琼脂糖凝胶的合适浓度对于回收酶切后质粒载体的重要性

目的：探讨琼脂糖凝胶的不同浓度对回收不同大小的酶切后质粒载体纯度的影响。方法：将2种质粒载体酶切,并经不同浓度的琼脂糖凝胶电泳分析,通过比较酶切前和酶切后载体的相对位置,研究胶浓度对回收酶切后载体纯度的影响。结果：不同浓度的琼脂糖凝胶中,酶切前和酶切后载体的相对位置会发生变化,对能否成功回收到纯度高的酶切后DNA片段有重要影响;质粒大小不同,胶浓度的影响也不同。结论：合适的胶浓度对于回收酶切后质粒载体具有重要意义,应选择合适的胶浓度回收酶切后质粒载体。     

用透析膜从琼脂糖胶中回收DNA片段

分离与回收DNA片段是基因操作的重要环节之一。本文介绍了一个用透析膜从琼脂糖胶中回收 DNA 片段的改进方法。利用本法回收DNA片段洗脱容易、节约时间、回收率在80％ 左右。回收的 DNA片段可用于酶切反应、连接反应和用缺口位移反应制备32p标记DNA探针。     

基于物质流分析的废纸回收利用体系生态成本研究

废弃物回收利用在一定程度上对缓解资源和环境危机起到积极的作用,已经成为可持续发展的重要举措,但生产过程中消耗的资源、能源,排放的污染物同样也会对自然环境产生负面影响。为解决此问题,以废纸回收利用体系为例,基于物质流分析方法构建了生态成本核算模型,为废弃物回收利用体系优化提供基础。在对生态成本相关研究归纳总结的基础上,定义了生态成本的概念,界定了生态成本的研究内容,并分析基于物质流核算生态成本的可行性。生态成本是对生态负荷的价值化,主要分为资源耗减成本、污染产生和环境保护成本以及生态环境损害成本3部分。污染产生和环境保护成本可以通过将总成本按比例分配给正、负产品的方式求得,资源耗减成本和环境损害成本借助LIME方法核算,总生态成本是回收利用体系内部各项生态成本的总和。生态成本核算是评价生态负荷的重要手段,在废纸回收利用体系物质流动图的基础上,分析各生产流程生态成本的构成情况。提出的生态成本核算模型不仅适用于废纸回收利用体系,其他废弃物也同样适用。通过生态成本的核算,寻找到对生态环境影响较大的工序、流程,为废弃物回收利用体系经济与环境的双赢提供理论与实践指导。     

一株中华绒螯蟹呼肠孤病毒RNA1 cDNA文库构建及其RNA聚合酶基因部分序列分析

在中华绒螯蟹体内分离到一株呼肠孤病毒(命名为EsRV905株).采用Trizol试剂提取病毒核酸,经聚丙烯酰胺凝胶电泳,碎胶法回收基因组各节段.随机引物法合成第一节段的cDNA文库,胶回收试剂盒去除小片段,平端连接于载体,化学转化,利用蓝白斑筛选阳性克隆子,酶切鉴定重组质粒.从基因组第一节段的重组质粒中选择2个插入片段约为1.5kb的质粒测序,结果得到包括RNA聚合酶主要特征性结构的一段序列.结果说明,这株蟹呼肠孤病毒的RNA聚合酶定位于基因组第一节段.     

烟草天蛾几丁质酶的表达、纯化及多克隆抗体制备

将烟草天蛾Manduca sexta几丁质酶基因克隆入融合表达载体pET-28a,并在大肠杆菌E.coli BL21(DE3)中进行诱导表达。表达菌株经0.5 mmol/L IPTG诱导6～8 h后,几丁质酶表达并形成包涵体。在Ni²⁺-NTA亲和柱上经变、复性和纯化,得到可溶的几丁质酶。表达产物经Western 印迹鉴定。采用切胶回收的方法切割回收包涵体,并将回收产物免疫新西兰大白兔,ELISA检测抗体效价达1∶20 000。Western 印迹检测证明抗体特异性良好。     

