无花果曲霉发酵生产β-葡萄糖苷酶的动力学分析

在7L生物反应器的分批发酵中,通过对无花果曲霉UV-29液态发酵茵丝体的生长、基质消耗（以总糖计）及β-葡萄糖苷酶产生的特性研究,发现总糖是无花果曲霉生长的限制性基质;β-葡萄糖苷酶的增长趋势明显滞后于细胞生长的增长趋势,其发酵过程属于部分相关模型,即Ga—den提出的Ⅱ型发酵;基于logistic方程,建立了发酵动力学模型,同时对实验数据与模型进行了验证比较,模型计算值与实验数据拟合良好。在7L生物反应器的最大茵体生物量（干重）达到1．17g／100mL,β-葡萄糖苷酶最高酶活达到22．25IU／mL。     

灰绿曲霉β-葡萄糖苷酶的分离及特性

目的：利用灰绿曲霉EU7-22发酵产纤维素酶,从中分离到β-葡萄糖苷酶,分析其理化特性,确定其最佳活性条件。方法：灰绿曲霉EU7-22发酵液离心后,上清液经硫酸铵沉淀、Phenyl 6 Fast Flow（highsub）疏水层析和Sephacryl S-200凝胶层析,获得纯化的β-葡萄糖苷酶。结果：纯酶的比活性为5.1 IU/mg,得率为13.89%。SDS-PAGE凝胶电泳分析表明该酶是单亚基蛋白,其分子量为56.2 kDa。在pH4.0~6.0范围内,β-葡萄糖苷酶具有较高的稳定性,该酶的最适酶促反应pH为5.0。当β-葡萄糖苷酶在温度低于60℃的缓冲液中温育1 h后,酶活损失不大,表现了较好的稳定性;当该酶在温度高于60℃的缓冲液中温育1 h后,酶活迅速丧失。β-葡萄糖苷酶在70℃时具有最大催化活性。结论：灰绿曲霉EU7-22发酵产生的β-葡萄糖苷酶具有较高活性,具有分子量较小、最佳催化温度较高的特点。     

絮凝剂产生菌培养条件的研究及在废水处理中的应用

通过菌种富集、分离、纯化,从含有大量微生物菌群的土壤和活性污泥中筛选到一株对高岭土悬浊液具有较高絮凝活性的絮凝剂产生菌,将其命名为B23.通过研究该菌株在不同培养时间的生长情况和发酵液的絮凝活性,从而得出发酵液絮凝活性与菌体生长量呈正相关.通过对该菌株培养条件的研究表明,在培养时间为24h、培养温度为30℃、培养基初始pH值为8.0,以葡萄糖为碳源、(NH4)2SO4为氮源时,发酵液絮凝活性最强.用B23菌株所产絮凝剂处理废水后,废水中CODCr的去除率为62.48%,SS的去除率为84.47%,表明该菌株有良好的实际应用前景.     

不产桔霉素的红曲霉菌种深层发酵生产莫纳可林K

对三株在YES培养基中不产桔霉素的红曲霉菌种,在摇瓶中研究了它们液体发酵生产莫纳可林K的情况。在大米粉培养基中,红色红曲霉不产莫纳可林K,但是紫色红曲霉和烟灰色红曲霉均能产莫纳可林K,前者产量高于后者。在葡萄糖．甘油培养基中,后两者的产量均很低,但是如果在该培养基中添加酵母膏,紫色红曲霉能产生较为可观的莫纳可林K。在2L的发酵罐中,利用添加了酵母膏的葡萄糖-甘油培养基,紫色红曲霉在第13d的莫纳可林K产量可达104mg/L,培养过程中无桔霉素产生。     

长双歧杆菌TTF菌株增强机体免疫活性研究

通过长双岐杆菌TTF活菌、菌体破碎物和发酵上清液3种处理物灌胃正常小鼠和免疫功能低下小鼠试验发现,长双歧杆菌TTF的3种受试物对环磷酰胺（Cy）造成的免疫功能低下小鼠模型（IDMM）3项免疫指标均有显著（P〈0．05）或极显著（P〈0．01）影响,且在本试验选取的3个剂量范围内基本呈量效关系。B．longum TTF的3种受试物对IDMM的非特异性免疫功能的影响普遍大于对小鼠的细胞免疫和体液免疫的影响;对正常小鼠免疫功能影响的水平低于其对IDMM各项免疫指标的影响,且无论活菌、菌体破碎物、发酵上清液均不会改变小鼠自身正常的免疫功能。急性毒性试验表明,B．longum,TTF3种处理物对健康小鼠无急性毒性。就3种处理物来讲,发酵上清液组的效果高于菌体破碎物和活菌。     

