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Biocatalysts and Enzyme Technology
By Klaus Buchholz V. Kasche U. T. Bornscheuer
John Wiley & Sons Copyright © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
All right reserved.
Chapter One Introduction to Enzyme Technology
Biotechnology offers an increasing potential for the production of goods to meet various human needs. In enzyme technology - a sub-field of biotechnology - new processes have been and are being developed to manufacture both bulk and high added-value products utilizing enzymes as biocatalysts, in order to meet needs such as food (e.g., bread, cheese, beer, vinegar), fine chemicals (e.g., amino acids, vitamins), and pharmaceuticals. Enzymes are also used to provide services, as in washing and environmental processes, or for analytical and diagnostic purposes. The driving force in the development of enzyme technology, both in academia and industry, has been and will continue to be:
the development of new and better products, processes and services to meet these needs; and/or
the improvement of processes to produce existing products from new raw materials as biomass.
The goal of these approaches is to design innovative products and processes that are not only competitive but also meet criteria of sustainability. The concept of sustainability was introduced by the World Commission on Environment and Development (WCED, 1987) with the aim to promote a necessary "... development that meets the needs of the present without compromising the ability of future generations to meet their own needs". To determine the sustainability of a process, criteria that evaluate its economic, environmental and social impact must be used (Gram et al., 2001; Raven, 2002; Clark and Dickson, 2003). A positive effect in all these three fields is required for a sustainable process. Criteria for the quantitative evaluation of the economic and environmental impact are in contrast with the criteria for the social impact, easy to formulate. In order to be economically and environmentally more sustainable than an existing processes, a new process must be designed to reduce not only the consumption of resources (e.g., raw materials, energy, air, water), waste production and environmental impact, but also to increase the recycling of waste per kilogram of product.
1.1.1 What are Biocatalysts?
Biocatalysts are either proteins (enzymes) or, in a few cases, they may be nucleic acids (ribozymes; some RNA molecules can catalyze the hydrolysis of RNA. These ribozymes were detected in the 1980s and will not be dealt with here; Cech, 1993). Today, we know that enzymes are necessary in all living systems, to catalyze all chemical reactions required for their survival and reproduction - rapidly, selectively and efficiently. Isolated enzymes can also catalyze these reactions. In the case of enzymes however, the question whether they can also act as catalysts outside living systems had been a point of controversy among biochemists in the beginning of the twentieth century. It was shown at an early stage however that enzymes could indeed be used as catalysts outside living cells, and several processes in which they were applied as biocatalysts have been patented (see Section 1.3).
These excellent properties of enzymes are utilized in enzyme technology. For example, they can be used as biocatalysts to catalyze chemical reactions on an industrial scale in a sustainable manner. Their application covers the production of desired products for all human material needs (e.g., food, animal feed, pharmaceuticals, fine and bulk chemicals, fibers, hygiene, and environmental technology), as well as in a wide range of analytical purposes, especially in diagnostics. In fact, during the past 50 years the rapid increase in our knowledge of enzymes - as well as their biosynthesis and molecular biology - now allows their rational use as biocatalysts in many processes, and in addition their modification and optimization for new synthetic schemes and the solution of analytical problems.
This introductory chapter outlines the technical and economic potential of enzyme technology as part of biotechnology. Briefly, it describes the historical background of enzymes, as well as their advantages and disadvantages, and compares these to alternative production processes. In addition, the current and potential importance - and the problems to consider in the rational design of enzyme processes - are also outlined.
1.2 Goals and Potential of Biotechnological Production Processes
Biomass - that is, renewable raw materials - has been and will continue to be a sustainable resource which is required to meet a variety of human material needs. In developed countries such as Germany, biomass covers [approximately equal to] 30% of the raw material need - equivalent to ~7000 kg per person per year. The distribution of biomass across different human demands is shown schematically in Figure 1.1. This distribution of the consumption is representative for a developed country in the regions that have a high energy consumption during the winter. However, the consumption of energy (expressed as tons of coal equivalent per capita in 1999) showed a wide range, from 11.4 in the United States, to 5.5 in Germany and the UK, 0.8 in China, and 0.43 in India (United Nations, 2002). This is mainly due to differences in energy use for housing, transport and the production of other material needs. In less-developed countries, although the fraction of biomass as raw material to meet human demands is higher than in the developed countries, the total consumption is smaller.
Biomass - in contrast to non-renewable raw materials such as metals, coal, and oil - is renewable in a sustainable manner when the following criteria are fulfilled:
the C-, N-, O-, and salt-cycles in the biosphere are conserved; and
the conditions for a sustainable biomass production through photosynthesis and biological turnover of biomass in soil and aqueous systems are conserved.
