植酸酶及其基因工程研究进展

植酸酶可添加于食品与饲料中,能消除因不能降解的植酸所引起的抗营养作用,提高机体对蛋白质及多种微量元素的利用率,降低粪便中磷的含量,从而减少环境中磷的积累污染,有利于保护生态环境。弄清植酸酶的性质、基因结构和功能,了解其基因工程的研究进展,不仅具有重要的理论意义,而且在开发研究植酸酶制剂用于饲料、食品和医药等方面具有重要的实践价值。本文谨对植酸酶的性质、基因结构和功能及其基因工程的研究进展做一综述,并探讨了植酸酶的应用前景。     

植酸酶基因工程研究进展

植酸酶是催化植酸及植酸盐水解成肌醇和无机磷酸的一类酶的总称.添加于食品和饲料中,能消除植酸所引起的抗营养作用,可提高蛋白质和矿物质的生物利用率.对植酸酶的生物学特性、基因结构和基因工程的研究进展做了综述.     

Phytase activity in Cryptococcus laurentii ABO 510

Ten Cryptococcus strains were screened for phytase activity, of which the Cryptococcus laurentii ABO 510 strain showed the highest level of activity. The cell wall-associated enzyme displayed temperature and pH optima of 62 degrees C and 5.0, respectively. The enzyme was thermostable at 70 degrees C, with a loss of 40% of its original activity after 3 h. The enzyme was active on a broad range of substrates, including ATP, D-glucose 6-phosphate, D-fructose 1,6-diphosphate and p-nitrophenyl phosphate (p-NPP), but its preferred substrate was phytic acid (K(m) of 21 microM). The enzyme activity was completely inhibited by 0.5 mM inorganic phosphate or 5 mM phytic acid, and moderately inhibited in the presence of Hg(2+), Zn(2+), Cd(2+) and Ca(2+). These characteristics suggest that the Cry. laurentii ABO 510 phytase may be considered for application as an animal feed additive to assist in the hydrolysis of phytate complexes to improve the bioavailability of phosphorus in plant feedstuff.     

甲基营养微生物的甲醛代谢途径及其在环境生物技术中的应用

甲醛是一种毒性很高的一碳化合物,甲基营养菌是一类能在有高浓度甲醛的环境中生存的微生物,它们体内有多种降解甲醛的氧化途径和将甲醛转化为细胞组分的同化途径。丝氨酸途径和酮糖单磷酸途径是同时存在于甲基营养型细菌中的两种甲醛同化途径,木酮糖单磷酸途径是甲基营养型酵母菌中独有的甲醛同化途径。为了充分挖掘甲基营养型微生物在环境生物技术中的潜在应用价值,最近有很多研究尝试利用甲基营养微生物的细胞及其甲醛代谢途径关键酶开发甲醛污染检测方法和生物治理技术,对这方面的研究进展进行综述。     

Dunaliella biotechnology: methods and applications

The microalga Dunaliella salina is the best commercial source of natural β-carotene. Additionally, different species of Dunaliella can accumulate significant amounts of valuable fine chemicals such as carotenoids, glycerol, lipids, vitamins, minerals and proteins. They also have a large potential for biotechnological processes such as expressing of foreign proteins and treatment of wastewater. In this review, we discussed several biotechnological aspects of the mass cultivation of D. salina like strain selection, carotenoid induction, culture conditions, culture systems and downstream processes. We also discuss several traditional and new applications of the genus.     

