黑曲霉单宁酶产生菌的筛选及处理滇橄榄汁的研究

利用实验室已有的17株黑曲霉单宁酶活性菌株为起始菌。经活化分离初筛,液体摇瓶复筛,选出具有高单宁酶活性的No．12菌株;对该菌株进行液体培养,提取单宁酶并固定化;以固定化单宁酶处理滇橄榄汁。结果表明,处理后的滇橄榄汁,固体悬浮物下降90％,说明本工艺具有潜在的工业开发价值。     

利用固定化黑曲霉单宁酶制备没食子酸的研究

用海藻酸钙载体包埋单宁酶,制备出转化五倍子单宁成没食子酸能力较好的固定化酶。研究了固定化条件和固定化单宁酶的部分性质,结果表明：最佳固定化条件为海藻酸钠90mg包埋单宁酶546u(3mL,182u/Ml),在1%～2%CaCl₂中作硬化处理;固定化单宁酶的最适温度为45℃,在10～50℃范围内稳定;其最适Ph值为6.5,在Ph5～7之间基本稳定;在此基础上,进行了没食子酸实验室克量级酶法制备实验,3次实验没食子酸产品的平均产率达到61%。和目前所用工业生产没食子酸的硫酸水解法相当,具有潜在的工业开发价值。     

黑曲霉单宁酶基因Tan2克隆与表达

单宁酶(Tannase,EC 3.1.1.20)能水解单宁中的酯键和羧酚酸键,产生没食子酸以及对应醇,在食品、饮料、饲料、制药、医药、化妆品等各类工业中应用广泛,也在普洱茶发酵中具有重要作用.从普洱茶发酵中分离的黑曲霉菌株PU001中克隆得到单宁酶基因Tan2,并连接到表达载体pCold-Ⅰ构建BL21-pCdd Ⅰ原...     

黑曲霉单宁酶高活性菌株的诱变选育*

以黑曲霉（Ａｓｐｅｒｇｉｌｌｕｓ ｎｈｉｇｅｒ）Ｎｏ．１３为出发菌株,经紫外线诱变处理,获得一株制备原生质体的起始菌,该菌株单宁酶活性比Ｎｏ．１３提高５５％,并对其制备原生质体的条件进行了研究,在优化方案基础上,紫外诱变原生质体,诱变株经筛选,最后得到一株具有稳定遗传性的单宁酶高活性菌株,在摇瓶培养基中进行生物转化实验,连续传代１０次,结果显示发酵液中没食子酸浓度始 维持在２２．８－２３．９ｍｇ／     

单宁酶基因在黑曲霉ST31中的克隆与表达

利用PCR扩增得到米曲霉(Aspergillusoryzae)单宁酶(tannase)基因的编码序列,经DNA测序证实单宁酶基因已成功克隆,然后将其连接到黑曲霉的表达载体ANED2-SP2上构建单宁酶基因表达载体。将构建好的单宁酶基因表达载体通过原生质转化法导入黑曲霉菌株ST31中进行表达研究。结果表明重组菌株的单宁酶活力最高为104.02U/ml发酵液,比原始出发菌株米曲霉提高2~3倍。研究构建了黑曲霉的高效转化体系,提高了黑曲霉表达系统的应用水平,为其它新酶的研究提供有价值的参考。     

黑曲霉固态发酵生产单宁酶的条件优化

研究采用响应面法优化黑曲霉固态发酵生产单宁酶的培养条件。应用Plackett—Burman试验筛选出重要影响因子：五倍子粉含量、（NH4）2SO4浓度以及接种孢子量,最陡爬坡试验逼近最大响应区域。应用Box．Behnken响应面试验对重要影响因子进一步优化。得到最佳培养条件：每250mL三角瓶中装入1．0g五倍子粉、4．4g稻壳和0．5g麸皮、液固比（mL／g）2：1且营养盐溶液组成为（NH4）2s0421g/L、MgSO4·7H2O1g／L、NaCl1g／L,培养基pH自然,接种5．7×10^7个孢子后在30℃温度下培养4d。在此条件下,单宁酶产量从40U／g提高到114U／g,3次重复验证性试验平均值为115U／g,验证了模型的可靠性。     

