植酸酶的纯化及其酶学性质初步研究

从绿色木霉LH374固态发酵产物中提取植酸酶。粗酶液经硫酸铵沉淀、凝胶过滤、离子交换层析纯化后,提纯倍数可达13.3,回收率为27.1%。酶学性质研究表明,植酸酶最适作用温度为55℃、最适作用pH为6.0,米氏常数Km为0.15mmol/L。     

米曲霉蛋白酶的分离纯化及酶学性质研究

【目的】获得米曲霉蛋白酶主要成分及其酶学性质。【方法】利用硫酸铵盐析,DEAE-Sepharose FF阴离子交换层析、Phenyl-Sepharose HP疏水层析和Superdex-G75/200凝胶层析对米曲霉所产蛋白酶系进行分离纯化,SDS-PAGE检测蛋白酶纯度和分子量,采用高效液相凝胶色谱分析两种蛋白酶酶解产物。【结果】从米曲霉所产蛋白酶系中分离纯化获得两种蛋白酶组分P1和P2,分子质量分别约为37 kD和45 kD。以酪蛋白为底物时,P1的Km=8.36 g/L,Vm=12.95μg/(mL·min),最适反应条件为pH 8.0、45°C;P2的Km=4.11 g/L,Vm=4.86μg/(mL·min),最适反应条件为pH 7.0、45°C。两种蛋白酶均对酪蛋白水解活性最高,而对牛血清蛋白的水解活性很低。P1和P2分别酶解大豆分离蛋白后水解产物中肽分子质量分布呈现出一定的差异。【结论】两种蛋白酶的酶学性质存在差异;两者对疏水氨基酸构成的肽键具有选择性,但其作用基团存在特异性。这些研究结果将为米曲霉所产蛋白酶在食品上的应用提供指导。     

枯草芽孢杆菌中性植酸酶的纯化和酶学性质

从土壤中分离到了产中性植酸酶的枯草芽孢杆菌菌株并对所产植酸酶进行了分离纯化。此中性植酸酶的反应最适 pH为 7 5,最适温度为 55℃ ,在 37℃下以植酸钠为底物的Km值为 0 1 9mmol/L ,植酸酶活性依赖Ca2 +的存在。酶蛋白的分子量大小约为 45kD ,纯酶蛋白N端序列为Lys His Lys Leu Ser Asp Pro Tyr His Phe Thr。     

弗氏柠檬酸杆菌植酸酶的分离纯化及其酶学性质研究

从弗氏柠檬酸杆菌(Citrobacter freundii)中分离纯化了一种植酸酶并进行了酶学性质研究,其反应最适pH为4.0~4.5,最适温度为40℃,在37℃下以植酸钠为底物的Km值为0.85nmol/L,Vmax为0.53IU/(mg.min),具有较好的抗胰蛋白酶的能力。酶蛋白的分子量大小约为45kDa,成熟酶蛋白N端序列为QCAPEGYQLQQVLMM。     

枯草芽孢杆菌中怀植酸酶的纯化和酶学性质

从土壤中分离到了产中性植酸酶的枯草芽孢杆菌菌株并对所产植酸酶进行了分离纯化,此中性植酸酶的反应最适pH为7．5,最适温度为55度,在37度下以植酸钠为底物的Km值为0．19mmol/L,植酸酶活性依赖Ca^2 的存在,酶蛋白的分子量大小约为45kD,纯酶蛋白N端序列为Lys-His-Lys-Leu-Ser-Asp-Pro-Tyr-His-Phe-Thr。     

红曲霉丙酮酸脱羧酶基因克隆及酶学性质分析

目的:研究生物乙醇发酵关键酶丙酮酸脱羧酶(Pyruvate decarboxylase,PDC)的性质和功能.方法:从SMART技术构建的红曲霉(Monascus anka CICC 5031 )cDNA文库中筛选pdc基因,亚克隆到表达载体pET-21b(+),原核表达、亲和层析纯化重组PDC.分析重组PDC和野生型PDC的酶学性质.结果:pdc基因的开放阅读框长1 713bp,编码一个570个氨基酸残基的蛋白质.重组PDC在E.coli BL21( DE3)中的表达量为菌体总蛋白的32.7％.重组PDC与野生型PDC比活分别为20.2U/mg和30.1U/mg.二者的最适反应条件均为pH6.0、30℃.重组PDC和野生型PDC的Km值分别为2.6mmol/L和0.56mmol/L.结论:红曲霉pdc基因可在大肠杆菌中高效表达,重组PDC稳定性较好,可用作生物乙醇生产的候选资源.     

