产中性纤维素酶特异腐质霉H31-3复合诱变研究*

中性纤维素酶在纺织、食品、饲料和制药行业均具有广泛的应用。采用离子束注入技术对中性纤维素酶产生菌特异腐质霉(Humicolainsolens)H31-3进行诱变,经发酵筛选获得较高酶活力且传代稳定的正突变菌株H14,制备其原生质体后进行紫外诱变。筛选后得到正突变菌株H14-2,最终CMC酶、滤纸酶活力分别达82.56IU/mL和5.77IU/mL,较原始菌株提高了78.57%和106.81%。     

斜卧青霉A10产纤维素酶的酶学性质研究

以斜卧青霉（Penieillium decumbens）A10为出发菌株,经450Gy ^60Coγ-射线诱变处理,选育出一株具有较高纤维素酶活力且传代稳定的正突变株A50,发酵60h后,其CMC酶活和滤纸酶活分别为27．28IU／mL和1．98IU／mL,较出发菌株A10分别提高了33．2％和45．59％。对突变株A50的产酶组分进行研究,通过SDS—PAGE电泳分析,从蛋白质水平上证明突变株A50确实是A10在遗传物质上发生改变的菌株,其CMC酶活最适作用pH值为4．0,最适作用温度为60℃;而滤纸酶活的最适作用pH值为5．2,最适作用温度45℃,二者在一定范围内具有较高的稳定性。     

黑曲霉原生质体诱变选育果胶酶高产菌株

通过UV和NTG诱变筛选获得了2株高产果胶酶突变株。以果胶酶产生菌黑曲霉EIM6为诱变材料,采用1．5％的溶壁酶和1．5％的纤维素酶处理其对教生长期菌丝体2h获得高质量的原生质体。采用UV25S或50μg／mL NTG诱变30min,构建原生质体突变库,经刚果红果胶平板筛选获得果胶酶突变株,通过液体深层培养复筛获得高产突变株EIM6-U11、EIM6-N5,酶活力分别从46598．08、46598．08U／mL提高至68596．57、68879．56U／mL,分别提高了47．21％、47．82％。连续8次传代经发酵测酶活力表明高产突变株EIM6-U11、EIM6-N5具有较高的遗传稳定性。     

拟康氏木霉（Trichoderma pseudokoningii）UV Ⅲ产纤维素酶液体发酵研究

以拟康氏木霉（Trichoderma pseudokoningii)TH为出发菌株,经紫外诱变获得一抗高浓度葡萄糖阻遏突变株UV Ⅲ,其液体发酵最适产酶培养基为（W／V）：豆皮粉3％,硝酸铵0．6％,磷酸二氢钠0．65％,硫酸镁0．25％,氯化钙0．15％,pH5.0;最佳发酵条件为：30℃,125r/min。发酵7d CMCase活力可达103．55IU／mL,滤纸酶活可达5．51IU／mL,β-葡萄糖苷酶活可达0．96IU／mL,分别比出发菌株TH提高了1．40、2．34、0．60倍。     

高产耐热脂肪酶嗜热脂肪地芽孢杆菌的选育

采用氮离子注入技术对耐热脂肪酶产生菌嗜热脂肪地芽孢杆菌（Geobacillus stearothermophilus）L4进行诱变,筛选获得酶活力有较大提高且传代稳定的正突变菌株L4-3;再对L4-3进行紫外线诱变,得到脂肪酶活力提高的正突变菌株L4-3-2,其脂肪酶活力达25．71U／mL,较原始菌株M提高511．9％。高产突变株L4-3-2所产脂肪酶的最适作用温度为50℃,70℃保温60min的剩余酶活为82％,最适作用pH为7．0～8．0,为一种耐热碱性脂肪酶。     