海枣曲霉β—D—岩藻糖苷酶的研究

Although beta-D-fucosidase (beta-D-fucoside fucohydrolase, EC 3.2.1.38) has been isolated from various sources, the identity of this enzyme is still not settled. We have purified a specific beta-D-fucosidase in electrophoretically homogeneous form crude extracts of Aspergillus phoenicis by polyethyleneglycol 6000-phosphate buffer aqueous two-phase separation, and successive chromatography on DEAE-Sephadex A-50, hydroxyapatite and Sephadex G-100 columns. The molecular weight of the enzyme was estimated to be 57000 by SDS-polyacrylamide gel electrophoresis and 50000 to 60000 by gel filtration on Sephadex G-100. The enzyme showed optimum coside were 2.4mmol/L, and 1.28 mumol min-1 the pH range 5.5-6.5 and below 35 degrees C. The Km and the Vmax values for pNP-beta-D-fucoside were 2.4mmol/L, and 1.28 mumol.min-1.mg-1 respectively. The enzyme was strongly inhibited by sulfhydryl group reagents, PCMB-NEM and iodoacetate. It was also inhibited by EDC, DEP and NBS. Thus, -SH, -COOH groups, histidyl and tryptophyl residues were essential for enzyme activity. The purified beta-D-fucosidase showed high specificity toward p-nitrophenyl beta-D-fucoside. The enzyme was inhibited by D-fucose and D-fucono-gamma-lactone, but not by D-galactose, D-galactono-gamma-lactone, D-glucose or D-glucono-gamma-lactone; the latter compounds are specific inhibitors of beta-D-galactosidase and beta-D-glucosidase respectively. Thus, this enzyme is the most strictly specific beta-D-fucosidase when compared with those previously reported.     

建立以TK基因为标记将外源基因导入臭曲霉（Asper—gillus...

A new system for selection of transformed Aspergillus foetidus was reported. In this system, TK- A. foetidus which were constructed by homologous recombination of mutated TK gene of vaccinia virus with TK gene of A. foetidus were screened by adding BUdR in agar plates. Conditions for screen of TK+ A. foetidus strain, transformation of A. foetidus and selection of transformed TK- A. foetidus have been studied. By using this system, several transformed A. foetidus which contained HBsAg gene derived bf a promoter H8 cloned from genomic DNA of A. foetidus were isolated. It was demonstrated that HBsAg gene was integrated into the chromosome DNA of A. foetidus by Southern blot after many passages of spores. ELISA showed that HBsAg was positive in the growth medium (p/n = 20). The 22 nm particles which were very similar to the HBsAg particles in human serum were found in the growth medium by immunoelectromicroscope. Western blot also gave the specific bands. All these data showed that HBsAg gene was expressed in A. foetidus and the products were secreted into the growth medium. The selection system using TK gene as marker could generally be used to study the expression of foreign gene in A. foetidus.     

重组毕赤酵母高密度发酵生产碱性果胶酶的策略

重组Pichia pastoris GS115表达碱性果胶酶的诱导阶段, 最佳初始菌体浓度和甲醇诱导浓度分别为122 g/L和20 g/L, 两者之间最佳比值范围是0.16~0.20 g/g (甲醇/菌体浓度). 在此基础上通过生长阶段甘油的指数流加, 以及诱导阶段基于甲醇比消耗速率和溶氧等参数进行甲醇流加的方式, 将甲醇与菌体浓度比例控制在0.171~0.195 g/g之间. 此时, 酶活达到430 u/mL, 生产强度为4.34 u/mL/h, 实现了碱性果胶酶高效生产。     

珠海市土石场空间分布特征与景观生态影响评价

人为水土流失是一种人类干扰下的典型景观退化现象．将人为侵蚀在空间上的表现称之为人为侵蚀景观．在遥感和GIS技术支持下,结合大量的实地调查资料,对珠海最主要的人为侵蚀景观类型——土石场开展空间分布分析和景观生态影响评价．调查范围内珠海土石场有235个,其中三灶、井岸两镇(区)分布最为集中,分别为28、29个．缓冲区分析表明,土石场的分布与距交通道路的远近呈对数线性相关,其中在距交通道路100～200m距离范围内分布最为集中．设计景观生态影响度评价指数,对面积大于5000m^2的152个土石场进行景观生态影响度评价结果表明,对周边景观生态环境影响极强的土石场有9个,属于强一级的有17个,应首先对这些土石场开展景观生态恢复．     

菌体表面表达与活菌疫苗

展示表达是将目的蛋白基因与细胞表面结构蛋白融合,使目的蛋白表达并锚定于细胞表面的一项技术,微生物特别是细菌常用作展示表达的宿主。在大肠杆菌中,多种外膜蛋白、鞭毛蛋白、菌毛蛋白、脂蛋白等均已被用于表达和锚定外源蛋白,革兰氏阴性菌中,较明确的是蛋白Ａ可用以细胞表面的整合,Ｍ６蛋白也被尝试用于乳酸菌的展示表达,酵母表达体系中,将目的蛋白与凝集素融合可表达细胞表面,菌体表面表达的蛋白容易被免疫系统识别,能引起强烈的免疫反应。其中沙门菌、大肠杆菌、链球菌等展示表达的致病细菌或病毒的抗原和毒素用于免疫动物均能产生高滴度的中和抗体,用作疫苗大有前途。本文对表达体系和在疫苗方面的应用进行了概述