蜂房哈夫尼菌植酸酶基因appA的克隆、表达及酶学性质研究

从蜂房哈夫尼菌(Hafniaalvei)中克隆获得一个植酸酶编码基因appA,该基因全长1335bp,编码444个氨基酸,其中前33个氨基酸为信号肽,成熟蛋白的理论分子量为45.2kD。将基因appA克隆到大肠杆菌E.coli表达载体pET-22b( ),并在大肠杆菌中表达,表达产物具有植酸酶活性。对表达的酶蛋白进行纯化,并初步研究了该酶的酶学性质,结果表明:酶的作用最适pH值为4.5;在pH2.0~10.0范围内,酶活性保留80%以上;酶的作用最适温度为60℃;酶的比活性为356.7U/mg,酶动力学分析表明其Km为0.49mmol/L,Vmax为238U/mg;该酶对胰蛋白酶和胃蛋白酶有一定的抗性。该研究为哈夫尼菌属来源植酸酶的首次报道。     

蜂房哈夫尼菌植酸酶基因appA的克隆、表达及酶学性质研究

从蜂房哈夫尼菌（Hafnia alvei）中克隆获得一个植酸酶编码基因appA, 该基因全长1335bp,编码444个氨基酸,其中前33个氨基酸为信号肽,成熟蛋白的理论分子量为45.2kD。将基因appA 克隆到大肠杆菌E. coli表达载体pET-22b(+),并在大肠杆菌中表达, 表达产物具有植酸酶活性。对表达的酶蛋白进行纯化,并初步研究了该酶的酶学性质,结果表明：酶的作用最适pH值为4.5;在pH 2.0～10.0范围内, 酶活性保留80％以上;酶的作用最适温度为60℃;酶的比活性为356.7U/mg,酶动力学分析表明其K,/i>_m为0.49mmol/L,V_max为238U/mg;该酶对胰蛋白酶和胃蛋白酶有一定的抗性。该研究为哈夫尼菌属来源植酸酶的首次报道。     

蜂房哈夫尼菌植酸酶基因appA的克隆、表达及酶学性质研究

从蜂房哈夫尼菌（Hafnia alvei）中克隆获得一个植酸酶编码基因appA, 该基因全长1335bp,编码444个氨基酸,其中前33个氨基酸为信号肽,成熟蛋白的理论分子量为45.2kD。将基因appA 克隆到大肠杆菌E. coli表达载体pET-22b(+),并在大肠杆菌中表达, 表达产物具有植酸酶活性。对表达的酶蛋白进行纯化,并初步研究了该酶的酶学性质,结果表明：酶的作用最适pH值为4.5;在pH 2.0～10.0范围内, 酶活性保留80％以上;酶的作用最适温度为60℃;酶的比活性为356.7U/mg,酶动力学分析表明其K,/i>_m为0.49mmol/L,V_max为238U/mg;该酶对胰蛋白酶和胃蛋白酶有一定的抗性。该研究为哈夫尼菌属来源植酸酶的首次报道。     

产纤维素酶菌株宇佐美曲霉Y-11产酶条件及酶性质的研究

通过正交试验,对宇佐美曲霉纤维素酶固态发酵条件进行了研究,其较适培养基为：麸皮与稻草配比为２：３,氮源：尿素,含水量：２００％（液固比）,ｐＨ：５．５。２８－３０℃培养７２小时,ＣＭＣ酶活力为５．０５（ｕ／ｍｌ）,滤纸酶活力达到１．０４（ｕ／ｍｌ）。ＣＭＣ酶及滤纸酶最适ｐＨ分别为４．４１、６．０６;最适温度分别为６５℃、５５℃。酶的热稳定性与ｐＨ稳定性较高。     

烟曲霉菌热稳定性植酸酶在黑曲霉菌中的分泌性表达

在先前克隆获得烟曲霉菌植酸酶phyA基因并构建了重组质粒的基础上,将该质粒转化黑曲霉菌pyrG基因缺陷株M54;同时制备植酸酶多克隆抗体用于植酸酶的免疫学检测。SDS-PAGE和western-blot结果表明,phyA在黑曲霉菌中获得分泌性表达。表达产物活性测定结果显示,重组植酸酶的表达量为597.6 IU/mL。在90℃加热10 min和100℃加热20 min后,重组植酸酶残余酶活分别为74%和70%,具有较好的热稳定性。实现了烟曲霉菌植酸酶在黑曲霉菌中的分泌性表达,表达产物具较高的生物活性和耐热性。     

