嗜热真菌hermomyces lanuginosus A236热稳定葡萄糖淀粉酶的纯化…

嗜热真菌Ｔｈｅｒｍｏｍｙｅｓ ｌａｎｕｇｉｎｏｓｕｓＡ２３６在液体培养基中５０℃下静止培养１４天,粗提酶液经硫酸铵分级沉淀、ＤＥＡＥ－Ｔｏｙｏｐｅａｒｌ离子交换层析、Ｂｕｔｙｌ－Ｔｏｙｏｐｅａｒｌ疏水层析,ＳｅｐｈａｃｒｙｌＳ１００凝胶过滤和ＦＰＬＣＭｏｎｏＱ离子交换层析,得到了凝胶电泳均质的葡萄糖淀粉酶。酶促反应产物经ＴＬＣ分析为葡萄糖,证明纯化的酶葡萄糖淀粉酶（ＥＣ３．２．１．３）。ＳＤＳ－Ｐ     

嗜热真菌Thermomyces lanuginosus A_(236)热稳定葡萄糖淀粉酶的纯化及其特性

嗜热真菌ThermomyceslanuginosusA_236在液体培养基中50℃下静止培养14天,粗提酶液经硫酸铵分级沉淀、DEAE-Toyopearl离子交换层析、Butyl－Toyopearl疏水层析、SephacrylS100凝胶过滤和FPLCMonoQ离子交换层析,得到了凝胶电泳均质的葡萄糖淀粉酶。酶促反应产物经TLC分析为葡萄糖,证明纯化的酶为葡萄糖淀粉酶（EC3．2．1．3）。SDS－PAGE测定其分子量为72,000,不具亚基,PI为4．0,富含Val和Leu。酶反应最适温度和pH分别为70℃和5．0。在pH5．0条件下,酶在60℃保温1h,仍具有原酶活性。酶活性在70℃和80℃的半衰期分别为20min和6min。Ca2+对酶有激活作用,Fe3+、Al3+、Hg2+等金属离子对酶活力有一定的抑制作用。纯酶碳水化合物含量为12．4％。纯酶可水解可溶性淀粉、直链淀粉、支链淀粉、糊精、糖原、麦芽三糖和麦芽糖,其中可溶性淀粉为最适底物。     

热稳定α—淀粉酶稳定性工程菌的构建

本文以质粒pE194为载体亚克隆B.licheniformis热稳定α-淀粉酶基因,构建成重组质粒pNW102,通过噬菌体PBS1将它转导进入中温α-淀粉酶生产菌B.subtilis BF7658。B.subtilis BF7658(pNW102)经过长时间非许可温度处理,筛选得到2株热稳定α-淀粉酶稳定性表达的工程菌株。酶学分析显示同源重组具有热点,2株重组菌株B.subtilis BFNW产生的热稳定α-淀粉酶符合B.licheniformis产生的淀粉酶特性。     

嗜热真菌Thermomyces lanuginosus A236热稳足葡萄糖淀粉酶的纯化及其特性

嗜热真菌Thermomyces lanuginosus A2a6在液体培养基中50℃下静止培养14天,粗提酶液经硫酸铵分级沉淀、DEAE-Toyopearl离子交换层析、Butyl-Toyopearl疏水层析、Sephacryl S100凝胶过滤和FPLC Mono Q离子交换层析,得到了凝胶电泳均质的葡萄糖淀粉酶。酶促反应产物经TLC分析为葡萄糖,证明纯化的酶为葡萄糖淀粉酶(EC 3．2．1.3)。SDS-PAGE测定其分子量为72,000,不具亚基,pI为4．0,富含val和Leu。酶反应最适温度和pH分别为70℃和5．0。在pH5．0条件下,酶在60℃保温lh,仍具有原酶活性。酶活性在70℃和80℃的半衰期分别为20min和6min, Ca²⁺对酶有激活作用,Fe³⁺、Al³⁺、Hg²⁺等金属离子对酶活力有一定的抑制作用。纯酶碳水化合物含量为12．4％。纯酶可水解可溶性淀粉,直链淀粉、支链淀粉．糊精、糖原、麦芽三糖和麦芽糖,其中可溶性淀粉为最适底物。     

提高黑曲霉产葡萄糖淀粉酶活力的研究

本研究所设计新型结构的20M~3发酵罐,其搅拌电动机的功率较低,为22千瓦。发酵液的投料浓度略增高,为原发酵液增加含糖量<2％。在这种发酵条件下,以黑曲霉UV11 AN5—1变异菌株产葡萄糖淀粉酶的活力,平均达14601单位/毫升,最高达15115单位/毫升,提高产率41.2％。发酵周期平均为107.2小时,每小时产酶活力为136.2单位/毫升。生产1罐葡萄糖淀粉酶,可降低耗电量679.2千瓦,年产300罐,可降低耗电量20.38×10~4千瓦。     

