南极磷虾体内胰蛋白酶的纯化及性质研究

以南极磷虾(Euphausia superb)为研究对象,通过硫酸铵分级沉淀、Phenyl-Sepharose疏水层析、DEAE-Sepharose FF离子交换层析等方法,从南极磷虾体内分离纯化出胰蛋白酶。其纯化倍数为5.44倍,比活力为38.3 U/mg,得率为26%。SDS-PAGE电泳结果显示,该酶的分子质量为28 ku。蛋白酶最适温度为37℃、最适pH为7.5,Mg2+、Ca2+、Mn2+对南极磷虾蛋白酶具有激活性,Zn2+、Cu2+、Fe3+具有酶活抑制性,其中Cu2+的抑制性最强。酶的动力学实验结果表明,以BApNA为底物测得Km为0.073 mmol/L,Vmax为1.44×10-2mmol/L·s,kcat为0.6S-1,kcat/Km为8.22×103,PMSF作为蛋白酶抑制剂,对南极磷虾蛋白酶作用机制为不可逆抑制。     

普鲁兰短梗霉N2-3菌株碱性蛋白酶的纯化及性质研究

[目的]对普鲁兰短梗霉N2-3菌株的碱性蛋白酶进行纯化,并对其性质进行研究。[方法]利用层析柱Sephadex G-75与阴离子交换柱DEAE Sepharose Fast Flow纯化菌株N2-3的胞外碱性蛋白酶,并对纯化的酶进行SDS-PAGE凝胶电泳检测,同时对纯化蛋白酶的最适反应温度、温度稳定性、最适反应pH、pH稳定性进行测定,研究金属离子及各种化合物对蛋白酶活性的影响。[结果]试验中N2-3菌株的碱性蛋白酶纯化倍数和回收率分别为2.34和25.9%;SDS-PAGE凝胶电泳显示该酶的分子量约为33 kDa;该酶的最适反应温度和pH分别为52℃和9.0;Mn2+、Mg2+、Na+离子浓度在5 mmol/L时对纯化蛋白酶的酶活有激活作用,Zn2+、Ca2+、K+、Li+对酶活的影响不大,而当存在Fe2+、Fe3+、Cu2+、Co2+、Ag+、Hg2+离子时,蛋白酶活明显降低;苯甲基硫酰氟(PMSF)强烈抑制了该蛋白酶的活性,而乙二胺四乙酸(EDTA)与碘乙酸微弱抑制了该蛋白酶的活性。[结论]为普鲁兰短梗霉N2-3菌株碱性蛋白酶的工业化生产提供了一定的理论基础。     

内生细菌对芝麻枯萎病菌的拮抗作用及其特性

采集了2个种植区的8个品种健康芝麻苗作为样品,利用涂布平板法从芝麻样品根部共分离到421株内生细菌;平板对峙法测定了内生细菌对尖孢镰刀菌(Fusarium oxysporium)拮抗性,结果显示:拮抗性菌株共180株;不同品种芝麻样品拮抗性菌株的比例有显著性差异,菌株拮抗比例最高和最低分别为68%和16%;通过对内生细菌生理生化特征的检测,实验表明:不同品种芝麻样品根部拮抗性菌株类型呈多样性,芽孢杆菌、假单胞菌和土壤杆菌的比例较高;拮抗性菌株中具有蛋白酶活性的菌株比例为68%。     

Improvements in the qualities of gluten-free bread after using a protease obtained from Aspergillus oryzae

From a nutritional perspective, rice flour is one of the most valuable flours and it is suitable for preparing food for people suffering from wheat allergy. However, bread made from rice flour is very difficult to bake because it lacks gluten. We found that pre-fermenting of rice flour using Aspergillus oryzae facilitated a better formulation of gluten-free rice bread. Bread swelling was remarkably improved with a longer pre-fermenting period at 55 °C. The specific loaf volume (SLV) without polymeric thickeners after a 12 h fermentation was approximately 2.2-fold (2.98 ml/g) higher than that after 0 h (1.36 ml/g). An enzymatic assay of the batter indicated that protease activity during the pre-fermentation period increased from 0.38 to 1.44 U/ml and this activity correlated with bread swelling. Furthermore, a commercial protease from A. oryzae also produced similar results with an adequate SLV of 3.03 ml/g. Rheological analysis showed that batter treated with protease had an increased batter viscosity and decreased flour settling behavior because of the aggregation of flour particle after partial cleavage of storage proteins. These results indicated that the improved SLV was mainly because of an A. oryzae protease, which affected the batter rheology thereby improving gas retention before baking.     