斜卧青霉纤维素酶和木聚糖酶高产菌株的选育

以纤维素酶高产菌株斜卧青霉A50为出发菌株,通过紫外诱变原生质体获得1株木聚糖酶活力提高80%而纤维素酶活力没有改变的6号菌。蛋白质电泳和酶谱检测结果显示,纤维素酶谱基本无差别,而木聚糖酶谱显示6号菌比A50多了一条带。6号菌优化后的产酶培养基组成为:麸皮7%、葡萄糖0.1%,该条件下,纤维素酶活为19.7IU/mL,木聚糖酶活力为215.4IU/mL。     

菊粉酶产生菌的筛选及60Co诱变选育简

目的：从新疆石河子盐碱地菊芋生长根际土壤中分离筛选高产菊粉酶活力菌株。方法：通过稀释平板涂布法分离微生物;利用^60Co诱变选育,96孔板筛选突变菌株;采用3,5-二硝基水杨酸比色法测定菊粉酶酶活。结果：分离到12株具有菊粉酶活力的菌株,复筛得到1株高产菊粉酶活力菌株,将其命名为G-60;以此菌株为出发菌株进行^60Co诱变,利用96孔板对诱变菌株进行筛选,经摇瓶发酵酶活测定,得到1株高产菊粉酶酶活的突变株,酶活达46．62U/mL,是未诱变菌株酶活的2．72倍。结论：经诱变得到1株高产菊粉酶活力的突变菌株。     

内切葡聚糖酶高产菌株的诱变选育

【目的】建立里氏木霉(Trichoderma reesei)高产突变菌株的快速筛选方法,选育出高产内切葡聚糖酶的突变株。【方法】对里氏木霉T306菌株的初筛培养基进行优化,建立快速筛选方法;通过紫外诱变手段选育内切葡聚糖酶高产突变菌株,并对突变菌株的产酶培养基进行优化。【结果】在初筛培养基中添加浓度为0.1%(W/V)的乳糖、蛋白胨及脱氧胆酸钠有利于菌株的筛选。诱变后筛选出菌落形态发生明显变化的内切葡聚糖酶高产突变株0516,其羧甲基纤维素酶活力(CMC酶)较出发菌株提高了38.9%。其产酶培养基经优化后,得到最适碳、氮源分别为:乳糖1.50%、硫酸铵0.14%、尿素0.05%、蛋白胨0.10%,优化后CMC酶活力达64.2 U/mL,较优化前提高了2.3倍。【结论】建立了里氏木霉高产突变菌株的快速筛选方法,通过紫外诱变育种获得了产内切葡聚糖酶能力高且遗传稳定的突变株0516。     

低温纤维素酶菌株CNY086选育及发酵培养基优化(Ⅱ)

自渤海湾海泥中分离21株低温纤维素酶产生菌。其中菌株CNY01为绿色木霉(Trichoderma viride), 酶活力为67.30 U/mL。以该菌株为出发菌株, 经UV、DES等诱变, 选育出高产突变菌株CNY086, 酶活力为92.17 U/mL。该突变菌株低温纤维素酶发酵具有遗传稳定性。通过单因素和正交实验确定突变菌株CNY086低温纤维素酶发酵最适培养基: 秸秆粉1.20%、麸皮0.70%、硫酸铵0.50%、磷酸二氢钾0.55%, 上述条件下CNY086菌株酶活力达到108.55 U/mL。     

产胞外青霉素酰化酶菌株的选育

以短小杆菌（B．pumilus）B－97为出发菌株,经过连续两次紫外线诱变处理,分离得一突变株B－U－29。其酶活力为4．56U／ml,较出发菌株酶活力提高113．3％。对B－U－29菌株进行连续两次亚硝酸处理,分离得一正变稳定株B—H－29,酶活力为4．93u／ml,较出发菌株酶活力提高了20％。     

Cloning and expression of endo-β-glucanase II gene in Humicola insolens H31-3

Endo-β-glucanase Ⅱ (EG Ⅱ) gene cDNA was isolated from the fungus Humicola insolens H31-3 by RT-PCR.It was cloned into the expression vector pGAPZαA.The resultant recombinant plasmid was introduced into Pichia pastoris GS115 by electroporation after being lin-earized by BspHI digestion.The recombinant Pichia pas-toris strain was obtained and SDS-PAGE showed that the molecular weight of the expression protein was about 55 kD.The cultivation condition and the characteristics of the recombinant EG Ⅱ were also explored.     

