One-dimensional crosslinked enzyme aggregates in SBA-15: Superior catalytic behavior to conventional enzyme immobilization

α-Chymotrypsin (CT) and lipase (LP) were immobilized in SBA-15 mesoporous silica by crosslinking adsorbed enzymes. This simple approach resulted in one-dimensional crosslinked enzyme aggregates (CLEAs) in the linear pore channels of SBA-15, which was very effective in preventing the enzyme leaching and consequently improving the enzyme stability. Both CLEAs of CT and LP showed negligible activity decrease under harsh shaking condition for one week while the conventional approaches including adsorption and covalent attachment resulted in more than 50–90% enzyme inactivation under the same condition. This effective stabilization results from the bent pore structure of SBA-15 with a high aspect ratio, which prevents the leaching of one-dimensional CLEAs and thereby achieves the higher enzyme loading capacity. Along with the higher specific activity than that of adsorbed enzymes, this CLEA approach is much simpler than that of covalent attachment by obviating the tedious processes for silica functionalization and enzyme attachment.     

Heavy-metal removal from aqueous solution by fungus Mucor rouxii

Biosorption of lead, cadmium, nickel and zinc by live and dead Mucor rouxii biomass treated with NaOH was studied over a range of pH. In the case of dead biomass, low pH resulted in a decrease in the biosorption capacity. At pH 3.0 or less, the inhibition of biosorption of metal ions took place. At pH 4.0 or higher, the biosorption of metal ions increased sharply. Ho's pseudo-second-order model described the biosorption kinetics better than the Lagergren model. Live biomass had high biosorption capacity, i.e. 35.69, 11.09, 8.46 and 7.75 mg/g at pH 5.0 for Pb(2+), Ni(2+), Cd(2+) and Zn(2+), respectively. The dead biomass adsorbed metal ions in the order of Pb(2+), Zn(2+), Cd(2+) and Ni(2+), with the biosorption capability of 25.22, 16.62, 8.36 and 6.34 mg/g at pH 5.0, respectively. At pH 6.0, the capacity of the dead biomass increased to 53.75, 53.85, 20.31 and 20.49 mg/g, respectively. For bi- or multi-metal ion adsorption, biosorption capacity of individual metal ion was reduced in the presence of other metal ions, but the total biosorption capacity increased, indicating the capability of M. rouxii biomass in adsorbing multi-metal ions. In addition, M. rouxii biomasses cultured with different media exhibited the same level of capacity to bind metal ions. Metal ions adsorbed by the biomass could be eluted effectively with HNO(3), while distilled water demonstrated negligible metal elution capability. Regeneration of the biomass with NaOH regained or enhanced the biosorption capacity even after five cycles of adsorption-elution-regeneration.     

双霉菌混合发酵腐乳前发酵条件优化及酶活研究

八公山腐乳前发酵工艺对其总体风味具有重要影响,在总状毛霉和米根霉单独发酵腐乳的基础上,探索总状毛霉(Mucorracemosus)和米根霉(Rhizopusoryzae)混合发酵腐乳的发酵工艺。在单因素的基础上,以蛋白酶活力和糖化酶活力为评价指标,选取发酵时间、发酵温度和混合比例为影响因子,采用响应面法优化总状毛霉和米根霉混合发酵腐乳的前发酵条件,比较最优条件下混合发酵和总状毛霉与米根霉单独发酵的前发酵情况。研究结果表明总状毛霉和米根霉混合发酵的前发酵条件为:发酵时间56 h、发酵温度28℃、混合比例(总状毛霉和米根霉)1∶1,此时分泌产蛋白酶活力50.51 U/m L,糖化酶活力为15.15 U/m L。通过对比双菌和单菌发酵腐乳前发酵情况可知:总状毛霉和米根霉混合发酵较总状毛霉更能耐高温,且在菌丝的生长和酶系的分泌积累较单独发酵都更有优势,为后期发酵的一系列生化反应奠定了良好的基础,有利于改善腐乳的发酵周期,提高腐乳的品质。      

特香型大曲表面毛霉对大曲与基酒质量的影响

为控制特香型大曲表面毛霉的过度生长,在不改变传统特香型大曲制作工艺的前提下,从缩短低温培菌期和减少毛霉初始接种量两个方面对毛霉生长进行控制,从而探究毛霉生长对大曲质量和基酒质量的影响。结果表明,与对照组相比,试验组（处理一、处理二）大曲表面的毛霉生长量（19.8%、24.4%）明显减少;大曲的糖化力（822 mg/（g·h）、858 mg/（g·h））和液化力（4.00 g/（g·h）、4.80 g/（g·h））提高;基酒产量（571.7 kg、632.6 kg）无显著性差异（P＞0.05）;处理一的总酯、乙酸乙酯及丙酸乙酯含量显著升高（P＜0.05）,总酸含量及其他7个主体指标差异均不显著（P＞0.05）,基酒的综合感官评价更优。综上,控制冬春季大曲表面毛霉的生长,有助于改善大曲的外观,提高大曲和基酒的质量。     

