Isolation and identification of a black Aspergillus strain and the effect of its novel protease on the aroma of Moutai‐flavoured liquor

Among Chinese traditional distilled liquors, Moutai‐flavoured liquor is the most famous, owing to its complicated process as it is derived from fermented sorghum coupled with the use of a Moutai‐flavoured Daqu. In this study, a novel isolate, belonging to a black Aspergillus, was obtained and identified as Aspergillus hennebergii by ITS‐5.8S rRNA sequencing analysis and conventional morphologic identification. The influences of initial pH, carbon source, nitrogen source and metal ions on the production of an A. hennebergii protease were studied. The results revealed that metal ions exerted a significant effect on enzyme production and activity. Additionally, the potential application of the protease from A. hennebergii was investigated. The enzymatic hydrolysis resulted in the identification and quantification of 42 compounds, including alcohols, aldehydes, pyrazines and esters. These volatile compounds exhibited special flavour properties. Significant differences were observed between the enzyme treatments and the control sample. Samples from the enzyme treatments led to the highest amounts of alcohols, pyrazines and aromatics. These results suggest that A. hennebergii, or its protease, may have some application values for the enhancement of the quality of Chinese liquor and for the improvement of the liquor production process. Copyright © 2014 The Institute of Brewing & Distilling     

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。      

酱香白酒酿造环境曲霉的分离及Aspergillus hennebergii酶分泌胁迫条件

以茅台镇酱香白酒酿造过程关键环节样品为载体,对曲霉的生态结构分布进行分析;以新鲜麸皮为培养基模拟固态发酵方法对影响筛选优势菌株Aspergillums henneberguii的生长及分泌糖化酶、蛋白酶的主要发酵胁迫条件进行了分析。结果分离得到曲霉103株,分别鉴定为10个种属种,即A.niger、A.oryzae、A.terreus、A.tubingensis、A.flavus、A.henneberguii、A.fumigatus、A.niveus、A.candidus和Monascus;发酵基质初始水分在50%和初始p H在3.5~4.0条件下有利于Aspergillums henneberguii的生长和酶的分泌;Aspergillums henneberguii的适宜生长温度范围为30~35℃,酶分泌温度为32~35℃;高乙醇体积质量(8%)对Aspergillums henneberguii的生长和产酶存在抑制,低乙醇体积质量(2%)反而有利于其生长和酶的分泌;(NH4)2SO4有利于Aspergillums henneberguii糖化酶的分泌,Na NO2和酵母浸膏有利于其生长和蛋白酶的分泌;乳糖和麦芽糖对Aspergillums henneberguii的生长和产酶有利,山梨糖、蔗糖、果糖可促进菌株糖化酶的分泌;单独添加硫酸钠有利于Aspergillums henneberguii的生长和酶的分泌;新鲜麸皮富含碳源、氮源、无机盐、微量元素等,为Aspergillums henneberguii菌体生产及制酶的优质天然载体。Aspergillums henneberguii的应用可满足传统固态白酒酿造过程的双边发酵和风味形成机制需求。     

Production of a potential collagenolytic protease by nejayote fermentation with Aspergillus oryzae

Nejayote is the wastewater obtained from maize lime cooking. It was used for Aspergillus oryzae growth and enzyme production. Protein production and collagenolytic activity were evaluated to test the effects of pH, spore inoculum and nejayote content in a 3³ factorial design. No collagenolytic activity was observed in the supernatant of the fermentation without nejayote. Using nejayote at 5% increased 1.5 times the fungus growth in comparison with nejayote 1% and 3%; and the maximum collagenolytic activity was reached after 3 days at pH 8 (P < 0.05). After sequencing, a 32 kDa alkaline protease was identified. Along with the alkaline conditions produced by the inclusion of nejayote solids, this waste is an important source of carbohydrates that improved the growth of A. oryzae to produce a protease that has the potential to break collagen. This permits a prospective revalorisation of a waste material to get a product with multiple biotechnological uses.     