用葡萄糖酶电极法测定葡萄糖淀粉酶活性的研究

利用固定化葡萄糖氧化酶酶膜和过氧化氢电极组成酶电极测定葡萄糖淀粉酶的活性单位。用已知单位的葡萄淀粉酶作为测定标准定标后,在仪器上直接测出被测样品的葡萄糖淀粉酶活性单位,测定时间１４０ｓ,操作周期３ｍｉｎ,连续１０次测定ＣＶ值为０.６７％,５０～５００ｕ／ｍｌ的范围内线性良好,ｒ＝０.９９９９。     

黑曲霉突变株葡萄糖淀粉酶的底物特异性

黑曲霉(Aspergillus niger)突变株T-21葡萄糖淀粉酶(GAI)仅能水解多种淀粉及麦芽低聚糖生成唯一产物β-葡萄糖,其水解麦芽糖及麦芽三糖的速度分别为200和570mg葡萄糖·h^(-1)·mg^(-1).GAI水解α-1,4键的速度比水解α-1.6键快100多倍.除了马铃薯淀粉外,对其它淀粉及麦芽低聚糖几乎都能100%地水解,但不能水解环状糊精,其水解各麦芽低聚糖的最先产物都比原底物少一个葡萄糖单位,说明GAI为一外切型淀粉酶.GAI对麦芽糖、麦芽三糖、可溶性淀粉、糯米淀粉、糊精及糖原的Km值分别1.92mmol/L、0.38mmol/L、0.053%、0.045%、0.059%、及0.076%,V_(max)分别为590、1370、1270、1520、1120和1220mg葡萄糖·h^(-1)·mg^(-1).D-葡萄糖酸-δ-内酯及麦芽糖醇对此酶分别具有反竞争性抑制和混合性抑制.     

一种耐热几丁质酶的产生及其稳定性研究

疏绵状嗜热丝孢菌(Thermomyces lanuginosus)SY2在以胶状几丁质为唯一碳源的诱导培养基中产生了胞外几丁质酶。该酶在50℃保温1 h, 酶活稳定; 65℃时半衰期为25 min; 酶液在室温下保存到12周, 残余酶活性为45％左右。该酶有较宽的pH范围, 3.0~9.0之间保持稳定, pH值为2.5时, 仍具有70％的剩余酶活性。Ca2+ 对几丁质酶的活性有显著的激活作用; 高浓度变性剂对酶有抑制作用。结果表明该酶是一种热稳定性高且耐酸碱的新型几丁质酶, 能在酸性和高温环境中发挥作用, 这些特性赋予了T. lanuginosus几丁质酶在几丁质的生物转化及其它生物技术中极大的应用优势。     

Short Communication: Effect of agitation rate on the growth and production of xylanase free of cellulase by Thermomyces lanuginosus MH4 in bioreactor

Cellulase-free xylanase production by T. lanuginosus MH4 was investigated in a 3-litre stirred tank bioreactor under different agitation rates and an aeration rate of 1v/v/m. The cultivation time in the bioreactor was reduced significantly over that in shake culture conditions. A xylanase productivity of 0.1 mkat1–1h–1 was achieved on xylan in the bioreactor. This was nearly double to that obtained in shake culture. The agitation rates influenced both growth and enzyme secretion in the bioreactor. The highest level of biomass concentration and activities of both xylanase and -xylosidase were obtained at 150 revmin–1     

Purification and characterization of an extracellular polygalacturonase from the thermophilic fungus,Thermomyces lanuginosus

A polygalacturonase was purified from the thermophilic fungus, Thermomyces lanuginosus to apparent homogeneity by ultrafiltration, acetone precipitation and ion-exchange chromatography. The enzyme was maximally active at pH 5.5 and 60 °C. The apparent K_M with potassium pectate was 0.67 mg/ml and the V_max was 7.2 × 10⁵ mol/min/mg protein. The apparent molecular weight of the enzyme was 59 kDa and it contained approximately 10% carbohydrate. The enzyme was completely stable at room temperature (32 ± 3 °C) and retained about 50% activity at 50 °C for 6 h. The zymogram of the purified enzyme revealed two activity bands, one of which was a major one. Polyclonal antibodies raised against the enzyme did not show any immunological relatedness with other mesophilic polygalacturonases.     

Short Note: Production and properties of pectinolytic enzymes from the thermophilic fungus, Thermomyces lanuginosus

Maximal pectinolytic activity was detected in the culture filtrates of Thermomyces lanuginosus when grown in medium containing pectin and sucrose. The pectinolytic enzyme system was optimally active at pH 5.5 and at 70°C with potassium pectate and at pH 4.5 at 50°C with pectin as substrates. Zymogram analyses showed two activity bands with pectin and three with potassium pectate.     

