Separation,Purification and Characterization of Three Endo-polygalacturonases from a Newly Isolated Penicillum oxalicum

Three endo-polygalacturonases (endoPGs) from a newly isolated Penicillum oxalicum (CGMCC 0907) capable of utilizing waste biomass as growth substrate were separated and purified to homogeneity by ultra-filtration, affinity adsorption chromatography, CM-cellulose column chromatography, and Sephadex G-100 gel filtration chromatography with the overall yield of 64.5% from the crude enzyme. The specific activities and recovery rates of endoPG-1, endoPG-2 and endoPG-3 were 1120 U/mg and 21.6%, 1350 U/mg and 25.9%, and 1560 U/mg and 17.0%, respectively. The three purified endoPGs had a close molecular weight to 41 kDa as estimated by SDS-PAGE. The optimum temperature and pH for the function of them were 65℃ and 5.0, 55℃ and 5.0, 50℃ and 5.5, respectively. Their pI and Km values were 5.9 and 0.78 mg/mL, 6.0 and 1.2 mg/mL, and 6.1 and 2.0 mg/mL, respectively.     

Production of 6,8-Dihydroxy Fatty Acids by Recombinant Escherichia coli Expressing T879A Variant 6,8-Linoleate Diol Synthase from Penicillium oxalicum

Recombinant Escherichia coli expressing T879A variant 6,8-linoleate diol synthase (LDS) from Penicillium oxalicum showed 2.1-fold higher activity than recombinant E. coli expressing wild-type 6,8-LDS for the production of 6,8-dihydroxy fatty acids (DiHFA) from linoleic acid. The optimal conditions for the production of 6,8-DiHFA by recombinant E. coli expressing T879A variant 6,8-LDS were pH 6.5, 35°C, 50 g L⁻¹ cells, 10 g L⁻¹ (35.7 mM) substrate, and 5% (v/v) dimethyl sulfoxide. Under these optimized conditions, 6.6 g L⁻¹ (22.1 mM) 6,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid (DiHODE) and 7.1 g L⁻¹ (22.6 mM) 6,8-dihydroxy-9(Z)-octadecenoic acid (DiHOME) were produced from linoleic acid and oleic acid in 40 min, respectively. The volumetric productivities of 6,8-DiHODE and 6,8-DiHOME under these conditions were 9.9 and 10.7 mg L⁻¹ h⁻¹, respectively. The volumetric productivities of 6,8-DiHODE and 6,8-DiHOME were the highest values among those of all reported regiospecific DiHODE and DiHOME, respectively. To the best of our knowledge, this is the first quantitative biotechnological production of 6,8-DiHFA.     

草酸青霉Penicillium oxalicum菌体吸附活性染料的性能及微观过程

研究了草酸青霉(Penicillium oxalicum)在模拟染料废水中以边生长边吸附的方式对活性翠蓝KN-G, M-GB, K-GL,活性黑K-BR、活性艳蓝M-BR、活性红紫K-3R和活性深蓝K-R 7种水溶性活性染料的脱色性能及吸附过程. 结果显示,生长菌体对7种活性染料具有良好的脱色性能,染料初始浓度为200 mg/L时,平均脱色率可达93.0%;染料初始浓度为400 mg/L时,活性翠蓝KN-G和M-GB的脱色率仍达到了99.7%和99.9%. 上清液的紫外光谱图及染料分子中铜离子浓度的检测结果表明,染料通过吸附方式从废水中去除. 通过SEM, TEM观察发现,生长菌体在吸附过程中,菌丝发生肿胀膨大,细胞壁发生结构重组,厚度增加10~15倍. 细胞壁的结构变化是生长菌体吸附染料的重要机制,为染料吸附提供位点和进入细胞内部的通道.     

鹅源草酸青霉果胶酶提高苹果出汁率工艺

目的:以鹅源草酸青霉果胶酶为研究对象,优化鹅源草酸青霉制备的果胶酶提高苹果出汁率工艺。方法:以烟台红富士苹果为材料,以酶解温度(X1)、酶解时间(X2)、酶添加量(X3)和初始pH值(X4)为自变量(Y),出汁率为响应值,进行Box-Behnken设计,利用Design Expert软件进行响应面分析,建立回归模型。结果:结果表明,①利用响应面法筛选的鹅源草酸青霉果胶酶酶解苹果浆最佳条件为:温度32.91℃、酶解时间90.02min、酶添加量0.197mL/kg、pH3.96工艺条件下,苹果出汁率达到91.34%;②自变量与响应值关系的回归模型为:Y=91.72-0.4X1+0.58X2+0.60X3-0.32X4+0.42X2X3+0.50X2X4-1.75X12-0.85X22-1.17X32-1.50X42。结论:鹅源草酸青霉果胶酶能显著提高苹果出汁率;采用自变量与响应值建立的回归模型具有良好的拟合度,可以作为调控生产工艺的依据。     

产壳聚糖酶海洋真菌鉴定、寡营养培养基优化及廉价培养基研究

对筛选出的产壳聚糖酶海洋真菌进行了rDNA-ITS序列分析技术鉴定,确定该菌为Penicillium oxali-cum,这是首次报道该菌属具有产壳聚糖酶的能力。该研究旨在为工业化发酵生产壳聚糖酶提供实验指导。实验在已确定的寡营养培养基的基础上,探讨适宜碳源、氮源、耐盐性、耐酸性、摇床转速等发酵因素对产壳聚糖酶的影响,并探索不同种类的廉价培养基发酵产酶。结果显示黄豆粉培养基的产酶效果较为理想,最终确定培养基为(g/L海水):黄豆粉25 g,酶活力最高为8.5 U/mL,该培养基成本不足原发酵培养基成本的5%,为微生物工业化应用生产壳聚糖酶提供了直接的理论和实验基础。     

从长白山阔叶林中筛选β-葡萄糖苷酶产生菌及其酶学性质研究

利用马丁氏培养基富集、PDA培养基纯化和七叶灵培养基显色,从长白山阔叶林中初步筛选出6株产β-葡萄糖苷酶的真菌。对真菌发酵生成的β-葡萄糖苷酶进行提取和纯化,测得10号菌株的β-葡萄糖苷酶活力最高为18.34 U/mL,形态学和分子学结合方法鉴定该菌株为草酸青霉(Penicillium oxalicum)。该菌株所产β-葡萄糖苷酶最适反应温度约为55℃,在温度30、40℃和50℃时较为稳定。最适反应pH值约为5,在pH值为5~6之间相对较稳定。Cu~(2+)对酶活力有较强的抑制作用,而Ag~+有较强的激活作用。