Improving the remaining activity of lignocellulolytic enzymes by membrane entrapment

The production of bioethanol by the conversion of lignocellulosic waste has attracted much interest in recent years because of its low cost and great potential availability. However, the high cost of the enzyme required for this conversion is often considered to be the major bottleneck in the commercial lignocellulosic ethanol industry. In this work, the hydrolysis of rice straw by free and entrapped lignocellulolytic enzymes (cellulase, xylanase and laccase) was carried out at pH 5.5 and 37 °C. The hydrolysis of rice straw by enzymes entrapped in a membrane produced a higher monosaccharide content: 601.05 mg/g rice straw for entrapped enzymes vs. 465.46 mg/g rice straw for free enzymes. This study has shown that enzyme entrapment is an important technique for the efficient use and reuse of enzymes in industrial applications and also for the rapid separation of saccharide products from the reaction medium, thus improving the remaining enzymatic activities.     

Post-production modification of industrial enzymes

Industry has an increasing interest in the use of enzymes as environmentally friendly, highly efficient, and specific bio-catalysts. Enzymes have primarily evolved to function in aqueous environments at ambient temperature and pressure. These conditions however do not always correspond with industrial processes or applications, and only a small portion of all known enzymes are therefore suitable for industrial use. Protein engineering can sometimes be applied to convey more desirable properties to enzymes, such as increased stability, but is limited to the 20 naturally occurring amino acids or homologs thereof. Using post-production modification, which has the potential to combine desirable properties from the enzyme and the conjugated compounds, enzymes can be modified with both natural and synthetic molecules. This offers access to a myriad of possibilities for tuning the properties of enzymes. At this moment, however, the effects of post-production modification cannot yet be reliably predicted. The increasing number of applications will improve this so that the potential of this technology can be fully exploited. This review will focus on post-production modification of enzymes and its use and opportunities in industry.     

Industrial enzymes--developments in production and application

The utilization of gene technology and of new production technologies have made industrial enzymes with improved properties or better cost performance available. This has in turn opened important new areas of enzyme applications. The benefits to the customers are considerable: cost savings in the application process, improved product quality, and in most cases also a significantly reduced impact on the environment.

Gene technology offers several benefits to the enzyme industry. This technology enables the use of safe, well-documented host organisms easy to cultivate, the microbial production of enzymes of animal and plant origin, the realization of enhanced efficiency and high product purity, and also the production of enzymes with improved stability and activity.

Developments in production technology include advanced control methods, the use of expert systems, and the application of large-scale crystallization.

As case stories the development of a lipase and of a cellulase is described. The effect on environment of enzyme application and production is discussed.     

The search for the ideal biocatalyst.

While the use of enzymes as biocatalysts to assist in the industrial manufacture of fine chemicals and pharmaceuticals has enormous potential, application is frequently limited by evolution-led catalyst traits. The advent of designer biocatalysts, produced by informed selection and mutation through recombinant DNA technology, enables production of process-compatible enzymes. However, to fully realize the potential of designer enzymes in industrial applications, it will be necessary to tailor catalyst properties so that they are optimal not only for a given reaction but also in the context of the industrial process in which the enzyme is applied.     

Simultaneous secretion of seven lignocellulolytic enzymes by an industrial second-generation yeast strain enables efficient ethanol production from multiple polymeric substrates

A major hurdle in the production of bioethanol with second-generation feedstocks is the high cost of the enzymes for saccharification of the lignocellulosic biomass into fermentable sugars. Simultaneous saccharification and fermentation with Saccharomyces cerevisiae yeast that secretes a range of lignocellulolytic enzymes might address this problem, ideally leading to consolidated bioprocessing. However, it has been unclear how many enzymes can be secreted simultaneously and what the consequences would be on the C6 and C5 sugar fermentation performance and robustness of the second-generation yeast strain. We have successfully expressed seven secreted lignocellulolytic enzymes, namely endoglucanase, β-glucosidase, cellobiohydrolase I and II, xylanase, β-xylosidase and acetylxylan esterase, in a single second-generation industrial S. cerevisiae strain, reaching 94.5 FPU/g CDW and enabling direct conversion of lignocellulosic substrates into ethanol without preceding enzyme treatment. Neither glucose nor the engineered xylose fermentation were significantly affected by the heterologous enzyme secretion. This strain can therefore serve as a promising industrial platform strain for development of yeast cell factories that can significantly reduce the enzyme cost for saccharification of lignocellulosic feedstocks.     

