Future prospects of enzyme engineering and enzyme technology

Future prospects of enzyme engineering

Enzyme engineering is the recent technology growing rapidly due to its higher application in a lot of fields and due to having bright and clear future vision. A most exciting development over the last few years is the application of genetic engineering techniques to enzyme technology. There are a number of properties which may be improved or altered by genetic engineering including the yield and kinetics of the enzyme, the ease of downstream processing and various safety aspects. Enzymes from dangerous or unapproved microorganisms and from slow-growing or limited plant or animal tissue may be cloned into safe high-production microorganisms. The amount of enzyme produced by a microorganism may be increased by increasing the number of gene copies that code for it. For example; The engineered cells, aided by the plasmid amplification at around 50 copies per cell, produce penicillin – G – Amidase constitutively and in considerably higher quantities than does the fully induced parental strain. Such increased yields are economically relevant not just for the increased volumetric productivity but also because of reduced downstream processing costs, the resulting crude enzyme being that much purer. New enzyme structures may be designed and produced in order to improve on existing enzymes or create new activities. Much protein engineering has been directed at Subtilisin (from Bacillus amyloliquefaciens), the principal enzyme in the detergent enzyme preparation, Alcalase. This has been aimed at the improvement of its activity in detergents by stabilizing it at even higher temperatures, pH and oxidant strength. A number of possibilities now exist for the construction of artificial enzymes. These are generally synthetic polymers or oligomers with enzyme-like activities, often called synzymes. Enzymes can be immobilized i.e., an enzyme can be linked to an inert support material without loss of activity which facilitates reuse and recycling of the enzyme.Use of engineered enzyme to form biosensor for the analytical use is also recent activity among the developed countries. Some enzymes make use in diseases diagnosis so they can be genetically engineered to make the task easier. Thus it is obvious that there is huge scope of the enzyme technology in the future as well as in present.

Introduction

Enzymes are Organic compounds, produced in the living cells to speed up chemical reaction in the biological systems so that they can take place at relatively lower temperature, but themselves remain apparently unchanged during the process. Therefore enzymes are termed as 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. 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 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

Enzymes have become big business. They are used in many industrial processes to catalyze biological reactions. Enzymes are exploited in a variety of manufacturing processes such as food processing and for the synthesis of medicines such as antibiotics like artificial penicillin. They are also used to clean up factory effluents and pollution in water and soil. Many processes can be made faster and cheaper by using the right enzyme and conditions.

Optimum conditions are maintained during factory production by use of bioreactors. These are vessels which are designed to provide the ideal environment for reactions involving enzymes or living organisms. Source of enzymes used commercial production is plant, animal and microbial cells. Animal enzymes used currently are lipases, tripsin, rennets etc. Most prevalent plant enzymes are papain, proteases, amylases and soybean lipoxygenase. These enzymes are used in food industries, for example, papain extracted from papaya fruit is used as meat tenderizer and pancreatic protease in leather softening and manufacture of detergents. In addition microbial enzymes have gained much popularity. Production of primary and secondary metabolites by microorganism is possible only due to involvement of various enzymes. They are of two types: the extracellular and the intracellular enzymes. There is a wide range of extracellular enzymes produced by pathogenic and saprophytic microorganisms such as cellulose, polymethylegalactouronase, pectinmethylesterase etc. These enzyme helps in establishment in host tissues or decomposition of organic substrates. The intracellular enzyme like invertase, uricoxidase, asparaginase are of high economic value and difficult to extract as they produced inside the cell. They can be extracted by breaking the cells by means of a homogenizer or a ball mill and extracted them through the biochemical process.

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. 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.

Sources of enzymes: Biologically active enzymes may be extracted from any living organism. A very wide range of sources are used for commercial enzyme production from Actinoplanes to Zymomonas, from spinach to snake venom. Of the hundred or so enzymes being used industrially, over a half are from fungi and yeast and over a third are from bacteria with the remainder divided between animal (8%) and plant (4%) sources. A very much larger number of enzymes find use in chemical analysis and clinical diagnosis. Non-microbial sources provide a larger proportion of these, at the present time. Microbes are preferred to plants and animals as sources of enzymes because:

  1. they are generally cheaper to produce.