Phytase production and decrease of phytic acid content in canola meal byAspergillus carbonarius in solid-state fermentation

Solid-state fermentation (SSF) usingAspergillus carbonarius with canola meal as a substrate showed that production of phytase was associated with growth; maximum activity was achieved after 72 h. Apparent 25% and 10% increases in the protein content of the canola meal were noticed after 48 h and 72 h, respectively but total carbohydrate concentration had fallen by 25% by the end of fermentation. The rate of decrease of phytic acid content was optimum with a moisture content between 53% and 60%; homogenization of the inoculum for 120 s led to the greatest biomass and lowest phytic acid content. Inoculation of sterile meal led to lower phytic acid contents than inoculation of non-sterile meal.The authors are with the Department of Chemical Engineering, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada     

产植酸酶菌株的筛选及产酶条件的研究

通过初筛和复筛,得到一株产植酸酶较高的黑曲霉AN00101菌株,并对该菌种的产酶条件进行了研究.结果表明:配制加水量为35%的麸皮固体培养基,在37℃培养114h,用3%CaCl2进行提取,每g固体发酵物酶活高达1.3×104IU.经L9(34)正交实验表明,硫酸铵和硫酸镁对产酶有显著的促进作用,适宜添加量分别为4%和0.3%.     

黑曲霉N25株产植酸酶及酶促反应条件研究

从植物种子中研究筛选出高产植酸酶的黑曲霉N25,进行了最适液体培养基的筛选,研究了黑曲霉N25在玉米半合成液体培养基中所产植酸酶的最适酶促反应条件。结果表明：在四种培养基中,玉米半合成液体培养基为最适培养基,黑曲霉N25产植酸酶高峰期在96h,黑曲霉N25所产植酸酶的酶促反应最适pH为2.6和4.6,并具有很好的热稳定性,一定浓度的Ca^2 ,Mg^2 ,Mn^2 ,Cr^3 ,Li^ ,EDTA和高磷是植酸酶活性的抑制剂,1.0mmol/ml聚乙二醇1000,0.3nmol/mL Fe^2 和低磷对植酸酶活性具有激活作用。     

植酸酶的研究与应用进展

综述了植酸酶的生物学特性 ,产植酸酶的菌种来源 ,以及通过诱变和基因工程对菌种的选育 ,并阐述了植酸酶在饲料中的应用     

Concerted Action of Endogenous and Heterologous Phytase on Phytic Acid Degradation in Seed of Transgenic Wheat (Triticum aestivum L.)

Expression of heterologous phytases in crops offers a great potential for improving phosphate and mineral bioavailability in food and feed. In this context it is of relevance to describe the concerted action of endogenous and hetrologous phytases on the transgenic seed inositol phosphate profile. Here we report metal-dye detection HPLC analysis of inositol phosphate degradation in flour from transgenic wheat materials possessing wheat endogenous 6-phytase [EC 3.1.3.26] and Aspergillus 3-phytase [EC 3.1.3.8] activities under the control of the maize ubiquitin-1 promoter and the wheat high molecular weight glutenin subunit 1DX5 promoter respectively. During 50 min incubation there is an accumulation of InsP5 to InsP2 breakdown products in non-transgenic material. Aspergillus niger phytase specific breakdown products are transiently detected in transgenic material but after 50 min incubation virtually all InsP5, InsP4 and InsP3 isomers are hydrolysed.     

Production of high activity thermostable phytase from thermotolerant Aspergillus niger in solid state fermentation

The thermotolerant fungus, Aspergillus niger NCIM 563, was used for production of extracellular phytase on agricultural residues: wheat bran, mustard cake, cowpea meal, groundnut cake, coconut cake, cotton cake and black bean flour in solid state fermentation (SSF). Maximum enzyme activity (108 U g⁻¹ dry mouldy bran, DMB) was obtained with cowpea meal. During the fermentation phytic acid was hydrolysed completely with a corresponding increase in biomass and phytase activity within 7 days. Phosphate in the form of KH₂PO₄ (10 mg per 100 g of agriculture residue) increased phytase activity. Among various surfactants added to SSF, Trition X-100 (0.5%) exhibited a 30% increase in phytase activity. The optimum pH and temperature of the crude enzyme were 5.0 and 50°C respectively. Phytase activity (86%) was retained in buffer of pH 3.5 for 24 h. The enzyme retained 75% of its activity on incubation at 55°C for 1 h. In the presence of 1 mM K⁺ and Zn²⁺, 95% and 55% of the activity were retained. Scanning electron microscopy showed a high density growth of fungal mycelia on wheat bran particles during SSF. Journal of Industrial Microbiology & Biotechnology (2000) 24, 237–243. Received 07 June 1999/ Accepted in revised form 18 December 1999     