Currently, these criteria are not fulfilled on a global level, one example being the imbalance between the C[O.sub.2] production to meet energy requirements and its consumption by photosynthesis in the presently decreasing areas of rain forests. This leads to global warming and other consequences that further violate these criteria. International treaties - for example, the Kyoto convention - have been introduced in an attempt to counteract these developments and to reach a goal that fulfills the above criteria.
When the above sustainability criteria are fulfilled, biomass can be used as raw material to meet the human demands illustrated in Figure 1.1. The needs for human food and animal feed must be met completely by biomass, though when these needs of highest priority are met, biomass can be used to fulfill the other demands shown in Figure 1.1. This applies especially to those areas with lower total raw material consumption than for food. From this point, it also follows that a large consumption of biomass to meet energy demands is only possible in countries with a low population density and a high biomass production.
By definition, biotechnological processes are especially suited to the production of compounds from biomass as the raw material (Fig. 1.2). The economic importance of such processes is detailed in Table 1.1. This also involves the development of suitable equipment to obtain more sustainable processes. From the information provided in Figure 1.1 it also follows that biotechnology has a major potential in the development of sustainable processes to meet all human needs.
Enzyme technology is a part of biotechnology that the European Federation of Biotechnology 1989 defined as:
"Biotechnology is the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services."
However, the following amendment was added:
"It is a clear understanding that Biotechnology is directed to the benefit of mankind by obeying biological principles."
Some of these principles have been outlined above. Here, we add the requirement that traditional classical - as well as new biotechnological processes - must be improved and/or developed in order to be sustainable (Fig. 1.2). The fundamentals needed for the development of such processes in the interdisciplinary field of biotechnology require the close cooperation of biologists, chemists, bioengineers, and chemical engineers.
1.3 Historical Highlights of Enzyme Technology/Applied Biocatalysis
1.3.1 Early Developments
Applied biocatalysis has its roots in the ancient manufacture and preservation of food and alcoholic drinks, as can be seen in old Egyptian pictures. Cheese making has always involved the use of enzymes, and as far back as about 400 BC, Homer's Iliad mentions the use of a kid's stomach for making cheese.
With the development of modern natural science during the 18th and 19th centuries, applied biocatalysis began to develop a more scientific basis. In 1833, Payen and Persoz investigated the action of extracts of germinating barley in the hydrolysis of starch to yield dextrin and sugar, and went on to formulate some basic principles of enzyme action (Payen and Persoz, 1833):
small amounts of the preparation were able to liquify large amounts of starch,
the material was thermolabile,
the active substance could be precipitated from aqueous solution by alcohol, and thus be concentrated and purified. This active substance was called diastase (a mixture of amylases).
In 1835, the hydrolysis of starch by diastase was acknowledged as a catalytic reaction by Berzelius. In 1839, he also interpreted fermentation as being caused by a catalytic force, and postulated that a body - by its mere presence - could, by affinity to the fermentable substance, cause its rearrangement to the products (Hoffmann-Ostenhof, 1954).
The application of diastase was a major issue from the 1830s onwards, and the enzyme was used to produce dextrin that was used mainly in France in bakeries, and also in the production of beer and wines from fruits. The process was described in more detail, including its applications and economic calculations, by Payen (1874) (Fig. 1.3). Indeed, it was demonstrated that the use of malt in this hydrolytic process was more economic than that of sulfuric acid.
Lab preparations were also used to produce cheese (Knapp, 1847), and Berzelius later reported that 1 part of lab ferment preparation coagulated 1800 parts of milk, and that only 0.06 parts of the ferment was lost. This provided further evidence for Berzelius' hypothesis that ferments were indeed catalysts.
About two decades later, the distinction of organized and unorganized ferments was proposed (Wagner, 1857), and further developed by Payen (1874). These investigators noted that fermentation appeared to be a contact (catalytic) process of a degradation or addition process (with water), and could be carried out by two substances or bodies:
A nitrogen-containing organic (unorganized) substance, such as protein material undergoing degradation.
A living (organized) body, a lower-class plant or an "infusorium", an example being the production of alcohol by fermentation.
It is likely that the effect is the same, insofar as the ferment of the organized class produces a body of the unorganized class - and perhaps a large number of singular ferments. Consequently, in 1878, Kuhne named the latter class of substances, enzymes.