Characterization of a xylanase from the newly isolated thermophilic Thermomyces lanuginosus CAU44 and its application in bread making

AIMS: A xylanase from the newly isolated thermophilic fungus, Thermomyces lanuginosus CAU44, was characterized and evaluated for its suitability in bread making. METHODS AND RESULTS: Xylanase was purified 3.5-fold to homogeneity with a recovery yield of 32.8%. It appeared as a single protein band on SDS-PAGE gel with a molecular mass of c. 25.6 kDa. The purified xylanase had an optimum pH of 6.2, and it was stable over pH 5.6-10.3. The optimal temperature of xylanase was 75 degrees C and it was stable up to 65 degrees C at pH 6.2. Study was further carried out to investigate the effect of the purified xylanase on the properties of wheat bread and its staling during storage. CONCLUSIONS: The purified xylanase from T. lanuginosus CAU44 was stable up to 65 degrees C and had a broad pH range. The presence of thermostable xylanase during bread making led to an improvement of the specific bread volume and better crumb texture. Besides, addition of xylanase provided an anti-staling effect. SIGNIFICANCE AND IMPACT OF THE STUDY: The xylanase from the newly isolated Thermomyces lanuginosus CAU44 shows great promise as a processing aid in the bread-making industry.     

外源硅对干旱胁迫下烟草幼苗生长、叶片光合及生理指标的影响

采用营养液水培法,研究了施用不同浓度外源硅(0、0.5、1.0 mmol/L)对干旱胁迫(10%PEG、20%PEG)下烟草幼苗生长、叶片光合特性和生理指标的影响。结果表明:干旱胁迫严重抑制了烟草幼苗生长和光合作用,膜质稳定性降低和引起氧化应激反应;施用不同浓度外源硅有效改善了干旱胁迫下烟草幼苗生长,均表现为株高、叶面积、根系体积、根系干重和地上部干重等生长指标增加,提高叶绿素a、叶绿素b、叶绿素a+b和类胡萝卜素含量,显著提高净光合速率(Pn)、蒸腾速率(Tr)、气孔导度(Gs)并降低胞间CO2浓度(Ci),膜质过氧化产物MDA含量显著降低,提高叶片含水量、膜稳定性系数和渗透调节物质(脯氨酸、可溶性糖)含量,显著提高SOD、POD和CAT等抗氧化酶活性,而且1.0 mmol/L Si处理对干旱胁迫下烟草幼苗生长和生理特性的影响显著优于0.5 mmol/L Si处理。以上结果说明,施用外源硅能提高干旱胁迫下烟草幼苗光合作用、抗氧化和渗透调节能力,缓解干旱胁迫对烟草幼苗的伤害,促进其生长。     

Studies on Leaf Surface Lipid of Tobacco I. Changes in Leaf Surface Lipid and Duvatrienediol during Growth,Senescence and Curing of Tobacco Leaves

Duvatrienediol is a diterpene specifically occurred in tobacco plants and thought to be a precursor of tobacco aroma. Green tobacco leaves contained 0.2~1% of duvatrienediol per dry weight and it was corresponded to 30~60% of leaf surface lipid. Leaves on upper stalk position contained more of leaf surface lipid and duvatrienediol. In leaves on each stalk position, leaf surface lipid and duvatrienediol contents increased with leaf growth and decreased by over-maturation. Production of leaf surface lipid and duvatrienediol was affected by soil conditions or applied amount of nitrogen fertilizer. Both leaf surface lipid and duvatrienediol were decreased during curing of tobacco leaves, but the change in the latter was more drastic. Comparing to leaf surface lipid, changes in cytoplasmic lipid were less during growth and senescence of tobacco leaves.     

机械性损伤与氨基丁酸的交互作用对烟草防卫性酶活性的影响

研究β-氨苯丁酸（BABA）和剪叶对烟草（Nicotiana tabacum L．cv．Yunyan 85）防卫性酶活性和两者间互作对其抗性激发的影响。结果表明：喷施BABA或剪叶都能明显地提高烟草的苯内氨酸解氨酶（PAL）和多酚氧化酶（PPO）的活性,且这些酶活性的变化与BABA的浓度和诱抗时间有关。然而存剪叶后使用BABA,会削弱对PAL和PPO活性的诱导。BABA＋剪叶处理后烟草花叶病（TMV）症状明显较BABA单独处理的严重,这表明BABA和机械性损伤呈现拮抗效应。BABA及其它以水杨酸（SA）为信号分子的化学诱抗剂可能不适宜用于剪叶后的烟草幼苗。