  2. their enzyme contents are more predictable and controllable,

  3. reliable supplies of raw material of constant composition are more easily arranged, and

  4. plant and animal tissues contain more potentially harmful materials than microbes, including phenolic compounds (from plants), endogenous enzyme inhibitors and proteases.

Table 1 . Some important industrial enzymes and their sources.

Enzyme

EC number

Source

Intra/extra
-cellular

Scale of production

Industrial  use

Animal enzymes

Catalase

1.11.1.6

Liver

I

Food

Chymotrypsin

3.4.21.1

Pancreas

E

Leather

Lipase

3.1.1.3

Pancreas

E

Food

Rennet

3.4.23.4

Abomasum

E

+

Cheese

Trypsin

3.4.21.4

Pancreas

E

Leather

Plant enzymes

Actinidin

3.4.22.14

Kiwi fruit

E

Food

a-Amylase

3.2.1.1

Malted barley

E

+++

Brewing

b-Amylase

3.2.1.2

Malted barley

E

+++

Brewing

Bromelain

3.4.22.4

Pineapple latex

E

Brewing

b-Glucanase

3.2.1.6

Malted barley

E

++

Brewing

Ficin

3.4.22.3

Fig latex

E

Food

Lipoxygenase

1.13.11.12

Soybeans

I

Food

Papain

3.4.22.2

Pawpaw latex

E

++

Meat

Bacterial enzymes

a-Amylase

3.2.1.1

Bacillus

E

+++

Starch

b-Amylase

3.2.1.2

Bacillus

E

+

Starch

Asparaginase

3.5.1.1

Escherichia coli

I

Health

Glucose isomerase

5.3.1.5

Bacillus

I

++

Fructose syrup

Penicillin amidase

3.5.1.11

Bacillus

I

Pharmaceutical

Protease

3.4.21.14

Bacillus

E

+++

Detergent

Pullulanase

3.2.1.41

Klebsiella

E

Starch

Fungal enzymes

a-Amylase

3.2.1.1

Aspergillus

E

++

Baking

Aminoacylase

3.5.1.14

Aspergillus

I

Pharmaceutical

Glucoamylase

3.2.1.3

Aspergillus

E

+++

Starch

Catalase

1.11.1.6

Aspergillus

I

Food

Cellulase

3.2.1.4

Trichoderma

E

Waste

Dextranase

3.2.1.11

Penicillium

E

Food

Glucose oxidase

1.1.3.4

Aspergillus

I

Food

Lactase

3.2.1.23

Aspergillus

E

Dairy

Lipase

3.1.1.3

Rhizopus

E

Food

Rennet

3.4.23.6

Mucor miehei

E

++

Cheese

Pectinase

3.2.1.15

Aspergillus

E

++

Drinks

Pectin lyase

4.2.2.10

Aspergillus

E

Drinks

Protease

3.4.23.6

Aspergillus

E

+

Baking

Raffinase

3.2.1.22

Mortierella

I

Food

Yeast enzymes

Invertase

3.2.1.26

Saccharomyces

I/E

Confectionery

Lactase

3.2.1.23

Kluyveromyces

I/E

Dairy

Lipase

3.1.1.3

Candida

E

Food

Raffinase

3.2.1.22

Saccharomyces

I

Food

Once the enzyme has been purified to the desired extent and concentrated, the manufacturer’s main objective is to retain the activity. Enzymes for industrial use are sold on the basis of overall activity. To achieve stability, the manufacturer should follow the recent advanced technology even genetic engineering thechniques.Most industrial enzymes contain relatively little active enzyme (< 10% w/w, including isoenzymes and associated enzyme activities), the rest being due to inactive protein, stabilisers, preservatives, salts and the diluent which allows standardisation between production batches of different specific activities.The key to maintaining enzyme activity is maintenance of conformation, so preventing unfolding, aggregation and changes in the covalent structure. Three approaches are possible: use of additives, the controlled use of covalent modification, and enzyme immobilization. So if the genetic engineering along with the advanced technique for enzyme engineering are employed there might be the great possibility of increasing the half life of active protein and their stability as well as specificity which will certainly reduce conventional methods for stabilizing the enzymes.