特异腐质霉内切葡聚糖酶Ⅱ基因在毕赤酵母中的表达及酶学性质

【目的】在毕赤酵母中表达特异腐质霉Humicola insolens的中性内切葡聚糖酶Ⅱ,并对其性质加以研究。【方法】利用RT-PCR的方法,以特异腐质霉(Humicola insolens)NC3总RNA为模板,克隆到中性内切葡聚糖酶Ⅱ基因(egⅡ)的cDNA。将其插入表达载体pPIC9K,重组质粒经线性化后电击转化毕赤酵母(Pichia pastoris)菌株GS115。【结果】SDS-PAGE和酶活的检测结果均表明:egⅡ基因在毕赤酵母中成功表达。重组酶的部分酶学性质研究表明,该酶的最适反应温度为70°C,且在65°C以下具有较好的热稳定性。最适反应pH为6.5,在pH 6.0?7.0之间有较好的稳定性。【结论】用重组毕赤酵母可高效表达外源中性内切葡聚糖酶,为其今后在工业应用奠定了基础。     

Avicel-adsorbable endoglucanase production by the thermophilic fungus Scytalidium thermophilum type culture Torula thermophila

Scytalidium thermophilum type culture Torula thermophila was isolated from mushroom compost and the total cellulase, endoglucanase, Avicel-adsorbable endoglucanase activities, as well as the fungal biomass generation and cellulose utilisation were analyzed in shake flask cultures with Avicel (microcrystalline cellulose) as the carbon source. Results were compared with an industrial strain of Scytalidium thermophilum type culture Humicola insolens. The pH and temperature optima for endoglucanase activities during enzyme assays were also analyzed for both organisms and determined to be pH 6.0 and 65 degrees C for type culture Torula thermophila, and pH 6.5 and 60 degrees C for type culture Humicola insolens. Analysis of the effect of growth temperature showed that type culture T. thermophila can grow and produce cellulases in the range of 35 to 55 degrees C although 40 to 50 degrees C seemed to favor growth and cellulase production. Although 45 degrees C was found optimal for fungal growth, both the specific endoglucanase and Avicel-adsorbable endoglucanase activities (U/mg protein) as well as the percentage of Avicel-adsorbable endoglucanase activity reached maxima at 50 degrees C and were higher as compared to type culture H. insolens. Results indicate that type culture T. thermophila, with further optimisations, is of potential use in the industrial production of cellulases.     

Enzymatic hydrolysis of wheat arabinoxylan by a recombinant "minimal" enzyme cocktail containing beta-xylosidase and novel endo-1,4-beta-xylanase and alpha-l-arabinofuranosidase activities