Enhancement of operation and storage stability of glucoamylase from Aspergillus awamori by a protease inhibitor preparation

Glucoamylase was produced extracellularly by fermentation of strain Aspergillus awamori, which had been genetically modified to have high-level glucoamylase activity. Initial experiments showed that the enzyme deactivated quickly, with a half-life of less than 6 days even stored at 5°C. A possible reason for the rapid deactivation was the presence of proteases, attacking and degrading the glucoamylase. Therefore a liquid protease inhibitor cocktail (Sigma, USA) was selected and applied to enhance the stability of the enzyme. The activity of the enzyme (stored at 5°C) measured by the Schoorl-method with starch as substrate showed that the cocktail was effective with the enzyme maintaining 95% of its initial storage activity for almost one year. The enzyme preparation has been used for starch hydrolysis in a flat-sheet membrane bioreactor at 60°C to manufacture glucose solution and its operation stability extended by using the cocktail.     

黑曲霉N25株产植酸酶及酶促反应条件研究

从植物种子中研究筛选出高产植酸酶的黑曲霉N25,进行了最适液体培养基的筛选,研究了黑曲霉N25在玉米半合成液体培养基中所产植酸酶的最适酶促反应条件。结果表明：在四种培养基中,玉米半合成液体培养基为最适培养基,黑曲霉N25产植酸酶高峰期在96h,黑曲霉N25所产植酸酶的酶促反应最适pH为2.6和4.6,并具有很好的热稳定性,一定浓度的Ca^2 ,Mg^2 ,Mn^2 ,Cr^3 ,Li^ ,EDTA和高磷是植酸酶活性的抑制剂,1.0mmol/ml聚乙二醇1000,0.3nmol/mL Fe^2 和低磷对植酸酶活性具有激活作用。     

Enhancement of operation and storage stability of glucoamylase from Aspergillus awamori by a protease inhibitor preparation

Glucoamylase was produced extracellularly by fermentation of strain Aspergillus awamori, which had been genetically modified to have high-level glucoamylase activity. Initial experiments showed that the enzyme deactivated quickly, with a half-life of less than 6 days even stored at 5°C. A possible reason for the rapid deactivation was the presence of proteases, attacking and degrading the glucoamylase. Therefore a liquid protease inhibitor cocktail (Sigma, USA) was selected and applied to enhance the stability of the enzyme. The activity of the enzyme (stored at 5°C) measured by the Schoorl-method with starch as substrate showed that the cocktail was effective with the enzyme maintaining 95% of its initial storage activity for almost one year. The enzyme preparation has been used for starch hydrolysis in a flat-sheet membrane bioreactor at 60°C to manufacture glucose solution and its operation stability extended by using the cocktail.     

Isolation and characterisation of Aspergillus awamori BS05, a root-knot-nematode-trapping fungus

Nematode-trapping fungi are important biocontrol agents against parasitic nematodes through adhesive or mechanical hyphal traps. Aspergillus awamori, a root-knot-nematode-trapping fungus from tomato rhizosphere soil, was identified based on morphology and molecular characteristics of internal transcribed spacer DNA sequence. Conidial heads were white to black brown, loosely globose, and 72–127 μm in diameter. Conidiophores usually arose from the foot cell of basal mycelium, straight, and 960–1730 × 10.2–13.4 μm, hyaline to pale brown, not constricted below the vesicles; vesicles hemispherical to elongate, 43–56 μm in diameter, black brown, fertile over the upper half to two-thirds. Aspergilla were biseriate, and metulae were variable, 12–26 × 3.8–4.7 μm; phialides were 8.2–9.4 × 2.5–3 μm. Conidia were globose or subglobose, 3.6–4.8 μm in diameter, rough, grey brown and parallel in chains. A. awamori BS05 showed 44.9% control efficacy against Meloidogyne incogtina in pot experiments which suggests it as a potential biocontrol agent against Meloidogyne. This is the first report on A. awamori as nematode-trapping fungus.     