Progress in the knowledge of soluble ferments (enzymes) remained slow until the 1890s, mainly due to a scientific discussion where leading scientists such as Pasteur denied the existence of "unorganized soluble ferments" that had no chemical identity. Consequently, the subject of enzymatic catalysis remained obscure, and was considered only to be associated with processes in living systems. In the theory of fermentation, a degree of mystery still played a role: Some vital factor, "le principe vital", which differed from chemical forces, was considered to be an important principle in the chemical processes associated with the synthesis of materials isolated from living matter. But Liebing and his school took an opposite view, and considered fermentation simply to be a decay process.
In 1874, in Copenhagen, Denmark, Christian Hansen started the first company (Christian Hansen's Laboratory) for the marketing of standardized enzyme preparations, namely rennet for cheese making (Buchholz and Poulson, 2000).
Scientific Progress Since 1890: The Biochemical Paradigm; Growing Success in Application
From about 1894 onwards, Emil Fischer elaborated on the essential aspects of enzyme catalysis. The first aspect was specificity, and in a series of experiments Fischer investigated the action of different enzymes using several glycosides and oligosaccharides. For this investigation he compared invertin and emulsin. He extracted invertin from yeast - a normal procedure - and showed that it hydrolyzed the [alpha]-, but not the ss-methyl-D-glucoside. In contrast, emulsin - a commercial preparation from Merck - hydrolyzed the ss-, but not the [alpha]-methyl-D-glucoside. Fischer therefore deduced the famous picture of a "lock and key", which he considered a precondition for the potential of an enzyme to have a chemical effect on the substrate. In this way he assumed that the "geometrical form of the (enzyme) molecule concerning its asymmetry, corresponds to that of the natural hexoses" (sugars) (Fischer, 1909).
The second aspect referred to the protein nature of enzymes. In 1894, Fischer stated that amongst the agents which serve the living cell, the proteins are the most important. He was convinced that enzymes were proteins, but it took more than 20 years until the chemical nature of enzymes was acknowledged. Indeed, Willstatter, as late as 1927, still denied that enzymes were proteins (Fruton, 1979).
A few years after Fischer's initial investigations, Eduard Buchner published a series of papers (1897, 1898) which signalled a breakthrough in fermentation and enzymology. In his first paper on alcoholic fermentation without yeast cells, he stated, in a remarkably short and precise manner, that "... a separation of the (alcoholic) fermentation from the living yeast cells was not successful up to now". In subsequent reports he described a process which solved this problem (Buchner, 1897), and provided experimental details for the preparation of a cell-free pressed juice from yeast cells, that transformed sugar into alcohol and carbon dioxide. Buchner presented the proof that (alcoholic) fermentation did not require the presence of "... such a complex apparatus as is the yeast cell". The agent was in fact a soluble substance - without doubt a protein body - which he called zymase (Buchner, 1897). In referring to the deep controversy on his findings and theory, and in contradiction to the ideas of Pasteur (see above), Buchner insisted that his new experimental findings could not be disproved by older theories.
After a prolonged initial period of about a hundred years, during which time a number of alternative and mysterious theories were proposed, Buchner's elaborate results brought about a new biochemical paradigm. It stated - in strict contrast to the theories of Pasteur - that enzyme catalysis, including complex phenomena such as alcoholic fermentation, was a chemical process not necessarily linked to the presence and action of living cells, nor requiring a vital force - a vis vitalis. With this, the technical development of enzymatic processes was provided with a new, scientific basis on which to proceed in a rational manner.
The activity in scientific research on enzymes increased significantly due to this new guidance, and was reflected in a pronounced increase in the number of papers published on the subject of soluble ferments from the mid-1880s onwards (Buchholz and Poulson, 2000). Further important findings followed within a somewhat short time. In 1898, Croft-Hill performed the first enzymatic synthesis - of isomaltose - by allowing a yeast extract ([alpha]-glycosidase) to act on a 40 % glucose solution (Sumner and Somers, 1953). In 1900, Kastle and Loevenhart showed that the hydrolysis of fat and other esters by lipases was a reversible reaction, and that enzymatic synthesis could occur in a dilute mixture of alcohol and acid (Sumner and Myrback, 1950). This principle was subsequently utilized in the synthesis of numerous glycosides by Fischer and coworkers in 1902, and by Bourquelot and coworkers in 1913 (Wallenfels and Diekmann, 1966). In 1897, Bertrand observed that certain enzymes required dialysable substances to exert catalytic activity, and these he termed coenzymes.
Excerpted from Biocatalysts and Enzyme Technology by Klaus Buchholz V. Kasche U. T. Bornscheuer Copyright © 2005 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Excerpted by permission.
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