Improvement of tissue culture,genetic transformation,and applications of biotechnology to Brassica

Development of in vitro plant regeneration method from Brassica explants via organogenesis and somatic embryogenesis is influenced by many factors such as culture environment, culture medium composition, explant sources, and genotypes which are reviewed in this study. An efficient in vitro regeneration system to allow genetic transformation of Brassica is a crucial tool for improving its economical value. Methods to optimize transformation protocols for the efficient introduction of desirable traits, and a comparative analysis of these methods are also reviewed. Hence, binary vectors, selectable marker genes, minimum inhibitory concentration of selection agents, reporter marker genes, preculture media, Agrobacterium concentration and regeneration ability of putative transformants for improvement of Agrobacterium-mediated transformation of Brassica are discussed.     

Axenic cultures for microalgal biotechnology: Establishment,assessment, maintenance,and applications

The impact of microalgae (including blue-green algae or cyanobacteria) on human life can be both beneficiary and deleterious. While microalgae can be cultivated and used as feedstocks for the production of bioenergy and high value-added products in nutraceuticals, pharmaceuticals, and aquaculture feeds, some microalgae cause harmful algal blooms (HABs) that cause large-scale mortality in aquatic environments around the world. Thus, with the development of microalgal biotechnology and increasing concern about HABs, research on microscopic algae has increased significantly. However, this growth of academic research and application fields has been hindered by difficulties in obtaining axenic cultures. Therefore, this review provides a brief explanation of diverse establishment techniques, along with their strengths and weaknesses, with the hope of facilitating successful axenic cultures. A compilation of research fields and relevant important findings is also presented to clarify the importance of pure algal cultures. Finally, several controversial and sometimes overlooked issues related to the establishment, maintenance, and utilization of axenic cultures are discussed.     

2019环境生物技术专刊序言

环境生物技术,作为一门由现代生物技术与环境工程相结合的新兴交叉学科,已经在环境污染治理、环境监测中得到了广泛的应用,环境友好、高效地处理有机及无机污染,同时变废为宝生产高值化合物为从根本上解决环境问题提供了希望与支持。本专刊报道了环境生物技术在多环芳烃、抗生素、石油基塑料等环境污染物降解领域的基础与应用研究,介绍了吲哚、微生物铁载体等分子在生物修复中的应用,为全面认识环境污染现状、深入开展环境生物技术研究并制定综合治理策略等提供参考。     

海南红树林群系及分子生物技术在其研究中的应用

红树林是生长在热带亚热带海岸潮间带的木本植物群落。海南红树林是中国种类最多,分布和保存面积最大的区域之一,具有极为重要的研究价值。综述了海南红树林在种类、群落组成及红树林资源引种恢复以及分子生物技术等方面的研究,重点综述了分子生物技术在海南红树林植物研究中的应用,包括等位酶的应用、随机扩增多态DNA技术(RAPD)的应用和红树林植物耐盐性研究。     

环境生物技术信息学及其应用

通过介绍环境生物技术及生物信息学的产生发展及应用,概述了环境生物技术信息学的内容与应用前景。     

蜂房哈夫尼菌植酸酶基因appA的克隆、表达及酶学性质研究

从蜂房哈夫尼菌(Hafniaalvei)中克隆获得一个植酸酶编码基因appA,该基因全长1335bp,编码444个氨基酸,其中前33个氨基酸为信号肽,成熟蛋白的理论分子量为45.2kD。将基因appA克隆到大肠杆菌E.coli表达载体pET-22b( ),并在大肠杆菌中表达,表达产物具有植酸酶活性。对表达的酶蛋白进行纯化,并初步研究了该酶的酶学性质,结果表明:酶的作用最适pH值为4.5;在pH2.0~10.0范围内,酶活性保留80%以上;酶的作用最适温度为60℃;酶的比活性为356.7U/mg,酶动力学分析表明其Km为0.49mmol/L,Vmax为238U/mg;该酶对胰蛋白酶和胃蛋白酶有一定的抗性。该研究为哈夫尼菌属来源植酸酶的首次报道。     

