Subsite affinities of beta-glucosidase from Aspergillus sojae on various xylooligosaccharides

The subsite affinities of beta-glucosidase (EC 3.2.1.21) with high beta-xylosidase activity from Aspergillus sojae on various xylooligosaccharides (degree of polymerization: n = 2-6) were investigated by steady-state kinetic analysis. The molecular activity (k0) value of the enzyme for xylobiose was not markedly different from those of other substrates (n = 3-6). The arrangement of the subsite affinities (A(i), i = 1-6) was evaluated; A1 = 2.93 kcal/mol, A2 = 3.67 kcal/mol, A3 = 0.64 kcal/mol, A4 = 0.12 kcal/mol, A5 = -0.07 kcal/mol, A6 = -0.05 kcal/mol, and the intrinsic rate constant (K(int)) was 7.6 s(-1). The subsite structure was similar to those of beta-glucosidase from A. niger and alpha-glucosidases from A. niger and Mucor javanicus, where the values for A1 were much larger than those for A3.     

一种米曲霉蛋白酶的分离纯化及酶解特性研究

对米曲霉固态发酵所产蛋白酶分离纯化,采用硫酸铵盐析、DEAE-FF层析、Butyl-HP层析和Superdux 7510/300GL凝胶层析得到一种电泳纯的蛋白酶,SDS-PAGE显示分子量大小为27 ku左右。以酪蛋白为底物时,该蛋白酶Km=1.23 g·L-1,Vm=27.03μg·m L-1·min-1,最适反应条件为50℃,p H9.0。该蛋白酶对酪蛋白水解活性最高,而对牛血清蛋白的水解活性很低;对牛胰岛素B链上-Phe-Val-,-Cys-Gly-,-Glu-Ala-和-Arg-Gly-组成的肽键有较强的切割能力,酶切位点较多,对疏水性氨基酸具有较高的选择性,为米曲霉所产蛋白酶在食品上的应用提供有力的参考。      

酱油曲霉蛋白酶特性研究

通过对酱油曲霉2128生理特性和生长规律的研究,得出酱油曲霉产酶的高峰期为分泌蛋白酶最适温度为28℃、培养基最适水分为100%、最适pH值为7、最适盐分为4%。     

In vitro cosmeceutical activity of alcoholic extract from chestnut inner shell fermented with Aspergillus sojae

Food Science and Biotechnology - Chestnut inner shell was fermented in solid state with Aspergillus sojae, and then extracted using ethanol (95%) to analyze its cosmeceutical activity and phenolic...     

Cloning, sequencing and expression of an alpha-L-arabinofuranosidase from Aspergillus sojae

The arabinofuranosidase gene was cloned from the cDNA of Aspergillus sojae. It was found to contain an open reading frame composed of 984 base pairs (bp) and to encode 328 amino acid residues (aa). The cDNA sequence suggested that the mature enzyme is preceded by a 26-aa signal sequence and the molecular mass was predicted to be 32,749 Da. The A. sojae arabinofuranosidase consists of a single catalytic domain; it does not have a specific substrate-binding domain such as the xylan-binding domain reported in an arabinofuranosidase from Streptomyces lividans (Vincent, P. et al.: Biochem. J., 322, 845-852, 1997). The deduced amino acid sequence of the catalytic domain of the mature enzyme exhibits extensive identity with the catalytic domains of Streptomyces coelicolor (74%), Aspergillus niger (75%), S. lividans (74%), and Aspergillus tubingensis (75%), which are enzymes that belong to family 62 of the glycosyl hydrolases. The cloned AFdase gene was expressed in Escherichia coli BL21 (DE3) pLysS as a cellulose-binding domain tag fusion protein. The specific activity of the purified recombinant enzyme was 18.6 units/mg protein, which is one-fourth that of the enzyme purified from a solid-state culture of A. sojae.     

