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。      

香樟精油抑制灰绿曲霉的活性与机理研究

香樟精油(Cinnamomum camphora essential oil)作为一种绿色、健康且抑菌活性强的天然植物精油,在食品行业中有广泛应用.本研究分析了香樟精油对灰绿曲霉(Aspergillus glaucus)的抑菌效果及其抑菌机理.通过最小抑菌浓度、最小杀菌浓度,以及抑制灰绿曲霉菌丝生长和孢子萌发等实验评价...     

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)。     

Purification and characterisation of an acid protease from the Aspergillus hennebergii HX08 and its potential in traditional fermentation

A protease AP3 from Aspergillus hennebergii HX08 was purified by ammonium‐sulphate precipitation, followed by anion‐exchange chromatography and gel filtration. The molecular weight of acid protease AP3 was 33 kDa (SDS‐PAGE and MALDI‐TOF‐MS). The protease AP3 was identified as an acid protease with MALDI‐TOF/TOF tandem MS. Its optimal temperature and pH were 60°C and 4.0, respectively. Its K _max and V _max were 57.92 mg/mL and 32.57 U/mL, respectively. The enzyme was active over a broad pH and temperature range (pH 3.0–5.0 and 30–60°C), and exhibited high activity and stability in 2–12% (v /v) ethanol solvent. Subsequent studies suggest that the enzyme presents a relatively high substrate affinity to wheat protein (98% of total activity). Its application to solid‐state fermentation of wheat flour with Saccharomyces cerevisiae could increase the hydrolysis degree of wheat protein (28.26%) and amino acid nitrogen concentration of fermented grains (34.21%). Additionally, enhanced S. cerevisiae biomass (37.09%) and alcohol concentration (38.29%) were also observed during the process. Volatile compounds analysis of fermented grains by headspace solid‐phase micro‐extraction and GC‐MS revealed more flavour compounds. These results suggest its potential in food and alcohol industries. Copyright © 2017 The Institute of Brewing & Distilling     

两种生产用米曲霉的形状及蛋白酶性质研究

研究对比了两株米曲霉的形态特征以及成曲的蛋白酶酶学性质。结果显示,两株米曲霉的形态差异明显:米曲霉1号菌落较大、发芽和产孢子较快,种曲及成曲颜色偏黄,菌丝较短,孢子较多;而米曲霉2号菌落较小,孢子发芽时间及产孢子时间比1号晚,成曲孢子较1号少,但菌丝长且粗壮,种曲颜色偏绿,成曲颜色偏白,米曲霉2号的产酶能力强。蛋白酶性质的研究结果显示,两株米曲霉成曲的蛋白酶性质有差异,1号和2号的最适反应温度分别为45℃和50℃,最适反应pH分别为6.0和9.0。此外,两株米曲霉的成曲蛋白酶的耐盐性相当,随着盐浓度的增大,酶活力均逐渐降低,在20%氯化钠条件下,酶活力均降至无盐条件下的20%~30%。     

米曲霉Y6产中性蛋白酶和内切葡聚糖酶的研究

以提高中性蛋白酶和内切葡聚糖酶酶活为目的,优化了米曲霉Y6的制曲工艺。在单因素的试验基础上,采用正交试验优化的米曲霉Y6中性蛋白酶的最佳制曲工艺参数为温度30℃,接种量1.0×107个孢子/g干基,培养时间42h,含水量80%,其酶活达到3637U/g;内切葡聚糖酶的最佳制曲工艺参数为温度为30℃,接种量0.25×107个孢子/g干基,培养时间48h,含水量100%,其酶活达到810U/g。     

Release of short and proline-rich antihypertensive peptides from casein hydrolysate with an Aspergillus oryzae protease

Angiotensin-I converting enzyme inhibitory activities were measured after hydrolysis of casein by 9 different commercially available proteolytic enzymes. Among these enzymes, a protease isolated from Aspergillus oryzae showed the highest angiotensin-I converting enzyme inhibitory activity per peptide. The A. oryzae peptide also showed the highest antihypertensive effect in spontaneously hypertensive rats when the systolic blood pressure was measured 5 h after oral administration of 32 mg/kg of various enzymatic hydrolysates. Significant antihypertensive effects were observed with dosages of 9.6, 32, and 96 mg of the A. oryzae peptide/kg of body weight (BW), and the effects were dependent on these peptide dosages.Analysis of peptide length showed the A. oryzae hydrolysate was the shortest of all tested casein hydrolysates; the peptide mixture had an average value of 1.4 amino acids (AA) in the sequence. To further characterize the A. oryzae hydrolysate, we analyzed the AA sequence of the whole peptide mixture. Various AA were detected at the first AA position, however, an increased number of Pro residues were observed at the second and third position of the A. oryzae hydrolysate. No strong signals were detected after the fourth AA position of the A. oryzae hydrolysate. These results suggest that the casein hydrolysate of A. oryzae, which expressed potent antihypertensive effects in spontaneously hypertensive rats, mainly contain short peptides of X-Pro and X-Pro-Pro sequences.     