This study describes the identification of the key enzyme activities required in a "minimal" enzyme cocktail able to catalyze hydrolysis of water-soluble and water-insoluble wheat arabinoxylan and whole vinasse, a fermentation effluent resulting from industrial ethanol manufacture from wheat. The optimal arabinose-releasing and xylan-depolymerizing enzyme activities were identified from data obtained when selected, recombinant enzymes were systematically supplemented to the different arabinoxylan substrates in mixtures; this examination revealed three novel alpha-l-arabinofuranosidase activities: (i) one GH51 enzyme from Meripilus giganteus and (ii) one GH51 enzyme from Humicola insolens, both able to catalyze arabinose release from singly substituted xylose; and (iii) one GH43 enzyme from H. insolens able to catalyze the release of arabinose from doubly substituted xylose. Treatment of water-soluble and water-insoluble wheat arabinoxylan with an enzyme cocktail containing a 20%:20%:20%:40% mixture and a 25%:25%:25%:25% mixture, respectively, of the GH43 alpha-l-arabinofuranosidase from H. insolens (Abf II), the GH51 alpha-l-arabinofuranosidase from M. giganteus (Abf III), a GH10 endo-1,4-beta-xylanase from H. insolens (Xyl III), and a GH3 beta-xylosidase from Trichoderma reesei (beta-xyl) released 322 mg of arabinose and 512 mg of xylose per gram of water-soluble wheat arabinoxylan dry matter and 150 mg of arabinose and 266 mg of xylose per gram of water-insoluble wheat arabinoxylan dry matter after 24 h at pH 5, 50 degrees C. A 10%:40%:50% mixture of Abf II, Abf III, and beta-xyl released 56 mg of arabinose and 91 mg of xylose per gram of vinasse dry matter after 24 h at pH 5, 50 degrees C. The optimal dosages of the "minimal" enzyme cocktails were determined to be 0.4, 0.3, and 0.2 g enzyme protein per kilogram of substrate dry matter for the water-soluble wheat arabinoxylan, the water-insoluble wheat arabinoxylan, and the vinasse, respectively. These enzyme protein dosage levels were approximately 14, approximately 18, and approximately 27 times lower than the dosages used previously, when the same wheat arabinoxylan substrates were hydrolyzed with a combination of Ultraflo L and Celluclast 1.5 L, two commercially available enzyme preparations produced by H. insolens and T. reesei.     

里氏木霉表达异源中性内切纤维素酶蛋白以改善最佳酶活pH

为了提高里氏木霉中天然纤维素酶的最佳活性pH,本实验从特异腐质霉,灰腐质霉的变种,枯草芽孢杆菌LH中分别筛选并克隆了其含有的中性纤维素酶基因,将其置于里氏木霉cbh1启动子的启动下,在里氏木霉中进行异源表达。改造株在pH 6.0下酶活提升16%,pH 7.0下活性保留75%以上,而此时原始菌酶活残留20%。本实验所得的产中性纤维素酶里氏木霉基因改造株,由于其良好的中酸性活性表现,在食品,纺织,纸浆和造纸行业应也有良好的使用潜力。     

Structure of two fungal beta-1,4-galactanases: searching for the basis for temperature and pH optimum

beta-1,4-Galactanases hydrolyze the galactan side chains that are part of the complex carbohydrate structure of the pectin. They are assigned to family 53 of the glycoside hydrolases and display significant variations in their pH and temperature optimum and stability. Two fungal beta-1,4-galactanases from Myceliophthora thermophila and Humicola insolens have been cloned and heterologously expressed, and the crystal structures of the gene products were determined. The structures are compared to the previously only known family 53 structure of the galactanase from Aspergillus aculeatus (AAGAL) showing approximately 56% identity. The M. thermophila and H. insolens galactanases are thermophilic enzymes and are most active at neutral to basic pH, whereas AAGAL is mesophilic and most active at acidic pH. The structure of the M. thermophila galactanase (MTGAL) was determined from crystals obtained with HEPES and TRIS buffers to 1.88 A and 2.14 A resolution, respectively. The structure of the H. insolens galactanase (HIGAL) was determined to 2.55 A resolution. The thermostability of MTGAL and HIGAL correlates with increase in the protein rigidity and electrostatic interactions, stabilization of the alpha-helices, and a tighter packing. An inspection of the active sites in the three enzymes identifies several amino acid substitutions that could explain the variation in pH optimum. Examination of the activity as a function of pH for the D182N mutant of AAGAL and the A90S/ H91D mutant of MTGAL showed that the difference in pH optimum between AAGAL and MTGAL is at least partially associated with differences in the nature of residues at positions 182, 90, and/or 91.     