米曲霉与酱油曲霉高酶活菌株的诱变选育

酱油酿造过程中菌株所分泌的酶系直接影响原料的利用率和最终产品的品质.实验通过紫外诱变、亚硝基胍诱变以及紫外-亚硝基胍复合诱变方法分别对米曲霉和酱油曲霉菌株进行了诱变,采州酪蛋白透明圈法初筛以及福林酚法和DNS法复筛,根据碱性蛋白酶、中性蛋白酶、酸性蛋白酶、纤维素酶和α-淀粉晦酶活水平的变化,选育出数株米曲霉和酱油曲霉高酶活菌株.并对初筛方法的可行性和不同诱变方法对菌株改良的效果进行了探讨.     

Purification and characterization of an alkaline manganese peroxidase from Aspergillus terreus LD-1

We report that Aspergillus terreus LD-1 produces an extracellular ligninolytic enzyme, manganese peroxidase (MnP), that reacts under alkaline conditions. This MnP was purified 13.1-fold from the culture supernatant to elicit a single band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of this MnP was estimated as either 43 kDa by SDS-PAGE or 44 kDa by gel permeation chromatography, suggesting a monomeric structure. The optimum pH and temperature of this MnP are 12.5 and 37 degrees C, respectively. This MnP is stable in the pH range 11.0 to 12.5 and also up to 40 degrees C. The K(m) values of this MnP for hydrogen peroxide, 2,6-dimethoxyphenol (2,6-DMP) and Mn2+ were 320 microM, 20 microM and 33 microM at pH 12.5, respectively. The activity of the MnP is completely inhibited by Hg2+, Pb2+, Ag+ and lactate. On the other hand, the MnP is activated by oxalate, maleate and fumarate. Maleate at 5 mM increased the MnP activity 5-fold. EDTA at 1 mM inhibited the MnP activity completely, but this inhibition was not observed in the presence of 1 mM Fe2+.     

Purification and characterization of cis-aconitic acid decarboxylase from Aspergillus terreus TN484-M1

cis-Aconitic acid decarboxylase (CAD) was assumed to be a key enzyme in the production of itaconic acid by comparing the activity of CAD from Aspergillus terreus TN484-M1 with that of CAD from the low-itaconate yielding strain Aspergillus terreus CM85J. The constitutive CAD was purified to homogeneity from A. terreus TN484-M1 by ammonium sulfate fractionation, and column chromatography on DEAE-toyopearl, Butyl-toyopearl, and Sephacryl S200HR, and then characterized. A molecular mass of 55 kDa for the native enzyme was determined by SDS-PAGE. The enzymic activity was optimal at a pH of 6.2 and temperature of 45 degrees C. The K(m) value for cis-aconitic acid was determined as 2.45 mM (pH 6.2, 37 degrees C). The enzyme was completely inactivated by Hg+, Cu2+, Zn2+, p-chloromercuribenzoate, and 5,5'-dithio-bis(2-nitrobenzoate).     

Partial purification of a polygalacturonase from a new Aspergillus sojae mutant and its application in grape mash maceration

The use of polygalacturonase (PG) preparations in winemaking promotes the release of phenolic compounds. A PG from a new source, Aspergillus sojae mutant, was semi‐purified and tested for grape mash maceration. Crude extract (CE), a commercial pectinase, and two high PG activity semi‐purified preparations, F_I and F_II, were applied for maceration at PG activity of 3.5 U g^?1 of grape for 46 h. Enzyme‐assisted maceration significantly (P < 0.05) increased the total phenolic content from 255.8 to 916.3 ± 5.2, 5732.9 ± 9.9, 563.4 ± 6.7 and 620.6 ± 18.4 mg L^?1 for CE, commercial pectinase, F_I and F_II, respectively. The content of individual phenolics such as gallic, protocatechuic, chlorogenic and p‐coumaric acids was improved. Principal component and hierarchical clustering analyses suggested that CE has a better performance upon the release of phenols. Semi‐purified preparations acted similar to commercial pectinase. These findings open an opportunity for the potential use of PG from the mutant strain as an alternative macerating enzyme.     