亲和层析法分离纯化猪肺血管紧张素转换酶

亲和胶合成实验以双环氧化合物1,4-丁二醇-2-缩水甘油醚(1,4-butanediol diglycidyl ether)为活化体及连接臂,在硼氢化钠(NaBH)存在的碱性条件下,将载体Sepharose CL―4B与雷诺普利(lisinopril)共价连接在一起,成功合成亲和层析胶,并利用亲和层析胶对猪肺血管紧张素转换酶（angiotensin converting enzyme, ACE）进行分离提纯.猪肺组织匀浆经1.6～2.6 mol/L硫酸铵分级沉淀、透析平衡、亲和柱分离等步骤,从200 g猪肺中提纯得到0.79 mg ACE蛋白,酶活力回收11.9％,比活力38.8 U/mg.与层析前的酶液比较,亲和层析一步提纯可达264倍;与肺匀浆液比较提纯达808倍.SDS-聚丙烯酰胺凝胶电泳可见,提纯的猪肺ACE为一条带,分子质量约为180 ku.     

微乳液凝胶及其固定化脂肪酶研究进展

利用微乳液胶凝现象来固定化酶是90年代初建立起来的酶固定化新技术,该技术为胶束酶学在生物合成与转化领域的应用奠定了基础.就微乳液凝胶及其固定化脂肪酶的制备、性质、微观结构及其潜在应用作了带有知识介绍性质的综述.     

一种利用普通垂直电泳槽回收PAGE胶蛋白条带的简便方法

植物总蛋白样品经聚丙烯酰胺凝胶电泳分离之后,直接用考马斯亮蓝染色、切胶回收目的条带,再用聚丙烯酰胺凝胶电泳槽电洗脱纯化得到单一条带的目的蛋白.此法可得到有活性的黄瓜衰老叶片中被特异激活的DNA酶,对样品中含量少,特别是与其他分子量相近的蛋白质十分有效.     

蛋白质胶内酶切技术研究进展

胶内酶切是蛋白质组研究中衔接电泳分离和质谱鉴定的重要环节,对最终的蛋白质定性和定量分析结果有显著的影响。该技术自1992年初步建立以来,一直处于不断完善中,出现了种类繁多的改进方案。为了更有效地利用胶内酶切技术,从凝胶脱色、杂质去除、蛋白酶切、肽段提取4个方面归纳整理了近年来蛋白质胶内酶切技术的主要研究进展。     

一株中华绒螯蟹呼肠孤病毒RNA1 cDNA文库构建及其RNA聚合酶基因部分序列分析

在中华绒螯蟹体内分离一株呼肠孤病毒（命名为EsRV905株）,采用Trizol试剂提取病毒核酸,经聚丙烯酰胺凝胶电泳,碎胶法回收基因组各节段。随机引物法合成第一节段的cDNA文库,胶回收试剂盒去除小片段,平端连接于载体,化学转化,利用蓝白斑筛选阳性克隆子,酶切鉴定重组质粒。从基因组第一节段的重组质粒中选择2个插入片段为1.5kb的质粒测序,结果得到包括RNA聚合酶主要特征性结构的一段序列。结果说明,这株蟹呼肠孤病毒的RNA聚合酶定位于基因组第一节段。     

Recycling of lipid-extracted hydrolysate as nitrogen supplementation for production of thraustochytrid biomass

Efficient resource usage is important for cost-effective microalgae production, where the incorporation of waste streams and recycled water into the process has great potential. This study builds upon emerging research on nutrient recycling in thraustochytrid production, where waste streams are recovered after lipid extraction and recycled into future cultures. This research investigates the nitrogen flux of recycled hydrolysate derived from enzymatic lipid extraction of thraustochytrid biomass. Results indicated the proteinaceous content of the recycled hydrolysate can offset the need to supply fresh nitrogen in a secondary culture, without detrimental impact upon the produced biomass. The treatment employing the recycled hydrolysate with no nitrogen addition accumulated 14.86 g L^?1 of biomass in 141 h with 43.3 % (w/w) lipid content compared to the control which had 9.26 g L^?1 and 46.9 % (w/w), respectively. This improved nutrient efficiency and wastewater recovery represents considerable potential for enhanced resource efficiency of commercial thraustochytrid production.     