低温对黄瓜种子发芽期水解酶活性的影响

以北京截头和长春密刺这两个低温发芽能力有显著差异的黄瓜品种为试材,比较研究了在种子发芽期间低温对水解酶（包括蛋白酶和脂肪酶）活性的影响。结果表明：黄瓜低温种子发芽能力与水解酶的活性高低和水解酶活性高峰的出现有关,其中水解酶活性的高低起主导作用;长春密刺在低温发芽过程中蛋白酶和脂肪酶活性有突然上升的现象,可能有新的水解酶诱导合成,这可能是低温下长春密刺发芽能力高于北京截头的原因;低温处理早期脂肪酶活性受到抑制,而蛋白酶活性无此现象,说明脂肪酶比蛋白酶对低温更敏感。     

Dynamics of upward and downward N2O and CO2 fluxes in ploughed or no-tilled soils in relation to water-filled pore space, compaction and crop presence

Sharp peaks in nitrous oxide (N₂O) fluxes under no-tillage in wet conditions appear to be related to near surface soil and crop cover conditions. Here we explored some of the factors influencing tillage effects on short-term variations in gas flux so that we could learn about the mechanisms involved. Field investigations revealed that a cumulative emission of 13 kg N₂O–N ha⁻¹ over a 12-week period was possible under no-tillage for spring barley. We investigated how reducing crop cover and changing the structural arrangement of the water-filled pore space (WFPS) by short-term laboratory compaction influenced N₂O and carbon dioxide (CO₂) fluxes in upward and downward directions in core samples from tilled and untilled soil. Increasing the downward flux of N₂O within a soil profile by changing soil or moisture conditions may increase the likelihood of its further reduction to N₂ or dissolution. We took undisturbed cores from 3 to 8 cm depth, equilibrated them to −1 or −6 kPa matric potential, incubated them and measured N₂O and CO₂ fluxes from the upper and lower surfaces in a purpose-designed apparatus before and after compaction in an uniaxial tester. We also measured WFPS, air permeability, bulk density and air-filled porosity before and after compaction. Spring barley was tested in 1999 and winter barley in 2000.Fluxes of N₂O were from 1.5 to 35 times higher from no-tilled than ploughed even where the soil was of similar bulk density. Reduction of the crop cover increased CO₂ flux and could reduce N₂O flux. The effects of structural changes induced by laboratory compaction on the fluxes of N₂O and CO₂ were not influenced greatly by the tillage and crop cover treatments. Fluxes from the upper surfaces of cores (corresponding to 3 cm soil depth, upwards direction) could be up to 100 times greater (N₂O) or 8 times (CO₂) than from the lower surfaces (8 cm depth, downwards direction). These differences between surfaces were greatest when N₂O fluxes were very high in no-tilled soil (4.2 mg N₂O–N m⁻² h⁻¹) as occurred when WFPS exceeded 80% or became blocked with water, an effect that was increased by our compaction treatment. In general N₂O fluxes increased with WFPS. The production and emission of N₂O were strongly influenced by the soil physical environment, the magnitude of the water-filled pore space and continuity of the air-filled pore space in particular, produced in no-till versus plough cultivation.     

海洋芽孢杆菌N11-8产蛋白酶的发酵条件优化

本研究采用响应面法(RSM)对南极海洋芽孢杆菌(Bacillus sp.)N11-8液体发酵产蛋白酶PBN11-8的发酵条件进行快速优化。通过单因素实验初步确定南极海洋芽孢杆菌N11-8产蛋白酶的最佳碳源和氮源分别为可溶性淀粉和蛋白胨;并通过Plackett-Burman(PB)设计对影响其产酶相关因素进行评估,筛选出具有显著效应的3个因素：温度、氮源和碳源;以最陡爬坡实验逼近至上述因子最大响应区域,进而采用RSM法对其最佳水平范围进行研究,确定最优发酵条件为：可溶性淀粉3 g/L,蛋白胨13.1 g/L,酵母浸粉2.9 g/L,NaCl 5 g/L,KH2PO4 1 g/L,FeCl3·6H2O 2 mmol/L,初始pH值为7.0,温度为34.0℃,转速为200 r/min,装液量为50 ml/250 ml,接种量为4%,培养时间为60 h。最终优化后的酶活达到90.5 U/ml,比初始酶活提高了23.6%。     

罗汉果蛋白酶对酒糟中蛋白质的水解作用研究

[目的]为酒糟中蛋白质的水解提供新方法。[方法]采用酶解法,用罗汉果蛋白酶提取酒糟中的蛋白质,测定酶解酒糟上清液中蛋白质的含量,计算单位体积蛋白质增量。在加酶量、酶解温度、酶解时间、pH值单因子试验的基础上,采用正交试验确定酒糟中蛋白质的最佳提取条件。[结果]各因素对单位体积蛋白质增量的影响依次为:pH值>水解时间>加酶量>水解温度。酶解酒糟的最佳条件为:pH值8.0,水解时间10 min,加酶量0.75 ml/100 g湿酒糟,温度65℃。在最佳条件下,单位体积蛋白质增量可达98.78%。[结论]该研究确定了酶解法提取酒糟中蛋白质的最优条件,使酶解上清液中蛋白质含量增加了近1倍。