基于基因测序鱼露发酵橘青霉YL-1鉴定及安全性评价

目的：为弥补传统方法在检测菌株产毒素上的不足,用基因组方法分析橘青霉YL-1（Penicillium citrinum YL-1,P. citrinum YL-1）物质生物合成途径及关键基因,以此评价P. citrinum YL-1在鱼露发酵生产过程中的安全性。方法：应用ITS基因测序鉴定菌株,利用Illumina平台Hiseq测序技术对菌株YL-1进行基因组survey测序,通过生物信息分析产青霉毒素、黄曲霉毒素及典型丝状真菌毒素的非核糖体多肽合成代谢、聚合酮酶代谢、聚合酮酶-非核糖体多肽联合代谢、萜类化合物代谢和氨基酸相关代谢途径及基因,考察菌株YL-1产真菌毒素能力,判断其是否存在产真菌毒素的潜在危害。结果：ITS鉴定菌株YL-1与P. citrinum同源性99%,survey测序结果表明P. citrinum YL-1全基因组大小为31.92?Mb,GC为46.27%。利用Maker2基因预测技术得到预测基因11?980?个。其中,被KOG注释基因5?417?个,被COG注释基因4?946?个;比对KEGG数据库被注释通路323?条,注释基因3?525?个。代谢分析表明,注释到可能产真菌毒素相关的代谢途径有5?条,仅注释到1?种产黄曲霉毒素代谢途径的同源基因Afld及其他5?种相关基因,但并未注释到其完整代谢途径。结论：ITS基因测序鉴定菌株YL-1为P. citrinum,P. citrinum YL-1基因注释存在1 种产黄曲霉毒素的同源基因及其他5?种相关基因,虽不存在完整代谢链,但P. citrinum YL-1运用于鱼露及相关产品的发酵安全性仍值得进一步考察。     

Purification,characterisation and application of α‐l‐rhamnosidase from Penicillium citrinum MTCC‐8897

An α‐l ‐rhamnosidase secreted by Penicillium citrinum MTCC‐8897 has been purified to homogeneity from the culture filtrate of the fungal strain using ammonium sulphate precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The sodium dodecyl sulphate/polyacrylamide gel electrophoresis analysis of the purified enzyme gave a single protein band corresponding to the molecular mass 51.0 kDa. The native polyacrylamide gel electrophoresis also gave a single protein band confirming the enzyme purity. The K_m and V_max values of the enzyme for p‐nitrophenyl α‐l ‐rhamnopyranoside were 0.36 mm and 22.54 μmole min^?1 mg^?1, respectively, and k_cat value was 17.1 s^?1 giving k_cat/K_m value of 4.75 × 10⁴ m ^?1 s^?1. The pH and temperature optima of the enzyme were 7.0 and 60 °C, respectively. The purified enzyme liberated l ‐rhamnose from naringin, rutin, hesperidin and wine, indicating that it has biotechnological application potential for the preparation of l ‐rhamnose and other pharmaceutically important compounds from natural glycosides containing terminal α‐l ‐rhamnose and also in the enhancement of wine aroma.     

Purification and Characterization of Alkaline Proteinase from Atlantic Menhaden Muscle

Two alkaline proteinases (A and B) were isolated and found to be composed of homogeneous subunits. These proteinases, A and B, were concentrated 62.9-and 986.5-fold compared to the crude muscle extract, with molecular weights of 707,000 and 450,000, respectively. Both are probably serine type proteinases, and optimum caseinolytic activity was shown at pH 8.0 and 55 °C. Both degraded actomyosin under similar conditions. Enzyme A had higher thermal stability than B. The residual activities of A and B in 3.6% NaCl solution were 95% and 85%. These data suggest that these proteinases are involved in the softening of menhaden surimi gels which occurs during heating at 50 to 70 °C.     