Application of LCA as a decision-making tool for waste management systems

Aim, Scope and Background  When materials are recycled they are made available for use for several future life cycles and can therefore replace virgin material more than just once. In order to analyse the optimal waste management system for a given material, the authors have analysed the material flows in a life cycle perspective. It is important to distinguish this approach for material flow analysis for a given material from life cycle analysis of products. A product life cycle analysis analyses the product system from cradle to grave, but uses some form of allocation in order to separate the life cycle of one product from another in cases where component materials are recycled. This paper does not address allocation of burdens between different product systems, but rather focuses on methodology for decision making for waste management systems where the optimal waste management system for a given material is analysed. The focus here is the flow of the given material from cradle (raw material extraction) to grave (the material, or its inherent energy, is no longer available for use). The limitation on the number of times materials can be recycled is set by either the recycling rate, or the technical properties of the recycled material. Main Features  This article describes a mathematical geometric progression approach that can be used to expand the system boundaries and allow for recycling a given number of times. Case studies for polyethylene and paperboard are used to illustrate the importance of including these aspects when part of the Goal and Scope for the LCA study is to identify which waste management treatment options are best for a given material. The results and discussion examine the different conclusions that can be reached about which waste management option is most environmentally beneficial when the higher burdens and benefits of recycling several times are taken into account. Results  In order to assess the complete picture of the burdens and benefits arising from recycling the system boundaries must be expanded to allow for recycling many times. A mathematical geometric progression approach manages to take into account the higher burdens and benefits arising from recycling several times. If one compares different waste management systems, e.g. energy recovery with recycling, without expanding the system to include the complete effects of material recycling one can reach a different conclusion about which waste management option is preferred. Conclusions  When the purpose of the study is to compare different waste management options, it is important that the system boundaries are expanded in order to include several recycling loops where this is a physical reality. The equations given in this article can be used to include these recycling loops. The error introduced by not expanding the system boundaries can be significant. This error can be large enough to change the conclusions of a comparative study, such that material recycling followed by incineration is a much better option than waste incineration directly. Recommendations and Outlook  When comparing waste management solutions, where material recycling is a feasible option, it is important to include the relevant number of recycling loops to ensure that the benefits of material recycling are not underestimated. The methodology presented in this article should be used in future comparative studies for strategic decision-making for waste management. The approach should not be used for LCAs for product systems without due care, as this could lead to double counting of the benefits of recycling (depending on the goal and scope of the analysis). For materials where the material cycle is more of a closed loop and one cannot truly say that recycled materials replace virgin materials, a more sophisticated approach will be required, taking into account the fact that recycled materials will only replace a certain proportion of virgin materials.     

Enzymatic deinking of various types of waste paper: Efficiency and characteristics

The aim of the present study was to characterize the enzymatic deinking of various types of waste paper. Studies on the optimization of enzymatic deinking have been performed previously using commercially available enzyme preparations containing cellulase and hemicellulase. The enzymatic deinking of different types of waste paper demonstrated a high efficiency of 86.6% on laser-printed paper, but a low deinking efficiency of 12.9% was obtained with newspaper. All enzymatic treatments significantly improved the drainage rate of the deinked waste paper. Enzymatic deinking increased the tensile index of magazine paper but reduced the tensile index of bubble jet-printed paper, photocopy paper and newspaper. Enzymatic hydrolysis caused a 21.1% reduction in the tear index for bubble jet-printed paper, but a 3.1% increase in the tear index was obtained for laser-printed paper relative to respective blank. In addition, enzymatic hydrolysis increased the burst index by 4.7% relative to blank for laser-printed paper. However, photocopy paper showed the highest reduction (8.3%) in the burst index relative to blank. Taken together, these results suggest that enzymatic hydrolysis is both advantageous and detrimental to the mechanical properties of deinked paper. Thus, the proper regulation of enzymatic hydrolysis is crucial to improve the quality of recycled paper.     

餐厨废弃物资源化利用的微生物技术研究进展

简单介绍餐厨废弃物的特征和危害,综述微生物技术处理餐厨废弃物资源化的途径,如发酵提取生物降解塑料技术、厌氧发酵处理技术、微生物堆肥技术、微生物农药技术、微生物产电技术,介绍利用复合微生物菌剂降解餐厨废弃物的研究进展,分析这一新技术的发展趋势。     

New insights into polyurethane biodegradation and realistic prospects for the development of a sustainable waste recycling process