米曲霉(Aspergillus oryzae FAFU)淀粉酶的分离纯化及其酶学性质研究

从米曲霉(Aspergillus oryzae FAFU)发酵产物中提取淀粉酶,通过硫酸铵盐析、透析、DEAE-Sepharose离子交换层析和Sephacryl S-200凝胶柱层析等纯化步骤,回收活性组分,结合十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行纯度鉴定,共分离纯化出3种酶:酶Ⅰ、酶Ⅱ、酶Ⅲ。通过酶解淀粉实验,酶解产物经薄层层析(TLC)分析,鉴定出酶Ⅰ、酶Ⅱ为液化酶,酶Ⅲ为糖化酶,经SDS-PAGE电泳确定分子量大小分别为:48.6、49.7、93.5 k Da。对酶Ⅰ、酶Ⅱ的酶学性质进行初步的探究:酶Ⅰ的最适反应温度为50℃,温度在50℃以下稳定性好,55℃的半衰期是40 min,最适反应p H为6.0,在p H7.2~9.6范围内稳定性好;酶Ⅱ的最适反应温度为50℃,温度在45℃以下稳定性好,50℃的半衰期是30 min,最适反应p H为6.6,在p H8.6~10.0范围内稳定性好。     

一种米曲霉耐盐蛋白酶的纯化及酶学性质分析

采用硫酸铵沉淀、Q-HP阴离子交换柱和Superdux 75凝胶柱等技术,从酱油大曲中纯化得到一种耐盐蛋白酶,经飞行时间质谱鉴定为钙蛋白酶RIM13,属于一种半胱氨酸蛋白酶。该蛋白酶的最适温度为50?℃;最适pH值为6.5;稳定温度为40?℃;稳定pH值为7.0;Mn2＋促进蛋白酶活力,Fe3＋、Fe2＋、Cu2＋、Ca2＋、K＋、Na＋抑制蛋白酶活力,以上金属离子对蛋白酶二级结构也产生不同程度的影响;米氏常数Km为2.43?g/L,最大反应速率Vm为103.09?mg/（L·min）。在5、10?g/100?mL和15?g/100?mL的NaCl质量浓度条件下,蛋白酶保留的酶活力分别为77.22%、54.39%以及41.15%。该蛋白酶在高盐度环境下保持较高的酶活力,因此具有潜在的工业应用价值。     

Purification and characterization of Aspergillus oryzae LK-101 salt-tolerant acid protease isolated from soybean paste

A novel salt-tolerant acid protease was produced from Aspergillus oryzae LK-101 (AOLK-101). The AOLK-101 protease was purified to homogeneity by ammonium sulfate precipitation, DEAE-Sephadex A-50 and Sephadex G-100 chromatographies in order. The specific activity and the purification ratio of the purified protease were 2,301 unit/mg and 11.6 fold, respectively, with 25 kDa of molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrpphoresis (SDS-PAGE). Its optimal pH and temperature were pH 6.5 and 50°C, respectively. This protease was relatively stable at pH 4.5–7.5, below 40°C, and up to 10% salt concentration. The protease was moderately inhibited by Ag²⁺ and Zn²⁺, and strongly by ethylenediamide tetraacetic acid (EDTA) and phenylmethysulfonyl fluoride (PMSF), but activated by Cu²⁺ and Mn²⁺. Therefore, the AOLK-101 protease was a serine protease based on the influence of metal ions and inhibitors. K _m , V _max , k _cat , and k _cat /K _m values of AOLK-101 protease for hammastein milk casein were 1.04 mg/mL, 124.84 unit/L, 163.5/sec, and 3.9×10⁶/m·sec, respectively.     

宇佐美曲霉木聚糖酶的纯化及其性质

宇佐美曲霉E001固态发酵成熟曲经水浸提、硫酸铵盐析、Phenyl-SepharoseCL-4B疏水层析、SephadexG-75凝胶过滤层析、DEAESepharosefastflow阴离子交换层析等提纯步骤,获得了比酶活3047IU/mg蛋白的纯木聚糖酶,其SDS-PAGE呈单一条带。用SDS-PAGE和凝胶过滤测得木聚糖酶的相对分子质量分别为23.2kD和23.0kD,表明该酶蛋白为单亚基。纯木聚糖酶的最适作用温度和pH值分别为50℃和4.6;以燕麦木聚糖为底物的酶动力学常数Km和Vmax值分别为5.27mg/ml和6494μmol/(min·mg);Ca2+、EDTA对酶有激活作用,而Sn2+、Pb2+、Fe3+有强烈的抑制作用。     