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。     

南美白对虾虾头内源酸性蛋白酶的分离纯化及其酶学特性研究

通过10%～50%的硫酸铵盐析和两次柱层析,从南美白对虾虾头的匀浆液中分离纯化出一种内源酸性蛋白酶,经电泳分析测得其分子量为27.45ku,该酶的Km值、Vmax值、最适pH和最适反应温度分别为2.01g/L、26.39μg/min、3.0和30℃。内源酸性蛋白酶的热稳定性较差,Pepstatin A和EDTA对该蛋白酶表现出强烈的抑制作用,Hg+能完全抑制该蛋白酶,但Ca2+却能激活该蛋白酶。据此推断该蛋白酶是一种金属蛋白酶,该酶活性中心含有一些金属离子,其性质类似于胃蛋白酶类。     

Purification and Characteristics of Feruloyl Esterase from Aspergillus awamori G-2 Strain

ABSTRACT:  For food industry production processes and other uses, a mold that produces high levels of feruloyl esterase was obtained from laboratory mold collections and other sources. It was Aspergillus awamori G-2 that produces high levels of feruloyl esterase. The feruloyl esterase was purified using ion-exchange chromatography, size-exclusion chromatography, and HPLC chromatography. The enzyme was identified as a monomer protein using size-exclusion chromatography. Its optimum temperature and pH were, respectively, 40 °C and pH 5. Its activity was stable at pH 3 to 5. The enzyme was combined with xylan and starch, but it was absorbed by cellulose. The km of the feruloyl esterase was 0.0019% (0.01 mM). The enzyme showed stable activity at pH 3 and 50 °C, making this enzyme useful for food production.     

宇佐美曲霉酸性β-甘露聚糖酶的纯化及性质研究

采用硫酸铵分部盐析、Phenyl Sepharose CL-4B疏水层析、Sephadex G-75凝胶过滤层析、DEAE Sepharosefast flow阴离子交换层析等分离技术,从宇佐美曲霉(Aspergillus usamii)YL-01-78菌株固态曲浸出液中分离出1种SDS-PAGE电泳纯和HPLC色谱纯的酸性β-甘露聚糖酶组分,其最终回收率为12.6%,纯化倍数为32.3。经SDS-PAGE和凝胶过滤层析测得该纯化酶的分子质量分别为42 ku和40 ku,表明该酶以单体形式存在;其等电点pI为4.2;含糖量为23.2%。以角豆胶半乳甘露聚糖为底物,测得该酶的Km值和Vmax值分别为6.3 mg/mL和1 667μmol/(min.mg)。     

黑曲霉发酵液中脂肪酸化学成分的研究

目的：分析黑曲霉（Aspergillusnigerxj）发酵液中的脂肪酸成分。方法：液体发酵培养黑曲霉0spergillusnigerxj）,发酵液通过石油醚萃取,硅胶柱层析,石油醚-乙酸乙酯洗脱,得到的脂肪酸经甲酯化处理,通过气相色谱一质谱联用技术对其进行鉴定。结果：鉴定出4种脂肪酸成分。结论：黑曲霉（Aspergillusniger巧）发酵液中的脂肪酸成分主要为亚油酸、6-十八碳烯酸、14-甲基十五烷酸和硬脂酸。     

利用外源蛋白酶和曲霉菌YL001加速沙丁鱼鱼露的发酵

该研究旨在利用外源蛋白酶和曲霉菌YL001缩短沙丁鱼鱼露的发酵周期.将沙丁鱼鱼肉分为2组,一组仅用外源蛋白酶发酵(鱼露A),另一组用外源蛋白酶和曲霉菌YL001复合发酵(鱼露B).2组样品先在35℃下发酵30 d,然后在室温下继续发酵150 d.结果表明,发酵180 d后,鱼露A、B中氨基酸态氮含量分别为7.6和10....     

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。     

酒曲源曲霉的分离及其产蛋白酶条件优化

利用稀释平板法从曲样中分离获得4株曲霉1#、2#、3#和4#,将各菌株分别制米曲后测定酶活力,并通过正交实验及单因素实验确定蛋白酶发酵的培养基和最佳工艺条件。在优化的发酵培养基和发酵条件下(温度、培养时间、培养基起始pH、接种量),产蛋白酶活力分别达到:1#菌11545.7U/g,2#菌8240.4U/g,3#菌9146.0U/g,4#菌4527.7U/g。最后,将活力比较高的三株菌进行Biolog微生物系统鉴定,分别为栖土曲霉(Aspergillus terricola Marchal),寄生曲霉(Aspergillus parasiticus Speare)和杂色曲霉(Aspergillus versicolor)。     