Unexpected regioselectivity of Humicola insolens Cel7B glycosynthase mutants

Four Humicola insolens Cel7B glycoside hydrolase mutants have been evaluated for the coupling of lactosyl fluoride on O-allyl N(I)-acetyl-2(II)-azido-beta-chitobioside. Double mutants Cel7B E197A H209A and Cel7B E197A H209G preferentially catalyze the formation of a beta-(1-->4) linkage between the two disaccharides, while single mutant Cel7B E197A and triple mutant Cel7B E197A H209A A211T produce predominantly the beta-(1-->3)-linked tetrasaccharide. This result constitutes the first report of the modulation of the regioselectivity through site-directed mutagenesis for an endoglycosynthase.     

Crystal structure of MltA from Escherichia coli reveals a unique lytic transglycosylase fold

Lytic transglycosylases are bacterial enzymes involved in the maintenance and growth of the bacterial cell-wall peptidoglycan. They cleave the beta-(1,4)-glycosidic bonds in peptidoglycan forming non-reducing 1,6-anhydromuropeptides. The crystal structure of the lytic transglycosylase MltA from Escherichia coli without a membrane anchor was solved at 2.0A resolution. The enzyme has a fold completely different from those of the other known lytic transglycosylases. It contains two domains, the largest of which has a double-psi beta-barrel fold, similar to that of endoglucanase V from Humicola insolens. The smaller domain also has a beta-barrel fold topology, which is weakly related to that of the RNA-binding domain of ribosomal proteins L25 and TL5. A large groove separates the two domains, which can accommodate a glycan strand, as shown by molecular modelling. Several conserved residues, one of which is in a position equivalent to that of the catalytic acid of the H.insolens endoglucanase, flank this putative substrate-binding groove. Mutation of this residue, Asp308, abolished all activity of the enzyme, supporting the direct participation of this residue in catalysis.     

特异腐质霉纤维素酶的研究

由腐植土中分离到一株嗜热真菌,经鉴定为特异腐质霉(Humicola insolens Cooney etEmerson)。研究了这株菌纤维素酶的产生条件和一般性质。菌在含麦麸5％、NaNO0.3％的液体培养基(灭菌前pH7.5,灭菌后pH7.2)中,于45℃培养4天,以羧甲基纤维素钠为底物,每ml滤液酶活力为20个单位。酶作用的最适条件为:pH6.0,温度为65—70℃。该纤维素酶是一种耐热酶,热稳定性较强,70℃保温5分钟后,酶活力剩余88％。底物对该酶的热钝化有较强的保护作用,无底物存在条件下,70℃保温6小时后,酶活力仅剩余1％,而在同样的处理温度和时间,在有底物存在条件下,酶活力可剩余30％。该酶在45℃保温15小时的条件下,pH稳定范围为6.0—9.0。     

Crystal structure of a family 45 endoglucanase from Melanocarpus albomyces: mechanistic implications based on the free and cellobiose-bound forms

Cellulose, a polysaccharide of beta-1,4-linked D-glucosyl units, is the major component of plant cell walls and one of the most abundant biopolymers in nature. Cellulases (cellobiohydrolases and endoglucanases) are enzymes that catalyse the hydrolysis of cellulose to smaller oligosaccharides, a process of paramount importance in biotechnology. The thermophilic fungus Melanocarpus albomyces produces a 20 kDa endoglucanase known as 20K-cellulase that has been found particularly useful in the textile industry. The crystal structures of free 20K-cellulase and its complex with cellobiose have been determined at 2.0 A resolution. The enzyme, classified into the glycoside hydrolase family 45, exhibits the characteristic six-stranded beta-barrel found before in Humicola insolens endoglucanase V structure. However, the active site in the 20K-cellulase shows a closing of approximately 2.5-3.5A while a mobile loop identified previously in Humicola insolens endoglucanase V and implicated in the catalytic mechanism is well-defined in 20K-cellulase. In addition, the crystal structure of the cellobiose complex shows a shift in the cellobiose position at the substrate-binding cleft. It is therefore proposed that these alterations may reflect differences in the binding mechanism and catalytic action of the enzyme.