Polyurethanes are polymeric plastics that were first used as substitutes for traditional polymers suspected to release volatile organic hazardous substances. The limitless conformations and formulations of polyurethanes enabled their use in a wide variety of applications. Because approximately 10 Mt of polyurethanes is produced each year, environmental concern over their considerable contribution to landfill waste accumulation appeared in the 1990s. To date, no recycling processes allow for the efficient reuse of polyurethane waste due to their high resistance to (a)biotic disturbances. To find alternatives to systematic accumulation or incineration of polyurethanes, a bibliographic analysis was performed on major scientific advances in the polyurethane (bio)degradation field to identify opportunities for the development of new technologies to recondition this material. Until polymers exhibiting oxo- or hydro-biodegradative traits are generated, conventional polyurethanes that are known to be only slightly biodegradable are of great concern. The research focused on polyurethane biodegradation highlights recent attempts to reprocess conventional industrial polyurethanes via microbial or enzymatic degradation. This review describes several wonderful opportunities for the establishment of new processes for polyurethane recycling. Meeting these new challenges could lead to the development of sustainable management processes involving polymer recycling or reuse as environmentally safe options for industries. The ability to upgrade polyurethane wastes to chemical compounds with a higher added value would be especially attractive.     

The Multilevel Cycle of Anthropogenic Zinc

A comprehensive annual cycle for stocks and flows of zinc, based on data from circa 1994 and incorporating information on extraction, processing, fabrication, use, discard, recycling, and landfilling, was carried out at three discrete governmental unit levels—54 countries and 1 country group (which together comprise essentially all global anthropogenic zinc stocks and flows), nine world regions, and the planet as a whole. All of these cycles are available in an electronic supplement to this article, which thus provides a metadata set on zinc flows for the use of industrial ecology researchers. A "best estimate" global zinc cycle was constructed to resolve aggregation discrepancies. Among the most interesting results are the following: (1) The accumulation ratio, that is, addition to in-use stock as a function of zinc entering use, is positive and large (2/3 of zinc entering use is added to stock) (country, regional, and global levels); (2) secondary input ratios (fractions of input to fabrication that are from recycled zinc) and domestic recycling percentages (fractions of discarded zinc that are recycled) differ among regions by as much as a factor of six (regional level); (3) worldwide, about 40% of the zinc that was discarded in various forms was recovered and reused or recycled (global level); (4) zinc cycles can usefully be characterized by a set of ratios, including, notably, the utilization efficiency (the ratio of manufacturing waste to manufacturing output: 0.090) and the prompt scrap ratio (new scrap as a fraction of manufacturing input: 0.070) (global level). Because capturable discards are a significant fraction of primary zinc inputs, if a larger proportion of discards were recaptured, extraction requirements would decrease significantly (global level). The results provide a framework for complementary studies in resource stocks, industrial resource utilization, energy consumption, waste management, industrial economics, and environmental impacts.     

Degradation of organic contaminants found in organic waste

In recent years, great interest has arisen in recycling of the waste created by modern society. A common way of recycling the organic fraction is amendment on farmland. However, these wastes may contain possible hazardous components in small amounts, which may prevent their use in farming. The objective of our study has been to develop biological methods by which selected organic xenobiotic compounds can be biotransformed by anaerobic or aerobic treatment. Screening tests assessed the capability of various inocula to degrade two phthalates di-n-butylphthalate, and di(2-ethylhexyl)phthalate, five polycyclic aromatic hydrocarbons, linear alkylbenzene sulfonates and three nonylphenol ethoxylates under aerobic and anaerobic conditions. Under aerobic conditions, by selecting the appropriate inoculum most of the selected xenobiotics could be degraded. Aerobic degradation of di(2-ethylhexyl)phthalate was only possible with leachate from a landfill as inoculum. Anaerobic degradation of some of the compounds was also detected. Leachate showed capability of degrading phthalates, and anaerobic sludge showed potential for degrading, polycyclic aromatic hydrocarbons, linear alkylbenzene sulfonates and nonyl phenol ethoxylates. The results are promising as they indicate that a great potential for biological degradation is present, though the inoculum containing the microorganisms capable of transforming the recalcitrant xenobiotics has to be chosen carefully.     