曲霉羧肽酶的分离纯化研究

采用 (NH4) 2 SO4、酒精、DE52 和SephadexG1 0 0对AspergillusspCP 1发酵液进行分离纯化 ,并检测在分离纯化过程中酶的回收率和比活力 ,最后用SDS PAGE电泳检验酶的纯度。确定 (NH4) 2 SO4饱和浓度在 60 %～ 90 %之间 ,酒精浓度为 3 5 %～ 70 %之间用于粗酶液的提纯 ;电泳得到一个清晰的条带。它们的分子质量约为 15 942u。     

高蛋白酶酶活酱油发酵用米曲霉的复合诱变选育

以市售曲精分离菌株米曲霉CS3.03为出发菌株,经UV—DES复合诱变处理,采用酪素平板法及琼脂块法进行初筛,结合Folin—酚法复筛结果对比其初筛效果,低盐固态发酵试验测试高蛋白酶酶活突变株的发酵性能,传代试验测试其遗传稳定性。结果表明:琼脂块法的初筛效率明显高于酪素平板法;对初筛菌株进行制曲复筛,获得5株有生产潜力的高蛋白酶酶活突变株,酱油发酵试验结果证明突变株氨态氮含量、全氮利用率均高于出发菌,其中突变株P7、Y5发酵制得酱油氨态氮含量分别提高14.56%,11.26%,全氮利用率分别提高3.83%,4.09%;遗传稳定性测试证明P7具有良好工业应用价值且遗传稳定性好,其蛋白酶酶活平均为4 994.18U/g干基,比出发菌株提高173.92%。     

黑曲霉降酸菌株F1降解酒石酸关键酶的分离纯化

为得到黑曲霉菌株F1中具有降酸作用的蛋白,将黑曲霉菌株F1经酒石酸诱导发酵得到菌丝体,用液氮研磨破碎细胞壁,超声波辅助提取得到粗酶液,硫酸鱼精蛋白去除核酸,热变除杂蛋白,聚乙二醇浓缩,透析脱盐,DEAE-纤维素52阴离子交换层析,Sephadex G-75葡聚糖凝胶柱分子筛层析,最终得到该蛋白的单一组分,其纯化倍数为14.32,并确定该酶为脱氢酶类。     

Partial Purification and Characterization of Sulfhydryl Oxidase from Aspergillus niger

ABSTRACT: Sulfhydryl oxidase (SOX) was purified after extraction and the contaminating catalase activity was completely eliminated in the last chromatography step. A yield of 25% was obtained with a purification factor higher than 300. The isoelectric point was 3.7 and the molecular weight 110 kDa. SOX exhibited an optimal activity at pH 5.6 and its efficiency (V_mapp/K_mapp) increased from pH 4.5 to 6.5. At pH 5.6, the K_mappvalues were 0.5, 2.5, 10.5, 110, and 450 mM for GSH, cysteine, g-glu-cys, dithiothreitol, and homocysteine, respectively, and the V_mappvalues represented 2, 34, 24, and 44% of the V_maPPvalue found for GSH, respectively. Cys-gly was not oxidized by SOX. In the presence of GSH, SOX is able to catalyze the oxidation of cysteine and cys-gly at a significant rate.     

新疆酸马奶中乳酸菌的乙醇耐受性及蛋白酶性质研究

通过对新疆酸马奶中乳酸菌以传统方式分离鉴定后,筛选得到不同种属菌株,并进一步对其乙醇耐受性及蛋白酶活性差别进行研究。结果表明:在分离的21株菌株中,其不同种属的5株菌株在不同浓度乙醇胁迫12 h后,在耐胁迫能力上,由高到低分别为发酵乳杆菌(Lactobacillus fermentum)、戊糖片球菌(Pediococcus pentosaceus)、植物乳杆菌(Lactobacillus plantarum)、屎肠球菌(Enterococcus faecium)、乳酸片球菌(Pediococcus acidilactici),其中最高能够耐受浓度为13%的乙醇胁迫。对蛋白酶水解能力的测定结果表明,菌株均能产生蛋白酶,其中乳酸片球菌(Pediococcus acidilactici)的水解圈直径最大,为13.1 mm,其菌株自溶度为36.4%。菌株的乙醇耐受性与其蛋白酶水解能力间无显著关联性。