Von der Krebsschale in die Creme

From crustacean to cream Chitin is one of the most abundant biopolymers on earth and can be obtained in large quantities from shell waste from the fishing industry. However, established chemical technologies are hazardous to human health and the environment due to the use of highly acidic and alkali conditions. Microbiological fermentation and the use of enzymes for the extraction of the polymer have been proven valuable alternatives which also yield other added‐value compounds from this raw material. However, the variety of organisms and conditions used and the varying yields that are obtained make it difficult to evaluate and compare these methods for upscaling to industrial use. Nonetheless, chitin and its derivatives have shown great potential in their antimicrobial antioxidative and binding properties that find use in food technology and medicine as well as in agriculture and consumer goods. To drive the innovation forward in this field, an interdisciplinary approach joining biology and chemistry is required.     

Superoxide dismutase: an industrial perspective

The application of enzyme technologies to industrial research, development, and manufacturing has become a very important field. Since the production of crude rennet in 1874, several enzymes have been commercialized, and used for therapeutic, supplementary, and other applications. Recent advancements in biotechnology now allow companies to produce safer and less expensive enzymes with enhanced potency and specificity. Antioxidant enzymes are emerging as a new addition to the pool of industrial enzymes and are surpassing all other enzymes in terms of the volume of research and production. In the 1990s, an antioxidant enzyme--superoxide dismutase (SOD)--was introduced into the market. Although the enzyme initially showed great promise in therapeutic applications, it did not perform up to expectations. Consequently, its use was limited to non-drug applications in humans and drug applications in animals. This review summarizes the rise and fall of SOD at the industrial level, the reasons for this, and potential future thrust areas that need to be addressed. The review also focuses on other industrially relevant aspects of SOD such as industrial importance, enzyme engineering, production processes, and process optimization and scale-up.     

Impact of microbial proteases on biotechnological industries

The demand of enzymes in industrial sectors is increasing rapidly due to their economical and ecological advantages. Micro-organisms produce different types of extracellular enzymes for maintaining their own metabolism, defense, and normal physiological condition. Among several enzymes, proteases have gained special attention in industrial sectors. Several sources of extracellular enzymes are reported by various researchers, but enzymes obtain from microbial sources have high demand in industries due to lower cost, high production rate, availability, stability, and diversity. Among micro-organism, bacteria and fungi are reported to be good sources of different types of proteases such as alkaline protease, cysteine protease, aspartate protease, and metallo protease. In this review, we have summarized the available information about the sources of bacterial and fungal proteases, their purification strategies and their temperature and pH optima. Due to huge competition, companies are trying to reduce their manufacturing cost and that’s why microbial sources of enzymes are important. However, genetically engineered strains or engineered proteases have much more importance over natural isolates/protease in industries due to higher production rate and other advantages. Here we have also summarized the important applications of protease in different industries such as, paper mill, starch degrading sector, food processing factories, and detergent making companies.     

Binary immobilization of tyrosinase by using alginate gel beads and poly(acrylamide-co-acrylic acid) hydrogels

The use of the immobilized and the stable enzymes has immense potential in the enzymatic analysis of clinical, industrial and environmental samples. However, their widespread uses are limited due to the high cost of their production. In this study, binary immobilization of tyrosinase by using Ca-alginate and poly(acrylamide-co-acrylic acid) [P(AAm-co-AA)] was investigated. Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for the free and binary immobilized enzymes. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized tyrosinase were also examined. For the free and binary immobilized enzymes on Ca-alginate and P(AAm-co-AA), optimum pH was found to be 7 and 5, respectively. Optimum temperature of the free and immobilized enzymes was observed to be 30 and 35 degrees C, respectively. Reuse number, storage and thermal stability of the free tyrosinase were increased by a result of binary immobilization.     