枯草芽孢杆菌WHNB02植酸酶的酶学性质研究

从118份样品中分离到1株产植酸酶的枯草芽孢杆菌(Bacillus subtilis,WHNB02),其发酵液经乙醇沉淀、硫酸铵分级沉淀及Sephadex G-100柱层析等步骤后分离纯化了该酶,纯化倍数约为31.5倍,回收率为13.0%。该酶为单体酶,SDS-PAGE测得的分子量约为43ku,以植酸钠为底物的Km值为0.5mmol/L,酶反应的最适温度为60℃,80℃作用10min酶活保存61%,最适pH为7.0,在pH6.0~10.0范围内稳定,酶活性及稳定性都需Ca2 存在。EDTA、Mn2 、Ba2 (5mmol/L)对酶活具有很大的抑制作用。     

Rosmarinic acid and its derivatives: biotechnology and applications

Rosmarinic acid (RA) is one of the first secondary metabolites produced in plant cell cultures in extremely high yields, up to 19% of the cell dry weight. More complex derivatives of RA, such as rabdosiin and lithospermic acid B, later were also obtained in cell cultures at high yields. RA and its derivatives possess promising biological activities, such as improvement of cognitive performance, prevention of the development of Alzheimer’s disease, cardioprotective effects, reduction of the severity of kidney diseases and cancer chemoprevention. The TNF-α-induced NF-κB signaling pathway has emerged as a central target for RA. Despite these impressive activities and high yields, the biotechnological production of these metabolites on an industrial scale has not progressed. We summarized data suggesting that external stimuli, the Ca²⁺-dependent NADPH oxidase pathway and processes of protein phosphorylation/dephosphorylation are involved in the regulation of biosynthesis of these substances in cultured plant cells. In spite of growing information about pathways regulating biosynthesis of RA and its derivatives in cultured plant cells, the exact mechanism of regulation remains unknown. We suggest that further progress in the biotechnology of RA and its derivatives can be achieved by using new high-throughput techniques.     

Appearance of different phosphatase forms and phosphorus partitioning in nodules of chickpea (Cicer arietinum L.) during development

Acid and alkaline phosphatase and phytase activities were determined in the bacteroid free fractions of chickpea (Cicer arietinum L.) nodules at 15 days intervals, from 40 days after sowing (DAS) to 85 DAS. In general, the activities and specific activity of both the acid and alkaline phosphatases declined at 55 DAS. Out of the various substrates studied, ATP was the best substrate for both phosphatases. Activities of phosphatases with glucose-6-phosphate and fructose-6-phosphate were low in comparison to these with fructose 1,6 bisphosphate. The efficiency of acid phosphatase for utilizing fructose 1,6 bis phosphate as a substrate increased with nodule development. A fructose 1,6 bis phosphate specific acid phosphatase with elution volume to void volume (Ve/Vo) ratio of around 2.0 was observed in mature nodules (80 DAS). Acid phosphatase at 40 DAS was resolved into two peaks which were eluted at Ve/Vo of about 1.5 and 1.8. However, at 60 DAS the peak with Ve/Vo of 1.5 could not be detected. With ATP as substrate, a high (Ve/Vo of 1.2) and low MM form (Ve/Vo of 2.1) alkaline phosphatases were observed at 40 DAS however at 60 DAS stage only one peak with Ve/Vo of 1.7 was detected. Although, a low activity of acid phytase was observed in nodules at all stages of development but neither alkaline phytase nor phytic acid could be detected. It appears that the nodules acquire inorganic phosphate from the roots. The higher content of water soluble organic phosphorus in mature nodules could be due to the low activities of phosphatases at maturity.