黑曲霉B0201利用五倍子固体发酵产单宁酶的条件研究

通过比较常规灭菌发酵和生料发酵, 研究黑曲霉B0201利用五倍子固体发酵产单宁酶的条件。结果表明, 在生料发酵过程中, 采用20%五倍子并且以(NH4)2SO4为氮源制备单宁酶的最佳条件为: 液固比=1.6:1、温度30°C、初始pH 6.0。在该条件下通过96 h的培养, 单宁酶的活力可达51.2 U/gds, 是常规灭菌发酵的3.6倍。以上结果显示, 生料发酵生产单宁酶是一种高效可行的方法。     

微生物单宁酶研究现状

主要介绍了微生物单宁酶的来源、生产、分离纯化及酶的生物学性质的研究,同时概括了有关单宁酶的分子生物学研究进展,为微生物单宁酶广泛的应用提供科学依据。     

利用黑曲霉单宁酶酶法制取没食子酸的研究

利用已有的 10株高单宁酶活性的菌株为起始菌 ,经活化分离选择 ,借助Ⅱ级发酵培养程序、生物转化、结合TLC分析进行筛选实验。最后选出具有高单宁酶活性的 1号和 5 0号菌株 ,开展了没食子酸 (GA)克量级生物转化法制备实验 ,结果表明 ,本酶法工艺是可行的 ,在发酵液中GA的浓度分别达到2 0 .6mg/ml和 2 1 3mg/ml,产品产率 (以从五倍子提取的单宁酸计 )达到 41 2 %和 42 6 % ,具有潜在的工业开发价值     

棉花植株中的单宁测定方法研究

通过 3种方法测定棉花组织中单宁含量比较表明,Folin酚还原法测定的 4个品种不同组织和不同生育期顶叶的含量显著高于正丁醇盐酸法 (即花色素反应,专门用于缩合单宁的测定)近 2倍,说明这种方法测定出的是相对总酚含量,用于表达棉花缩合单宁的含量是不合适的,而香草醛法测定结果与正丁醇盐酸法差异不显著,可用于棉花组织单宁含量的测定.在棉花各个组织中,花萼、铃皮和叶片缩合单宁含量较高,陆地棉中一般达 5%～10%;花瓣、花柱子房和铃心中含量较低 (2%左右).顶端嫩叶缩合单宁含量从苗期 (1%以下)起不断增加,至吐絮期达最高 (10%左右),表明缩合单宁含量与植物组织成熟衰老和木质化程度密切相关.     

Isolation,purification and properties of tannase from Aspergillus niger van Tieghem

Tannase (tannin acyl hydrolase E.C. 3.1.1.20) has been isolated from Aspergillus niger van Tieghem and purified 29-fold. The enzyme had a temperature optimum of 60^°C, pH optimum of 6.0 with a second peak at pH 4.5, K_m of 0.20mM and V_max of 5.0mol min^–1 mg^–1protein.     

Effects of temperature, pH and additives on the activity of tannase produced by a co-culture of Rhizopus oryzae and Aspergillus foetidus

Summary Tannase was produced by modified solid-state fermentation (MSSF) of tannin rich substrates by a co-culture of the two filamentous fungi, Rhizopus oryzae and Aspergillus foetidus. The enzyme thus produced was then partially purified by solvent precipitation and DEAE-Sephadex column chromatography. A study on the effects of temperature and pH was made on the activity of tannase so purified. The optimum values of incubation time, reaction temperature and pH for tannase activity were 5 min, 40 °C and 5.0 respectively. The half-life period thermal stability and kinetic constants (K _m 0.21 mM, V _max 4.9×10⁻² M min^-1 at 40 °C) of this tannase were determined and the effects of different metal ions, surfactants, chelators, denaturants and inhibitors on the enzyme activity were also studied.     

黑曲霉纤维素酶的纯化及酶学性质研究

黑曲霉(Aspergillusniger)固态发酵后粗酶液经硫酸铵盐析,2次SephadexG-200柱层析后可提纯8倍左右.CMC酶最适作用温度为60℃,最适作用pH为3.5,30℃～70℃区间酶活力较稳定,在pH3.0～5.0范围内,50℃保温30min能保持80%的酶活力.CMC酶的Km、Vmax值分别为7.69%CMCg/ml、0.33mg/ml·     