Screening for novel enzymes: One of the major skills of enzyme companies and suitably funded academic laboratories is the rapid and cost-effective screening of microbial cultures for enzyme activities. Natural samples, usually soil or compost material found near high concentrations of likely substrates, are used as sources of cultures.

Preparation of enzymes: After the screening of the novel enzyme having great commercial as well as industrial use, enzyme is prepared by optimizing the condition of higher production with available resources. Purification of enzyme after preparation depends upon its future use. Often the enzyme may be purified several hundred-fold but the yield of the enzyme may be very poor, frequently below 10% of the activity of the original material. In contrast, industrial enzymes will be purified as little as possible, only other enzymes and material likely to interfere with the process which the enzyme is to catalyze, will be removed.                         Fig.1 Flow diagram for the preparation of enzymes.

Genetic Protein Engineering of Enzymes


A most exciting development over the last few years is the application of genetic engineering techniques to enzyme technology. Recombinant DNA technology has allowed the transfer of useful enzyme genes from one organism to another. Thus, when an enzyme has been identified as a good candidate enzyme for industrial use, the relevant gene can be cloned into a more suitable production host microorganism and an industrial fermentation carried out. In this way, it becomes possible to produce industrial enzymes of very high quality and purity. A recent example of this technology is the detergent enzyme Lipolase produced by Novo Nordisk A/S, which has improved removal of fat stains in fabrics. The enzyme was first identified in the fungus Humicola languinosa at levels inappropriate for commercial production. The gene DNA fragment for the enzyme was cloned into the fungus Aspergillus oryzae and commercial levels of enzyme achieved. The enzyme has proved to be efficient under many wash conditions. The enzyme is also very stable at a variety of temperature and pH conditions relevant to washing.

There are a number of properties which may be improved or altered by genetic engineering including the yield and kinetics of the enzyme, the ease of downstream processing and various safety aspects. Enzymes from dangerous or unapproved microorganisms and from slow-growing or limited plant or animal tissue may be cloned into safe high-production microorganisms.

All proteins, including enzymes, are based on the same 20 different amino acid building blocks arranged in different sequences. Enzyme proteins typically comprise sequences of several

hundred amino acids folded in a unique three-dimensional structure. Only the sequence of these 20 building blocks determines the three-dimensional structure, which in turn determines all properties such as catalytic activity, specificity and stability. Nature has been performing ‘protein engineering’ for billions of years since the very start of evolution. Natural spontaneous mutations in the DNA coding for a given protein result in changes of the protein structure and hence its properties. This natural variation is part of the adaptive evolutionary process continuously taking place in all living organisms, allowing them to survive in continuously changing environments. Natural variants of enzyme proteins are adapted to perform efficiently in different environments and conditions. This explains why in nature enzymes belonging to the same enzyme family but isolated from different organisms and environments often show a variation in amino acid sequence of more than 50%. The properties of enzymes used for industrial purposes sometimes also require some adaptations in order to function more effectively in applications for which they were not designed by nature. Traditionally, such enzyme optimization is performed by screening naturally occurring microorganisms, followed by classical mutation and selection. The disadvantage of this method is, however, that it may take a very long time until the enzyme with the desired properties is found. This is why protein engineering was developed.

Assumptions for Protein Engineering

While attempting protein engineering, one should recognize the following properties of enzymes:

(i) many amino acid substitutions, deletions or additions lead to no change in enzyme activity, so that they are silent mutations;

(ii) proteins have a limited number of basic structures and only minor changes are superimposed on them leading to variation;

(iii) similar patterns of chain folding and domain structure can arise from different amino acid sequences, which show little or no homology (although same amino acid sequence never gives different folding and domain structures).

The above properties suggest that while many major changes sometimes may lead to no alteration in function, some of the minor changes at specific positions may lead to the desired favourable change.

For example, a single amino acid replacement (glycine to aspartic acid) in E. coli asparate transcarbamylase leads to

(i) loss of activity and to

(ii) an alteration in the binding of catalytic and regulatory subunits. Another example involved the engineering of a single chain biosynthetic antibody binding site (BARS), which is though only one sixth of the size of the complete antibody, but retains its antigen binding specificity.

This synthetic fragment has heavy and light chain variable regions (V H and V J connected by a 15 – amino acid linker. A synthetic gene has also been prepared for the fragment, which expressed in E. coli. This fragment binds to digoxin, a cradiac glycoside. Single amino acid replacements in BABS fragment have sometimes led to major changes in its binding affinity.