毛霉蛋白酶在腐乳成熟中的作用

采用SDS—PAGE与ESI—MS相结合的方法研究了毛霉蛋白酶和Alcalase蛋白酶对SPI的酶促降解作用,研究结果表明,Alcalase是一种典型的内切型蛋白酶,它能在1h内催化大豆蛋白降解为分子量10000D以下的肽段,但降解产物容易相互聚合而沉淀,毛霉蛋白酶则具有较强的肽酶活性,能够以大豆蛋白或多肽为底物酶促降解生成多量的水溶性肽,为后期嗜盐菌的生长提供了可供利用的速效氮素营养,毛霉蛋白酶还具有消除苦味、形成鲜味物质的作用,与腐乳风味形成有关。     

不同光照模式对卷枝毛霉类胡萝卜素合成及相关基因表达的影响

为探究不同光照模式对卷枝毛霉（Mucor circinelloides）类胡萝卜素合成的影响,在持续恒定光照、光暗交替和逐步提高光强3种不同蓝光照射条件下培养卷枝毛霉菌株CBS 277.49,观察不同培养时间（0、6、12、18 h和24 h）的菌落形态,测定生物量和类胡萝卜素含量,并通过实时荧光定量聚合酶链式反应（quantitative real-time polymerase chain reaction,qRT-PCR）确定蓝光感受器编码基因mcwc-1c以及类胡萝卜素合成结构基因carB和carRP的表达情况,以黑暗处理作为对照组。结果表明：3种光照模式均能促进卷枝毛霉CBS 277.49的生长,并且提高其类胡萝卜素产量。但持续恒定蓝光照射使卷枝毛霉在12 h后产生“光适应”,蓝光感受器编码基因mcwc-1c以及类胡萝卜素合成结构基因carB和carRP表达量均明显下降,类胡萝卜素合成停滞。光暗交替和逐步提高光强两种光照模式则因为在培养过程中光强随时间不断变化,避免了\"光适应\"的产生,因此,能够保持结构基因以较高水平表达,使类胡萝卜素持续积累。在该研究考察光强和培养时间范围内...     

碎囊毛霉产β-1,3-1,4-葡聚糖酶的分离纯化及其酶学性质

基于碎囊毛霉M-28固态发酵方式,采用单因素试验对其所产β-1,3-1,4-葡聚糖酶进行提取纯化条件优化,并开展部分酶学性质研究。结果表明:用pH 5.5的乙酸-乙酸钠缓冲溶液在200 r/min浸提固态发酵基质30 min,所得粗酶液经饱和度80%硫酸铵盐析、24 h透析浓缩、Sephadex G-100柱层析分离后,经紫外分光光度计检测,得到2个蛋白峰,酶活力测定发现有一个峰具有酶活性,收集该酶活组分并采用聚乙二醇高度吸附浓缩,再用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法进行纯度检验,纯化酶蛋白呈单一谱带,分子质量约为17.2 kD,酶比活力达到225.02 U/mg,纯度较粗酶液提高了2.48倍。酶的最适反应温度为55℃、在40~50℃时相对稳定,其最适pH值为5.5、酶活力在pH 4.5~5.5条件下相对稳定。Fe3+和Al3+对该酶具有明显抑制作用,Fe2+对其有激活作用,其他金属离子影响不大。     

毛霉蛋白酶特性研究及在大豆肽生产中的应用

通过对毛霉蛋白酶活力的测定研究了毛霉蛋白酶的特性。研究表明,毛霉在培养的第5d～6d为旺盛期,毛霉蛋白酶的最适反应pH值为4.0～6.0,且在pH4.0～6.0时稳定性较好;毛霉蛋白酶的最适反应温度为45℃～55℃,50℃时酶活力最大,且在40℃以下热稳定性较好。1.0%NaCl、1.0%CaCl2、1.0%MnSO4和1.0%MgSO4对蛋白酶有激活作用,1.0%KCl、1.0%ZnSO4和1.0%CuSO4则对蛋白酶有不同程度的抑制作用。用毛霉所产的蛋白酶对大豆分离蛋白进行水解所得的大豆肽具有较好的质量品质。     

重组微小毛霉凝乳酶的分离与纯化

用硫酸铵分级沉淀分离提取重组微小毛霉凝乳酶(GS115/pPICZαA-Mucor pusillus rennet),经过Sephadex G-75分子筛和DEAE-52离子交换2次柱层析纯化,酶的比活力由2078U/mg提高为10526U/mg,纯化倍数超过5倍,纯化的重组凝乳酶经SDS-PAGE电泳分析纯化的重组凝乳酶分子质量约为46kDa。