Manipulating corn germplasm to increase recombinant protein accumulation

Using plants as biofactories for industrial enzymes is a developing technology. The application of this technology to plant biomass conversion for biofuels and biobased products has potential for significantly lowering the cost of these products because of lower enzyme production costs. However, the concentration of the enzymes in plant tissue must be high to realize this goal. We describe the enhancement of the accumulation of cellulases in transgenic maize seed as a part of the process to lower the cost of these dominant enzymes for the bioconversion process. We have used breeding to move these genes into elite and high oil germplasm to enhance protein accumulation in grain. We have also explored processing of the grain to isolate the germ, which preferentially contains the enzymes, to further enhance recovery of enzyme on a dry weight basis of raw materials. The enzymes are active on microcrystalline cellulose to release glucose and cellobiose.     

Autodisplay of enzymes--molecular basis and perspectives

To display an enzyme on the surface of a living cell is an important step forward towards a broader use of biocatalysts. Enzymes immobilized on surfaces appeared to be more stable compared to free molecules. It is possible by standard techniques to let the bacterial cell (e.g. Escherichia coli) decorate its surface with the enzyme and produce it on high amounts with a minimum of costs and equipment. Moreover, these cells can be recovered and reused in several subsequent process cycles. Among other systems, autodisplay has some extra features that could overcome limitations in the industrial applications of enzymes. One major advantage of autodisplay is the motility of the anchoring domain. Enzyme subunits exposed at the cell surface having affinity to each other will spontaneously form dimers or multimers. Using autodisplay enzymes with prosthetic groups can be displayed, expanding the application of surface display to the industrial important P450 enzymes. Finally, up to 10?-10? enzyme molecules can be displayed on a single cell. In the present review, we summarize recent achievements in the autodisplay of enzymes with particular attention to industrial needs and process development. Applications that will provide sustainable solutions towards a bio-based industry are discussed.     

Aspects for the future of biotransformations

Biotransformations have gained extensive importance in practical use as a support for chemical synthesis or in the conversion of natural products. Biotransformations may present an enlargement, a sequential degradation or a specific modification of synthetic or natural compounds. The tools for biotransformations are principally mammalian, plant or microbial cells and their cell-free enzymes. In technical practice the biocatalysts are so far limited to the use of microorganisms and some cell-free enzymes of low cost. Although numerous microbial or enzymatical reactions were already developed for industrial processes, the capacities of biotransformations offer a broad field of inexhaustible possibilities for the future.

     

Technical enzymes produced in transgenic plants

Technical enzymes are used in many industrial applications. Nowadays technical enzymes are often produced in transgenic host organisms. The use of transgenic plants with respect to high level of expression at low costs as a prerequisite for successful commercial production of technical enzymes is discussed. This review summarises recently published examples for production of technical enzymes in plants. In addition, plastid transformation and viral vectors are discussed as methods which might be useful for obtaining high expression level of recombinant proteins in plants.     

Industrial relevance of thermophilic Archaea

The dramatic increase of newly isolated extremophilic microorganisms, analysis of their genomes and investigations of their enzymes by academic and industrial laboratories demonstrate the great potential of extremophiles in industrial (white) biotechnology. Enzymes derived from extremophiles (extremozymes) are superior to the traditional catalysts because they can perform industrial processes even under harsh conditions, under which conventional proteins are completely denatured. In particular, enzymes from thermophilic and hyperthermophilic Archaea have industrial relevance. Despite intensive investigations, our knowledge of the structure-function relationships of their enzymes is still limited. Information concerning the molecular properties of their enzymes and genes has to be obtained to be able to understand the mechanisms that are responsible for catalytic activity and stability at the boiling point of water.     