Identification and toxigenic potential of the industrially important fungi, Aspergillus oryzae and Aspergillus sojae

Mold strains belonging to the species Aspergillus oryzae and Aspergillus sojae are highly valued as koji molds in the traditional preparation of fermented foods, such as miso, sake, and shoyu, and as protein production hosts in modern industrial processes. A. oryzae and A. sojae are relatives of the wild molds Aspergillus flavus and Aspergillus parasiticus. All four species are classified to the A. flavus group. Strains of the A. flavus group are characterized by a high degree of morphological similarity. Koji mold species are generally perceived of as being nontoxigenic, whereas wild molds are associated with the carcinogenic aflatoxins. Thus, reliable identification of individual strains is very important for application purposes. This review considers the pheno- and genotypic markers used in the classification of A. flavus group strains and specifically in the identification of A. oryzae and A. sojae strains. Separation of A. oryzae and A. sojae from A. flavus and A. parasiticus, respectively, is inconsistent, and both morphologic and molecular evidence support conspecificity. The high degree of identity is reflected by the divergent identification of reference cultures maintained in culture collections. As close relatives of aflatoxin-producing wild molds, koji molds possess an aflatoxin gene homolog cluster. Some strains identified as A. oryzae and A. sojae have been implicated in aflatoxin production. Identification of a strain as A. oryzae or A. sojae is no guarantee of its inability to produce aflatoxins or other toxic metabolites. Toxigenic potential must be determined specifically for individual strains. The species taxa, A. oryzae and A. sojae, are currently conserved by societal issues.     

Purification and characterization of two types of chitosanase from Aspergillus sp. CJ22-326

Aspergillus CJ22-326, a fungi strain capable of utilizing chitosan as a carbon source, was isolated from soil samples. Two types of chitosanase (ChiA and ChiB) produced from the culture supernatant of Aspergillus CJ22-326 were purified to an apparent homogeneity identified by SDS–PAGE through ammonium sulfate precipitation, CM-Sepharose FF chromatography, and Sephacryl S-200 gel filtration. Molecular weights of the enzymes were 109 kDa (ChiA) and 29 kDa (ChiB). Optimum pH values and temperature of ChiA were 4.0 and 50 °C, respectively, those of ChiB were 6.0 and 65 °C. The enzyme activities of ChiA and ChiB were increased by about 0.5-fold and 1.5-fold, respectively, by the addition of 1 mM Mn²⁺. However, 2.5 mM Ag⁺, Hg²⁺ and Fe³⁺strongly inhibited ChiA and ChiB activities. Viscosimetric assay and analysis of reaction products of these enzymes, using chitosan as a substrate, by TLC indicated endo- and exo-type cleavage of chitosan by ChiB and ChiA, respectively. ChiB catalysed the hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, and chitosan with a low degree of acetylation (0–30%), and formed chitotriose with chitohexaose as the major products. ChiA released a single glucosamine residue from chitosan and glucosamine oligomers. Both of the activities of ChiA and ChiB increased with the degree of deacetylation of chitosan. The enzyme ChiB had a useful reactivity and a high specific activity for producing functional chitooligosaccharides with high degree of polymerization.     

饲用黑曲霉纤维素酶的纯化及酶学特性的研究

从饲用黑曲霉纤维素酶的粗酶粉中，可以分离纯化4种内切β-1，4-萄葡聚糖酶(EG1、EG2、EG3和EG4)。经SDS—PAGE凝胶电泳分析，分子量分别为67500、59300、47700和38100u。EG4酶的最适作用温度为55℃，其他3种酶均为50℃。而其酶活相对稳定温度范围为35-65℃。EG3和EG4的耐热性优于EG1和EG2。它们的最适PH值均为5．0，在PH值为2．5-7．5情况下都较稳定。在PH值大于8．0时，相对酶活力下降较快，其中EG1和EG4失活程度高于EG2和EG3。