In view of the above, it is necessary to examine not only the crystal structure but also the active sites therein, so that the gene may be modified or artificially synthesized for protein engineering to meet the desired needs.

Methods for Protein Engineering

A variety of methods have been used and proposed for future use in protein engineering. In this connection mutagenesis, selection, and recombinant DNA are being used and will be increasingly utilized in future.

1. Mutagenesis and Selection for Protein Engineering – Mutagenesis and selection can be effectively utilized for improving a specific property of an enzyme. Following are some of the examples of selection of mutant enzymes:

(i) E. coli anthranilate synthetase enzyme is normally sensitive to tryptophan inhibition due to feedback inhibition. An MTR 2 mutation of E. coli was found to possess an altered form of enzyme anthranilate synthetase that is insensitive to tryptophan inhibition. They may help in continuous synthesis of tryptophan without any inhibition by tryptophan accumulated as a product.

(ii) Xanthine dehydrogenase enzyme oxidizes 2 hydroxy-purine at position 8, but a mutant has been inolated which oxidizes 2 hydroxy-purine at position 6.

(iii) Lactate dehydrogenase (LDU) from a bacterial system was modified to malate dehydrogenase able a natural mutation leading to a single amino acid substitution (Gln 02… Arg; see later m thIS chapter).

In the above and other cases of naturally occurring mutant enzymes, single amino acid modification or addition/deletion has been observed.

However, if improvement requires changes in several amino acids, such a mutant will be rare or nonexistent and modifications of this type will be possible only through gene modification techniques discussed in the following section.

2. Production of Artificial Semi Synthetic Oxido Reductases – Flavo Enzymes – Artificial oxido reductases can be prepared by covalently attaching redoxactive prosthetic groups to existing sites. Linking of 10-methyilsoalloxazine derivatives (as redox-active groups) to specific sites of several proteins has been achieved. The efficiency of these semisynthetic enzymes (e.g. flavopapain) compares favourably with that of naturally occurring flavoenzymes.

3. Modification of Proteases into Peptide Ligases -Peptide ligation to native enzymes may lead to high specificity and stereoselecitivity, and may suppress side reactions. Therefore, synthesis of any enzyme that may catalyze peptide ligation will be most welcome.

Protease ‘subtilisin’ has been modified (by converting a serine into cysteine or seleno-cysteine) into thiol-and selenolsubtilisin, the two semi synthetic enzymes (they are damaged proteases), which can catalyse peptide ligation. Both these damaged proteases are efficient peptide ligases. Similarly histidine residue can also be modified to yield peptide ligases.

4. Enzyme PEG Conjugates – An enzyme L- asparaginase (isolated from microbes) has antitumour properties, but is toxic with a life time of less then 18hr thus reducing its utility. It has been shown that E. coli L­-asparaginase can be modified by polyethylene glycol derivatives to produce PEG-asparaginase conjugates , which differ from the native enzyme in following features:
(i) it retains only 52% of the catalytic activity of native enzyme;
(ii) it becomes resistant to proteolytic degradation; (Hi) it does not cause allergy. In view of this, PEG-asparaginase has been used to treat malignant murine (mouse), canine (cats, etc.) and human tumours. PEG conjugates of a large number of enzymes (adenosine deaminase, uricase, catalase, etc.) have been prepared and will be utilized in industry also.

5. Production of Site Specific Nucleases – Restriction Enzymes – The DNA recognition and binding properties of proteins can be combined using chemical cleavage agents. Cys178 of E. coli CAP protein; has been modified using ‘S-iodoacetamide -1, 10- phenanthroline’ yielding a DNA cleaving agent that recognized and cleaved DNA at the centre of the recognition site (22 bp) for CAP.

This may give restriction enzymes recognizing upto 20 bases instead of 6 or 8 bases and may, therefore, be useful for isolating long DNA fragments needed for sequencing and mapping. Nucleases may also be produced by fusion of non-specific phosphodiesterases to oligonucleotides of defined sequence.