多元混菌发酵对纤维素酶活性的影响

研究了两种曲霉(UF2和UA8)二元混菌体系和两种曲霉与1种酵母菌组成的三元混菌体系混合发酵对纤维素酶系三种酶组分活性的影响。结果表明：两种霉菌按一定比例接种进行混合发酵时三种纤维素酶组分的活性较单菌发酵大幅度提高,滤纸酶(FPA)、微晶纤维素酶(AVI)和羧甲基纤维素酶(CMC)活性分别较UA8单菌发酵提高2.2％～51.1％、20．7％～332．6％和29．4％～299．6％;向由两种霉菌组成的二元混菌发酵体系中接入酵母菌可显著降低3种纤维素酶组分的活性;三菌混合发酵能使纤维素酶3组分的产酶高峰出现时间较双菌混合发酵滞后约24h,但三菌与双菌混合发酵3种纤维素酶组分的酶活峰值无明显差异;双菌混合发酵有利于缩短纤维素酶生产发酵周期。     

黑曲霉D2-26高温乳糖酶的酶学性质研究

为研究黑曲霉来源的高温乳糖酶的酶学性质,对黑曲霉D2-26发酵液进行分离纯化使酶蛋白达到电泳纯,并对其进行酶学性质研究.结果表明:乳糖酶的最适温度70℃,在30℃～60℃有较高的耐受性;最适pH为2.5,pH稳定范围在2.0～9.0;Mn2 对乳糖酶活性有显著的激活作用,Hg2 、Pb2 对酶活有较强的抑制作用;SDS严重抑制了酶活性;以ONPG为底物采用双倒数做图法测得Vmax为97 nmol/min,Km为8.77 mmol/L;单亚基蛋白的分子量为116.978 kD;糖基化程度为11.3%.     

京尼平的微生物转化及其交联特性的测定分析

从土壤中富集筛选获得一株产β-葡萄糖苷酶的菌株,经菌落的形态和18S rDNA鉴定确定为黑曲霉。将筛选出的黑曲霉菌株接种于发酵培养基,利用含有京尼平苷的栀子粉作为底物发酵,通过对发酵条件优化,得到在装液量50/250 mL,栀子粉浓度为10%,转速为180 r/min,发酵时间为96 h时,京尼平的微生物转化率达到最大22%。这种微生物转化法简化了京尼平的生产工艺,大大降低了生产成本。利用微生物转化获得的京尼平交联胶原蛋白材料,研究表明其具有较好的交联特性,是一种在食品、医药等领域都具有应用前景的生物交联剂。     

Immobilization of cross-linked tannase enzyme on multiwalled carbon nanotubes and its catalytic behavior

Immobilization of cross-linked tannase on pristine multiwalled carbon nanotubes (MWCNT) was successfully performed. Cross-linking of tannase molecules was made through glutaraldehyde. The immobilized tannase exhibited significantly improved pH, thermal, and recycling stability. The optimal pH for both free and immobilized tannase was observed at pH 5.0 with optimal operating temperature at 30°C. Moreover, immobilized enzyme retained greater biocatalytic activities upon 10 repeated uses compared to free enzyme in solution. Immobilization of tannase was accomplished by strong hydrophobic interaction most likely between hydrophobic amino acid moieties of the glutaraldehyde-cross-linked tannase to the MWCNT.     

Modelling the effect of temperature and water activity on growth of Aspergillus niger strains and applications for food spoilage moulds

AIMS: To develop a model for the combined effect of water activity (a(w)) and temperature on growth of strains of Aspergillus niger, and comparison with data on food spoilage moulds in the literature. METHODS AND RESULTS: An extended combined model describing the growth of two strains of A. niger, as a function of temperature (25-30 degrees C) and a(w) (0.90-0.99) was developed. The growth rate (micro) was expressed as the increase in colony radial growth per unit of time. This extends the previous square root model showing the relationship between temperature and bacterial growth rate developed by Ratkowsky et al. (1983) and the parabolic relationship between the logarithm of the growth rate and a(w) developed by Gibson et al. (1994). A good correlation between the experimental data and the model predictions was obtained, with regression coefficients (r(2)) > 0.99. In addition, the use of this model allowed predictions of the cardinal a(w) levels: a(w(min)), and a(w(opt)). The estimation of the minimum a(w) levels (a(w(min))) was in accordance with data in the literature for similar and a range of other Aspergillus and related species, regardless of the solutes used for a(w) modification. The estimation of the optimal a(w) (a(w(opt))) and the optimal growth rate (micro(opt)) were in good agreement with the experimental results and data from the literature. CONCLUSIONS: This approach enables accurate prediction of the combined effects of environmental factors on growth of spoilage fungi for rapid prediction of cardinal limits using surface response curves. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach is a rapid method for predicting optimal and marginal conditions for growth of a wide range of spoilage micro-organisms in relation to interacting environmental conditions and will have applications for improving shelf-life of intermediate moisture foods.