Application of Markov Chain Model to Calculate the Average Number of Times of Use of a Material in Society. An Allocation Methodology for Open-Loop Recycling. Part 1: Methodology Development (7 pp)

- Preamble. In this series of two papers, a methodology to calculate the average number of times a material is used in a society from cradle to grave is presented and applied to allocation of environmental impact of virgin material. Part 1 focuses on methodology development and shows how the methodology works with hypothetical examples of material flows. Part 2 presents case studies for steel recycling in Japan, in which the methodology is applied and allocation of environmental impact of virgin steel is conducted. - Abstract Goal, Scope and Background. It has been recognized that LCA has a limitation in assessing open cycle recycling of materials because of inevitable subjective judgments in setting system boundary. According with the enforcement of recycling laws, there has been a rapid increase in recycling ratio of materials at the end-of-life of products in many industrialized countries. So, materials' life cycle is getting more complicated, which makes it difficult to quantify the environmental impacts of materials used in a product in an appropriate way. The purpose of this paper is to develop a methodology to calculate the average number of times a material is used in a society from cradle to grave. The method developed in this paper derives the average number of times material is used; this value could be used for allocation of environmental burdens of virgin material as well as an indicator for assessing the state of material use in a certain year, based on material flow of material in that year. Main Features Our methodology is based on Markov chain model using matrix-based numerical analysis. A major feature of this method is that it creates transition probability matrices for a material from the way in which the material is produced, consumed, and recycled, making it possible to simply elicit indicators that assess the status of material use in products in society. Our methodology could be an alternative method to derive the average number of times material is used, which could be used for allocation of environmental burdens of virgin material. Results and Discussions The methodology was applied to hypothetical examples of material flows, in which a virgin material was produced and used in products, recycled and finally landfilled. In some cases, closed loop and open loop recycling of materials existed. The transition probability matrix was created for each material flow, and how many times a virgin material is used in products until all of the elements are ultimately landfilled. Conclusions This methodology is applicable to a complicated material flow if the status of residence of a material and its flow in a society can be figured out. All the necessary data are the amount of virgin material production, amount of the material used in products, recycling rate of the material at the end of life of each product, the amount of scrap of the material that are used for products. In Part 2 of this paper, case studies for steel were conducted.     

Engineered Humicola insolens cutinase for efficient cellulose acetate deacetylation

Cutinases comprise a family of esterases with broad hydrolytic activity for chain and pendant ester groups. This work aimed to identify and improve an efficient cutinase for cellulose acetate (CA) deacetylation. The development of a mild method for CA fiber surface deacetylation will result in improved surface hydrophilicity and reactivity while, when combined with cellulases, a route to the full recycling of CA to acetate and glucose. In this study, the comparative CA deacetylation activity of four homologous wild‐type (wt) fungal cutinases from Aspergillus oryzae (AoC), Thiellavia terrestris (TtC), Fusarium solani (FsC), and Humicola insolens (HiC) was determined by analysis of CA deacetylation kinetics. wt‐HiC had the highest catalytic efficiency (≈32 [cm² L^‐1]^‐1 h^‐1). Comparison of wt‐cutinase catalytic constants revealed that differences in catalytic efficiency are primarily due to corresponding variations in corresponding substrate binding constants. Docking studies with model tetrameric substrates also revealed structural origins for differential substrate binding amongst these cutinases. Comparative docking studies of HiC point mutations led to the identification of two important rationales for engineering cutinases for CA deacetylation: (i) create a tight but not too closed binding groove, (ii) allow for hydrogen bonding in the extended region around the active site. Rationally designed HiC with amino acid substitutions I36S, predicted to hydrogen bond to CA, combined with F70A, predicted to remove steric constraints, showed a two‐fold improvement in catalytic efficiency. Continued cutinase optimization guided by a detailed understanding of structure‐activity relationships, as demonstrated here, will be an important tool to developing practical cutinases for commercial green chemistry technologies.     

Role of micronutrients in activated sludge-based biotreatment of industrial effluents

Industrial processes often produce wastewaters that resist biological treatment owing to the unfamiliarity of some components to biological systems. Availability of nutrients determines the community structure of the activated sludge and hence the efficiency of the degradation process. Micronutrients influence the bacteria involved in waste degradation and also the species diversity within the sludge. The requirements for and toxicity of different micronutrients vary according to the nature of the waste and the ecology of the sludge. Adding micronutrients to biological treatment processes is one possible approach to upgrading an existing facility in order to deal with increasing volumes and strengths of industrial wastewaters and the tightening discharge legislation.