and the potential industrial applications of these enzymes will also be presented.     

Cellulase end xylanase activity of fungi in a collection isolated from the southern Caucasus

Fungi, comprising about 4000 cultures, were collected from different climatic zones of the southern Caucasus. Almost all cultures in the collection showed a high potential for degrading basic plant biopolymers such as cellulose and hemicellulose. Cultures of Penicillium canescens possessing comparatively low cellulase activity in their wild type were treated with ultraviolet light, which produced genetically stable mutants. Few of them had high xylanase and no cellulase activities. More than 6% of all cultures in the collection were thermophiles and, from these, 56 cultures with the highest cellulase and xylanase activity were selected. It was shown that under two different thermophilic growth conditions, 40 and 48°C, Allescheria terrestris formed two sets of endoglucanases and endoxylanases with different thermal stabilities. It was also shown that when cultivated on straw, Allescheria terrestris grows primarily in its internal part for an extended period of time.     

四种嗜热真菌的分离与鉴定

目的:在对嗜热真菌的资源调查中,分离到嗜热真菌20株。方法:通过形态学比较研究并结合分子分析方法。结果:鉴定出嗜热真菌4种,即杜邦青霉Penicillium dupontii、疏绵状嗜热丝孢菌Thermomyceslanuginosus、嗜热子囊菌Thermoascus aurantiacus、嗜热革节孢Scytalidium thermophilum。此外,还分离到耐热真菌1种,鉴定为不规则头梗霉Cephaliophora irregularis,为中国新记录种。结论:这些研究结果新增了嗜热真菌在中国的分布记载,丰富了我国西南地区嗜热真菌的菌种资源库,另外对分离获得的嗜热真菌进行木聚糖酶活性测试,发现嗜热子囊菌为高产木聚糖酶活力的菌株。     

Characterization of genes fromThermoanaerobacterium thermosulfurigenes EM1 that encode two glycosyl hydrolases with conserved S-layer-like domains

Two genes fromThermoanaerobacterium thermosulfurigenes EM1 were identified which are predicted to encode a xylanase (XynA) and a polygalacturonate hydrolase (PglA). ThexynA gene has the potential to encode a 1234-amino acid product consisting of a signal peptide followed by a repeated domain, a xylanase family F domain, two cellulose-binding domains and a triplicated sequence at its C-terminus. The genepglA is predicted to encode a product of 1148 amino acids consisting of a signal sequence followed by a fibronectin type III-like domain (Fn3 domain), the catalytic domain, a Gly/Thr/Ser/Asn-rich segment and a triplicated domain. The triplicated segments at the C-termini of deduced XynA and PglA are about 95% identical to each other and to the S-layer-like domains of the previously characterized pullulanase (AmyB) from the same organism. In contrast, sequence comparisons revealed only distant amino acid sequence similarities between the fibronectin type III-like domains of PglA and AmyB fromT. thermosulfurigenes EM1.     

Purification and biochemical characterization of β-xylosidase from Humicola grisea var. thermoidea

Abstract β-d-Xylosidase production was maximal for Humicola grisea var. thermoidea grown on xylan as the sole carbon source. The main β-d-xylosidase activity was localised in the periplasm. β-Xylosidase was purified from crude extracts by heat treatment, ammonium sulfate precipitation and chromatography on DEAE-cellulose and Sephadex G-100. The purified enzyme was a monomer of molecular mass estimated to be 43 kDa by SDS-PAGE and gel filtration. Optima of pH and temperature were 6.0 and 50 °C, respectively. The enzyme activity was stimulated by Ca²⁺, Fe²⁺, and Mg²⁺. The purified β-xylosidase did not exhibit xylanase, carboxymethylcelullase, galactosidase, glucosidase, fucosidase or arabinosidase activities. The purified β-xylosidase hydrolysed xylobiose and xylo-oligosaccharides of up to five monosaccharide units. The enzyme had a K _m of 0.49 mM for p -nitrophenyl- β -d-xylopyranoside and was not inhibited by its product, xylose.     

A highly-active endo-1,3-1,4-β-glucanase from thermophilic Talaromyces emersonii CBS394.64 with application potential in the brewing and feed industries

A 1245-bp endoglucanase gene of glycoside hydrolase (GH) family 7, egl7A, was cloned from the acidothermophilic fungus Talaromyces emersonii CBS394.64 and successfully expressed in Pichia pastoris. Sequence alignments indicated that Egl7A had highest identity of 62.7% at the amino acid level with the functionally characterized endoglucanase from Aspergillus terreus NIH2624. Purified recombinant Egl7A exhibited the maximum activity at pH 4.5 and 70 °C, retained stable over the pH range of 2.0–12.0 and at 65 °C, and was strongly resistant to acidic and neutral proteases, most metal ions and SDS. The enzyme exhibited the highest specific activity reported so far (11,299 U mg⁻¹) when using barley β-glucan as the substrate. Egl7A exhibited broad substrate specificity, including barley β-glucan, lichenin, CMC-Na, and xylan and had capacity to cleave cellopentaose and cellohexaose into smaller units rapidly. Under simulated mashing conditions, addition of Egl7A reduced the mash viscosity by 12.40%; when combined with a GH10 xylanase, more viscosity reduction (27.75%) was observed, which is significantly higher than that of the commercial enzyme Ultraflo XL (17.91%). All these properties make Egl7A attractive for potential applications in the feed and brewing industries.     

木聚糖酶高产菌株选育

以黑曲霉为出发菌,经紫外线和亚硝基胍（NTG）交替诱变处理,获得一株木聚糖酶高产菌,并初步研究了其固体发酵条件,在该条件下最高酶活力可达3181IU／min.gdw。     

Molecular cloning and expression of the xylanase gene of alkalophilic Bacillus sp. strain C-125 in Escherichia coli.

The gene for xylanase A of alkalophilic Bacillus sp. strain C-125 was cloned in Escherichia coli with pBR322. The plasmid pCX311 contained 2.6- and 2.0-kilobase-pair HindIII fragments. The characteristics of the purified pCX311-encoded xylanase were the same as those of purified xylanase A from alkalophilic Bacillus sp. strain C-125.     

耐碱性木聚糖酶产酶菌株的选育

目的:筛选出产碱性木聚糖酶酶活力高的菌株。方法:从碱性环境中采集样品,以自制木聚糖为惟一利碳源,采用刚果红透明圈法于摇瓶发酵法相结合筛选出18株产木聚糖酶酶活较高的菌株,其中1株酶活最高可达335.14 IU/ml。结果:通过培养特性及形态特征初步鉴定为芽孢杆菌。对部分酶学性质研究的结果表明:其作用最适温度60℃,最适pH8.0,在pH9.0条件下仍具有80%酶活力。结论:属于耐碱性木聚糖酶,具有很好的工业应用前景。