For a nuclease from Staphylococcus modified by this approach, it was shown that oligonucleotide component of fused product pairs with its complementary sequence and the hybrid enzyme hydrolyses single stranded DNA or RNA adjacent to the oligonucleotide binding site. This approach thus can also be used for developing artificial restriction enzymes.

Protein engineering and how it is applied to enzymes

A most exciting development over the last few years is the application genetic engineering techniques to enzyme technology. Protein engineering of enzymes is a faster, more controlled, more targeted and more accurate method to optimize the properties of enzymes for a specific industrial application than the traditional method described above. It makes it possible to sidestep the high number of natural isolate screenings that would otherwise be necessary to find the enzyme with the desired properties, and increases the likelihood that a suitable enzyme will be found. The protein engineering technique involves genetic modification by means of recombinant DNA technology of the enzyme producing microorganism, in particular the enzyme encoding gene, resulting in substitution of one or more amino acids in the amino acid sequence of the enzyme protein. Strategies for making such amino acid substitutions and developing protein engineered enzymes are based on the knowledge of the structure/function relationships of enzymes, computer modeling and techniques for creating and testing enzyme variants.

Enzyme technology is the application of modifying an enzyme’s structure (and thus its function) or modifying the catalytic activity of isolated enzymes to produce new metabolites, to allow new (catalyzed) pathways for reactions to occur, or to convert from some certain compounds into others (biotransformation). These products will be useful as chemicals, pharmaceuticals, fuel, food or agricultural additives. An enzyme reactor consists of a vessel containing a reactional medium that is used to perform a desired conversion by enzymatic means. Enzymes used in this process are free in the solution or immobilized in particulate, membranous or fibrous support. There are many directions in which enzyme technologists are currently applying their art and which are at the forefront of biotechnological research and development. Some of these have already been examined in some detail earlier. At present, relatively few enzymes are available on a large scale (i.e. > kg) and are suitable for industrial applications. These shortcomings are being addressed in a number of ways:

  1. New enzymes are being sought in the natural environment and by strain selection

  2. Novel enzymes are being designed and produce by genetic engineering;

  3. New organic catalysts are being designed and synthesized using the ‘knowhow’ established from enzymology; and

  4. More complex enzyme systems are being utilized.

Each of these areas has a extensive and rapidly expanding literature. Some advances possibly belong more properly to other areas of science. Thus, the development of genetically improved enzymes is generally undertaken by molecular biologists and the design and synthesis of novel enzyme-like catalysts is in the provenance of the organic chemists. Both groups of workers will, however, base their science on data provided by the enzyme technologist.

There are a number of properties which may be improved or altered by genetic engineering including the yield and kinetics of the enzyme, the ease of downstream processing and various safety aspects. Enzymes from dangerous or unapproved microorganisms and from slow growing or limited plant or animal tissue may be cloned into safe high-production microorganisms. In the future, enzymes may be redesigned to fit more appropriately into industrial processes; for example, making glucose isomerase less susceptible to inhibition by the Ca2+ present in the starch saccharification processing stream.

The amount of enzyme produced by a microorganism may be increased by increasing the number of gene copies that code for it. This principle has been used to increase the activity of penicillin-G-amidase in Escherichia coli. The cellular DNA from a producing strain is selectively cleaved by the restriction endonuclease HindIII. This hydrolyses the DNA at relatively rare sites containing the 5′-AAGCTT-3′ base sequence to give identical ‘staggered’ ends.

[Fig2]
intact DNA cleaved DNA

The total DNA is cleaved into about 10000 fragments, only one of which contains the required genetic information. These fragments are individual cloned into a cosmid vector and thereby returned to E. coli. These colonies containing the active gene are identified by their inhibition of a 6-amino-penicillanic acid-sensitive organism. Such colonies are isolated and the penicillin-G-amidase gene transferred on to pBR322 plasmids and recloned back into E. coli. The engineered cells, aided by the plasmid amplification at around 50 copies per cell, produce penicillin-G-amidase constitutively and in considerably higher quantities than does the fully induced parental strain. Such increased yields are economically relevant not just for the increased volumetric productivity but also because of reduced downstream processing costs, the resulting crude enzyme being that much purer.

The process starts with the isolation and characterisation of the required enzyme. This information is analysed together with the database of known and putative structural effects of amino acid substitutions to produce a possible improved structure. This factitious enzyme is constructed by site-directed mutagenesis, isolated and characterised. The results, successful or unsuccessful, are added to the database, and the process repeated until the required result is obtained.