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。     

米曲霉PRB-1蛋白酶系和淀粉酶系与高盐稀态酱油理化特性关系的研究

本文采用一株自行分离的米曲霉PRB-1菌株(AP)进行制曲和高盐稀态酱油发酵。结果表明:AP成曲的酸性和中性蛋白酶活力达到398.53 U/g干基和1539.98 U/g干基,较米曲霉3.042(AO)分别提高71.79%和59.93%。AP成曲淀粉酶活力比AO略低。AP成曲含有5个中性蛋白酶组分,2个酸性蛋白酶组分和4个淀粉酶组分。其中,蛋白酶Rf6号和淀粉酶Rf3号活力最大。随着发酵时间的延长,AP和AO酱醪总酸含量呈现上升趋势。p H值由中性持续下降至偏酸性。氨基态氮含量持续增加,还原糖含量均呈现先显著上升后缓慢降低的趋势。发酵55d AP头油的氨基态氮含量为0.86 g/100 m L,原料利用率为81.97%,氨基酸生成率为61.37%,较AO均有较高的优势。AP头油的还原糖含量为4.82 g/100 m L,低于AO。成曲的中性蛋白酶活力和发酵酱油的氨基酸生成率呈正相关(p0.05),对其他指标无显著影响(p0.05)。     

Purification and functional characterisation of an α‐l‐rhamnosidase from Penicillium citrinum MTCC‐3565

An extracellular α‐l ‐rhamnosidase from Penicillium citrinum MTCC‐3565 has purified to homogeneity from its culture filtrate using ethanol precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The purified enzyme gave a single protein band corresponding to molecular mass of 45.0 kDa in SDS‐PAGE analysis showing the purity of the enzyme preparation. The native PAGE analysis showed the monomeric nature of the purified enzyme. Using p‐nitrophenyl α‐l ‐rhamnopyranoside as substrate, K_m and V_max values of the enzyme were 0.30 mm and 27.0 μm min mg^?1, respectively. The k_cat value was 20.1 s giving k_cat/K_m value of 67.0 mm s^?1 for the same substrate. The pH and temperature optima of the enzyme were 8.5 and 50 °C, respectively. The activation energy for the thermal denaturation of the enzyme was 29.9 KJ mol^?1. The α‐l ‐rhamnosidase was able to hydrolyse naringin, rutin and hesperidin and liberated l ‐rhamnose, indicating that the purified enzyme can be used for the preparation of α‐l ‐rhamnose and pharmaceutically important compounds by derhamnosylation of natural glycosides containing terminal α‐l ‐rhamnose. The α‐l ‐rhamnosidase was active at the level of ethanol concentration present in wine, indicating that it can be used for improving wine aroma.     

一株从泡菜中分离的产细菌素乳杆菌的鉴定及细菌素特性研究

从泡菜中分离的329 株乳酸菌中,筛选出一株产细菌素的乳酸菌,编号为SD-22。经生理生化实验鉴定为短乳杆菌(Lactobacillus brevis)。在排除过氧化氢干扰和有机酸抑菌作用后,brevicin SD-22 对革兰氏阳性细菌和革兰氏阴性细菌具有较好的抑制作用,对部分真菌也有抑制作用,但对酵母菌和青霉无抑制作用,具有良好的热稳定性和pH 值稳定性,经胰蛋白酶、胃蛋白酶和蛋白酶K 处理后抑菌活性完全消失,对α- 淀粉酶不敏感。因此初步认为该菌株是一株产广谱细菌素的乳酸菌。     

酱香型大曲中分离到的阿姆斯特丹散囊菌产酶产香特性

为了探究酱香型大曲中霉菌的种类及其功能,采用稀释平板涂布法从遵义地区的6 个企业的酱香型大曲中分离筛选霉菌菌株,结合形态观察和真菌rDNA-ITS基因序列同源性比对进行分析。在其中5 个企业的大曲样品中均能筛选分离得到一株散囊菌,归类并命名为阿姆斯特丹散囊菌（Eurotium cristatus）。以茅台大曲筛选的阿姆斯特丹散囊菌（FBKL3.0120）为研究对象,该菌株产糖化型淀粉酶活力最高,达到（3 100.16±109.43） U/g,酸性蛋白酶活力为（225.69±2.69） U/g、果胶酶活力为（435.88±68.49） U/g、脂肪酶与纤维素酶活力分别为（13.56±1.73） U/g和（4.14±0.16） U/g。采用固相微萃取-气质联用技术对固态发酵的挥发性香味物质进行结果分析,该菌固态发酵具有很强花香和果蔬香,挥发性香味物质以高级醇、酮和呋喃类为主,其中L-芳樟醇、1-辛烯-3-醇、3-辛酮、2-戊基呋喃的相对含量较高,分别为挥发性物质的16.37%、41.23%、6.95%、6.75%。分离得到的这株阿姆斯特丹散囊菌（FBKL3.0120）是一株产多种酶并同时产多种香气成分的霉菌,是酱香大曲微生物组成中的重要霉菌之一。