Xi-3米曲霉菌株的保藏与复壮

介绍了Xi-3米曲霉菌株的保藏与复壮的具体做法。采用麸皮保藏法可以使Xi-3米曲霉保藏18年之久且菌种优良性状不变；行之有效的复壮措施，提高了保藏菌株的优良性状和生产性能，为种质资源的可持续发展作出了贡献。     

海鞘残渣对米曲霉沪酿3.042生长和酶活力的影响

研究了提取脂肪酸后的柄海鞘残渣对米曲霉沪酿3．042生长和蛋白酶活力的影响。结果表明：柄海鞘残渣代替一部分麸皮作为米曲霉沪酿3．042同体培养基中的主要成分，增加了菌体的生长量和蛋白酶活力，但当海鞘残渣浓度过高时，对米曲霉沪酿3．042的生长和胞外蛋白酶的分泌都有不利的影响。     

酿造激活剂对不同曲霉菌株蛋白酶组分及活性的影响

以活性电泳比较了不同来源酱油生产菌株的蛋白酶组分,研究了激活对蛋白酶组分表达及活性的影响.结果表明,不同来源菌株蛋白酶组分数量各不相同,其中米曲霉GM蛋白酶组分数量最多,其他菌株的蛋白酶组分均少于米曲霉GM.激活后多数蛋白酶组分的活性都有提高,其中对GM的组分4、AS3.324的组分4、336-2的组分1、3042-HL的组分4、3042-ZH的组分4和3.042KG的组分7激活作用明显.激活剂还能诱导336-2新的蛋白酶组分表达.     

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     

金鲳鱼内脏酸性蛋白酶的分离纯化及酶学性质研究

以金鲳鱼内脏为原料,依次采用p H7.0的磷酸钠缓冲液浸提,0⁵5%硫酸铵沉淀,Sephadex G-100和Sephadex G-50凝胶过滤法得到纯化的酸性蛋白酶,并研究了其酶学性质。结果表明,纯化酶的分子量为18.5 ku,最适p H为4.0,最适温度为35℃,具有较好的酸稳定性和耐盐能力;以血红蛋白为底物时,该蛋白酶的米氏常数Km为10.13 g/L;胃蛋白酶抑制剂（pepstatin A）对该酶活性有抑制作用,而乙二胺四乙酸（EDTA）、反-环氧丁二酰基-L-亮氨酰胺基（4-胍基）丁烷（E-64）和苯甲基磺酰氟（PMSF）对此酶活性基本无影响,据此推断该蛋白酶是一种天冬氨酸蛋白酶;此酶对罗非鱼鱼皮明胶的酶解能力强于猪胃蛋白酶,与木瓜蛋白酶和碱性蛋白酶的能力相近。      

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....     

曲霉M3蛋白酶酶解特性研究

用曲霉M3蛋白酶水解大豆分离蛋白,对最佳预处理条件和酶解工艺条件进行了研究。结果表明:其最佳预处理条件为:80℃水浴处理10min;最佳酶解条件为:底物浓度5%(W/V)、加酶量4200u/g蛋白、温度50℃、pH7.5。在此条件下水解,测定不同水解时间的水解度,并品尝苦味。研究发现:曲霉M3蛋白酶水解液的水解度随着水解时间的延长而增大,且在任何水解时间水解液都没有苦味。     

双水相萃取分离米曲霉中性蛋白酶

采用聚乙二醇(PEG)/盐双水相体系萃取分离米曲霉中性蛋白酶。探讨了不同成相盐及其浓度、成相聚合物PEG及其浓度、pH和NaCl浓度对中性蛋白酶萃取的影响。结果表明,适宜的双水相萃取体系为20%PEG1000/25%(NH4)2SO4(m/m)、pH8.0、不添加NaCl,此条件下中性蛋白酶上相酶活回收率和纯化倍数分别为99.88%和2.32。