Another extremely promising area of genetic engineering is protein engineering. New enzyme structures may be designed and produced in order to improve on existing enzymes or create new activities. An outline of the process of protein engineering is shown in Figure 2. Such factitious enzymes are produced by site-directed mutagenesis (Figure 3). Unfortunately from a practical point of view, much of the research effort in protein engineering has gone into studies concerning the structure and activity of enzymes chosen for their theoretical importance or ease of preparation rather than industrial relevance. This emphasis is likely to change in the future. Figure 2. The protein engineering cycle.

As indicated by the method used for site-directed mutagenesis (Figure 3), the preferred pathway for creating new enzymes is by the stepwise substitution of only one or two amino acid residues out of the total protein structure. Although a large database of sequence-structure correlations is available, and growing rapidly together with the necessary software, it is presently insufficient accurately to predict three-dimensional changes as a result of such substitutions. The main problem is assessing the long-range effects, including solvent interactions, on the new structure. As the many reported results would attest, the science is at a stage where it can explain the structural consequences of amino acid substitutions after they have been determined but cannot accurately predict them. Protein engineering, therefore, is presently rather a hit or miss process which may be used with only little realistic likelihood of immediate success. Apparently quite small sequence changes may give rise to large conformational alterations and even affect the rate-determining step in the enzymic catalysis. However it is reasonable to suppose that, given a sufficiently detailed database plus suitable software, the relative probability of success will increase over the coming years and the products of protein engineering will make a major impact on enzyme technology.

Much protein engineering has been directed at subtilisin (from Bacillus amyloliquefaciens), the principal enzyme in the detergent enzyme preparation, Alcalase. This has been aimed at the improvement of its activity in detergents by stabilising it at even higher temperatures, pH and oxidant strength. Most of the attempted improvements have concerned alterations to:

  1. the P1 cleft, which holds the amino acid on the carbonyl side of the targeted peptide bond;

  2. the oxyanion hole (principally Asn155), which stabilises the tetrahedral intermediate;

  3. the neighbourhood of the catalytic histidyl residue (His64), which has a general base role; and

  4. the methionine residue (Met222) which causes subtilisin’s lability to oxidation.

It has been found that the effect of a substitution in the P1 cleft on the relative specific activity between substrates may be fairly accurately predicted even though predictions of the absolute effects of such changes are less successful. Many substitutions, particularly for the glycine residue at the bottom of the P1 cleft (Gly166), have been found to increase the specificity of the enzyme for particular peptide links whilst reducing it for others. These effects are achieved mainly by corresponding changes in the Km rather than the Vmax. Increases in relative specificity may be useful for some applications. They should not be thought of as the usual result of engineering enzymes, however, as native subtilisin is unusual in being fairly non-specific in its actions, possessing a large hydrophobic binding site which may be made more specific relatively easily (e.g. by reducing its size). The inactivation of subtilisin in bleaching solutions coincides with the conversion of Met222 to its sulfoxide, the consequential increase in volume occluding the oxyanion hole. Substitution of this methionine by serine or alanine produces mutants that are relatively stable, although possessing somewhat reduced activity.

Figure 3. An outline of the process of site-directed mutagenesis, using a hypothetical example. (a) The primary structure of the enzyme is derived from the DNA sequence. A putative enzyme primary structure is proposed with an asparagine residue replacing the serine present in the native enzyme. A short piece of DNA (the primer), complementary to a section of the gene apart from the base mismatch, is synthesised. (b) The oligonucleotide primer is annealed to a single-stranded copy of the gene and is extended with enzymes and nucleotide triphosphates to give a double-stranded gene. On reproduction, the gene gives rise to both mutant and wild-type clones. The mutant DNA may be identified by hybridisation with radioactively labelled oligonucleotides of complementary structure.

An example of the unpredictable nature of protein engineering is given by trypsin, which has an active site closely related to that of subtilisin. Substitution of the negatively charged aspartic acid residue at the bottom of its P1 cleft (Asp189), which is used for binding the basic side-chains of lysine or arginine, by positively charged lysine gives the predictable result of abolishing the activity against its normal substrates but unpredictably also gives no activity against substrates where these basic residues are replaced by aspartic acid or glutamic acid.