工业蛋白质理性设计与应用

作为合成生物学与绿色生物制造等领域的底层核心技术,蛋白理性设计可有效解决天然功能元件性能不足等共性挑战,创制高性能人工酶元件。值此天津工业生物研究所(Tianjin Institute of Industrial Biotechnology, TIB)创立10周年之际,文中回顾了研究所在工业蛋白理性设计领域的系列重要工作进展。从酶设计方法学研究、新酶反应设计到生物催化应用等方面进行了分析讨论,并展望了本领域未来发展方向。望借此搭建学术界和产业界与酶理性设计的桥梁,促进新技术、新策略的开发应用,加速融合人工酶的基础研究与产业应用,推动我国生物制造领域的科技创新升级。     

Methanol,a potential feedstock for biotechnological processes

A wide variety of bacteria and yeasts is able to grow in inexpensive synthetic media with methanol as the sole or major source of carbon and energy. This is due to the presence of a few unique enzymes which enable these organisms to generate metabolic energy and synthesize cell constituents from this one-carbon substrate. In the chemical industry there is currently much interest in the production of fuels and chemicals from methanol. As a feedstock for industrial fermentations methanol is also attractive because of its low cost, ease of handling and abundant availability. In many countries methanol-utilizing microbes are being studied and their potential utility in biotechnological processes is explored. These studies are aimed at making use of their characteristic properties, exploiting known organisms and new strains for improving existing processes and developing novel products.     

Phospholipases and their industrial applications

Phospholipids are present in all living organisms. They are a major component of all biological membranes, along with glycolipids and cholesterol. Enzymes aimed at modifying phospholipids, namely, phospholipases, are consequently widespread in nature, playing very diverse roles from aggression in snake venom to signal transduction and digestion in humans. In this review, we give a general overview of phospholipases A₁, A₂, C and D from a sequence and structural perspective and their industrial application. The use of phospholipases in industrial processes has grown hand-in-hand with our ability to clone and express the genes in microbial hosts with commercially attractive amounts. Further, the use in industrial processes is increasing by optimizing the enzymes by protein engineering. Here, we give a perspective on the work done to date to express phospholipases in heterologous hosts and the efforts to optimize them by protein engineering. We will draw attention to the industrial processes where phospholipases play a key role and show how the use of a phospholipase for oil degumming leads to substantial environmental benefits. This illustrates a very general trend: the use of enzymes as an alternative to chemical processes to make products often provides a cleaner solution for the industrial processes. In a world with great demands on non-polluting, energy saving technical solutions—white biotechnology is a strong alternative. An erratum to this article can be found at     

Practical insights on enzyme stabilization

Enzymes are efficient catalysts designed by nature to work in physiological environments of living systems. The best operational conditions to access and convert substrates at the industrial level are different from nature and normally extreme. Strategies to isolate enzymes from extremophiles can redefine new operational conditions, however not always solving all industrial requirements. The stability of enzymes is therefore a key issue on the implementation of the catalysts in industrial processes which require the use of extreme environments that can undergo enzyme instability. Strategies for enzyme stabilization have been exhaustively reviewed, however they lack a practical approach. This review intends to compile and describe the most used approaches for enzyme stabilization highlighting case studies in a practical point of view.     

Metagenomics: an inexhaustible access to nature's diversity

The chemical industry has an enormous need for innovation. To save resources, energy and time, currently more and more established chemical processes are being switched to biotechnological routes. This requires white biotechnology to discover and develop novel enzymes, biocatalysts and applications. Due to a limitation in the cultivability of microbes living in certain habitats, technologies have to be established which give access to the enormous resource of uncultivated microbial diversity. Metagenomics promises to provide new and diverse enzymes and biocatalysts as well as bioactive molecules and has the potential to make industrial biotechnology an economic, sustainable success.