Increased thermostability of Asn182 --> Ala mutant Aspergillus awamori glucoamylase

Asn182 --> Ala Aspergillus awamori glucoamylase expressed in Saccharomyces cerevisiae had a first-order thermodeactivation coefficient 40% that of wild-type glucoamylase at pH 4.5 between 60 degrees and 65 degrees C, caused by the elimination of an Asn-Gly sequence subject to deamidation and eventual chain breakage. Above 70 degrees C, and at pHs 3.5 and 5.5, thermodeactivation coefficients of wild-type and mutant enzymes were roughly equal, because the fastest deactivation mechanism was no longer deamidation. The mutation had little effect on the enzyme's optimal pH for activity and subsite map, or on the glucose yield from starch dextrin hydrolysis. During enzyme production by yeast fermentation, highest cell densities and activities of wild-type and mutant glucoamylases were attained after a period of glucose starvation, followed by a second addition of glucose. (c) 1994 John Wiley & Sons, Inc.     

Enhancement of acid amylase production by an isolated Aspergillus awamori

AIMS: Evaluation of the influence of fermentation components on extracellular acid amylase production by an isolated fungal strain Aspergillus awamori. METHODS AND RESULTS: Eight fungal metabolic influential factors, viz. soluble starch, corn steep liquor (CSL), casein, potassium dihydrogen phosphate (KH(2)PO(4)) and magnesium sulfate (MgSO(4) x 7H(2)O), pH, temperature and inoculum level were selected to optimize amylase production by A. awamori using fractional factorial design of Taguchi methodology. Significant improvement in acid amylase enzyme production (48%) was achieved. The optimized medium composition consisted of soluble starch--3%; CSL--0.5%; KH(2)PO(4)--0.125%; MgSO(4) x 7H(2)O--0.125%; casein--1.5% at pH 4.0 and temperature at 31 degrees C. CONCLUSION: Optimization of the components of the fermentation medium was carried out using fractional factorial design of Taguchi's L-18 orthogonal array. Based on the influence of interaction components of fermentation, these could be classified as the least significant and the most significant at individual and interaction levels. Least significant factors of individual level have higher interaction severity index and vice versa at enzyme production in this fungal strain. The pH of the medium and substrate (soluble starch) showed maximum production impact (60%) at optimized environment. Temperature and CSL were the least influential factors for acid amylase production. SIGNIFICANCE AND IMPACT OF THE STUDY: Acid amylase production by isolated A. awamori is influenced by the interaction of fermentation factors with fungal metabolism at individual and interaction levels. The pH of the fermentation medium and substrate concentration regulates maximum enzyme production process in this fungal strain.     

Antifungal Activity of Thymol against Aspergillus awamori and Botrytis aclada Isolated from Stored Onion Bulbs

The antifungal activity of thymol against Aspergillus awamori F23 and Botrytis aclada F15 in onions was examined through direct treatment with amended media and gaseous treatment with I-plates (plastic plates containing central partitions). The protective and curative control efficacy of thymol was examined 24 h before and after the inoculation of onion bulbs with the fungal isolates. Mycelial growth, sporulation, and spore germination of the isolates were inhibited on potato dextrose agar amended with various concentrations of thymol or acetic acid (positive control). Overall, thymol produced a stronger inhibitory effect on the mycelial growth and development of the isolates than acetic acid. Following gaseous treatment in I-plates, mycelial growth, sporulation, and spore germination of the isolates were inhibited at higher concentrations of thymol or acetic acid; however, acetic acid showed a little effect on the sporulation and spore germination of the isolates. Following the treatment of onion bulbs with 1000 mg L⁻¹ of thymol 24 h before and after fungal inoculation, lesion diameter was greatly reduced compared with that following treatment with 0.5% ethanol (solvent control). Onion bulbs sprayed with thymol 24 h before fungal inoculation generally showed reduced lesion diameters by isolate F23 but not in isolate F15 compared with those sprayed 24 h after fungal inoculation. Collectively, thymol effectively inhibited the growth and development of A. awamori and B. aclada on amended media and in I-plates. In addition, spraying or fumigation of thymol is more desirable for effectively controlling these postharvest fungal pathogens during long-term storage conditions.     

激光诱变筛选高产植酸酶黑曲霉菌株的研究

用1.06um,15KHz高重复率声光调QNd：YAG激光以20W和15W的辐照功率分别照射产植酸酶的黑曲霉孢子液和原生质体液不同时间,检定再生菌落数并经透明圈初筛和摇瓶复筛,测定诱变菌株所产植酸酶酶活。结果表明：20W的功率辐照黑曲霉孢子1′以上,致死率近乎100…,15W功率辐照原生质体液20″-4′,存活率在8.571％-117.14％之间,正变率在27.45％-100％之间,正变辐度60.69％-124.96％,说明原生质体比孢子耐受激光诱变的能力强,并且诱变效果好。筛选到了一株酶活提高达3.75倍的单个变异菌株。