Considerable effort has been spent on engineering more thermophilic enzymes. It has been found that thermophilic enzymes are generally only 20-30 kJ more stable than their mesophilic counterparts. This may be achieved by the addition of just a few extra hydrogen bonds, an internal salt link or extra internal hydrophobic residues, giving a slightly more hydrophobic core. All of these changes are small enough to be achieved by protein engineering. To ensure a more predictable outcome, the secondary structure of the enzyme must be conserved and this generally restricts changes in the exterior surface of the enzyme. Suitable for exterior substitutions for increasing thermostability have been found to be aspartate , glutamate, lysine , glutamine, valine , threonine, serine , asparagine, isoleucine , threonine, asparagine , aspartate and lysine , arginine. Such substitutions have a fair probability of success. Where allowable, small increases in the interior hydrophobicity for example by substituting interior glycine or serine residues by alanine may also increase the thermostability. It should be recognised that making an enzyme more thermostable reduces its overall flexibility and, hence, it is probable that the factitious enzyme produced will have reduced catalytic efficiency.

Artificial enzymes:

A number of possibilities now exist for the construction of artificial enzymes. These are generally synthetic polymers or oligomers with enzyme-like activities, often called synzymes. They must possess two structural entities, a substrate-binding site and a catalytically effective site. It has been found that producing the facility for substrate binding is relatively straightforward but catalytic sites are somewhat more difficult. Both sites may be designed separately but it appears that, if the synzyme has a binding site for the reaction transition state, this often achieves both functions. Synzymes generally obey the saturation Michaelis-Menten kinetics . For a one-substrate reaction the reaction sequence is given by

synzyme + S (synzyme-S complex) synzyme + P

Some synzymes are simply derivatised proteins, although covalently immobilised enzymes are not considered here. An example is the derivatisation of myoglobin, the oxygen carrier in muscle, by attaching (Ru(NH3)5)3+ to three surface histidine residues. This converts it from an oxygen carrier to an oxidase, oxidising ascorbic acid whilst reducing molecular oxygen. The synzyme is almost as effective as natural ascorbate oxidases.

It is impossible to design protein synzymes from scratch with any probability of success, as their conformations are not presently predictable from their primary structure. Such proteins will also show the drawbacks of natural enzymes, being sensitive to denaturation, oxidation and hydrolysis. For example, polylysine binds anionic dyes but only 10% as strongly as the natural binding protein, serum albumin, in spite of the many charges and apolar side-chains. Polyglutamic acid, however, shows synzymic properties. It acts as an esterase in much the same fashion as the acid proteases, showing a bell-shaped pH-activity relationship, with optimum activity at about pH 5.3, and Michaelis-Menten kinetics with a Km of 2 mm and Vmax of 10-4 to 10-5 s-1 for the hydrolysis of 4-nitrophenyl acetate. Cyclodextrins (Schardinger dextrins) are naturally occurring toroidal molecules consisting of six, seven, eight, nine or ten a-1, 4-linked D-glucose units joined head-to-tail in a ring (a-, b-, g-, d- and e-cyclodextrins, respectively: they may be synthesised from starch by the cyclomaltodextrin glucanotransferase (EC 2.4.1.19) from Bacillus macerans). They differ in the diameter of their cavities (about 0.5-1 nm) but all are about 0.7 nm deep. These form hydrophobic pockets due to the glycosidic oxygen atoms and  inwards-facing C-H groups. All the C-6 hydroxyl groups project to one end and all the C-2 and C-3 hydroxyl groups to the other. Their overall characteristic is hydrophilic, being water soluble, but the presence of their hydrophobic pocket enables them to bind hydrophobic molecules of the appropriate size. Synzymic cyclodextrins are usually derivatised in order to introduce catalytically relevant groups. Many such derivatives have been examined. For example, a C-6 hydroxyl group of b-cyclodextrin was covalently derivatised by an activated pyridoxal coenzyme. The resulting synzyme not only acted a transaminase but also showed stereoselectivity for the L-amino acids. It was not as active as natural transaminases, however. Polyethyleneimine is formed by polymerising ethyleneimine to give a highly branched hydrophilic three-dimensional matrix. About 25% of the resultant amines are primary, 50% secondary and 25% tertiary:Ethyleneimine                      polyethyleneimine

The primary amines may be alkylated to form a number of derivatives. If 40% of them are alkylated with 1-iodododecane to give hydrophobic binding sites and the remainder alkylated with 4(5)-chloromethylimidazole to give general acid-base catalytic sites, the resultant synzyme has 27% of the activity of a-chymotrypsin against 4-nitrophenyl esters. As might be expected from its apparently random structure, it has very low esterase specificity. Other synzymes may be created in a similar manner.

Antibodies to transition state analogues of the required reaction may act as synzymes. For example, phosphonate esters of general formula (R-PO2-OR’)- are stable analogues of the transition state occurring in carboxylic ester hydrolysis. Monoclonal antibodies raised to immunising protein conjugates covalently attached to these phosphonate esters act as esterases. The specificities of these catalytic antibodies (also called abzymes) depends on the structure of the side-chains (i.e. R and R’ in (R-PO2-OR’)-) of the antigens. The Km values may be quite low, often in the micromolar region, whereas the Vmax values are low (below 1 s-1), although still 1000-fold higher than hydrolysis by background hydroxyl ions. A similar strategy may be used to produce synzymes by molecular ‘imprinting’ of polymers, using the presence of transition state analogues to shape polymerising resins or inactive non-enzymic protein during heat denaturation.

Coenzyme-regenerating systems

Many oxidoreductases and all ligases utilise coenzymes (e.g. NAD+, NADP+, NADH, NADPH, ATP), which must be regenerated as each product molecule is formed. Although these represent many of the most useful biological catalysts, their application is presently severely limited by the high cost of the coenzymes and difficulties with their regeneration. These two problems may both be overcome at the same time if the coenzyme is immobilised, together with the enzyme, and regenerated in situ.

A simple way of immobilising/regenerating coenzymes would be to use whole-cell systems and these are, of course, in widespread use. However as outlined earlier, these are of generally lower efficiency and flexibility than immobilised-enzyme systems. Membrane reactors (may be used to immobilise the coenzymes but the pore size must be smaller than the coenzyme diameter, which is extremely restrictive. Coenzymes usually must be derivatised for adequate immobilisation and regeneration. When successfully applied, this process activates the coenzymes for attachment to the immobilisation support but does not interfere with its biological function. The most widely applied synthetic routes involve the alkylation of the exocyclic N6-amino nitrogen of the adenine moiety present in the coenzymes NAD+, NADP+, NADH, NADPH, ATP and coenzyme A.

In some applications, such as those using membrane reactors it is only necessary that the coenzyme has sufficient size to be retained within the system. High molecular weight water-soluble derivatives are most useful as they cause less diffusional resistance than insoluble coenzyme matrices. Dextrans, polyethyleneimine and polyethylene glycols are widely used. Relatively low levels of coenzyme attachment are generally sought in order to allow greater freedom of movement and avoid possible inhibitory effects. The kinetic properties of the derived coenzymes vary, depending upon the system, but generally the Michaelis constants are higher and the maximum velocities are lower than with the native coenzymes. Coenzymes immobilised to insoluble supports presently have somewhat less favourable kinetics even when co-immobilised close to the active site of their utilising enzymes. This situation is expected to improve as more information on the protein conformation surrounding the enzymes’ active sites becomes available and immobilisation methods become more sophisticated. However, the cost of such derivatives is always likely to remain high and they will only be economically viable for the production of very high value products.

There are several systems available for the regeneration of the derivatised coenzymes by chemical, electrochemical or enzymic means. Enzymic regeneration is advantageous because of its high specificity but electrochemical procedures for regenerating the oxidoreductase dinucleotides are proving competitive. To be useful in regenerating coenzymes, enzymic processes must utilise cheap substrates and readily available enzymes and give non-interfering and easily separated products. Formate dehydrogenase and acetate kinase present useful examples of their use, although the presently available commercial enzyme preparations are of low activity:

Genetically Engineered Enzymes

Enzymes are naturally occurring proteins that speed up biochemical processes. They’re used to produce everything from wine and cheese to corn syrup and baked goods. Enzymes allow the manufacturer to produce more of a particular product in a shorter amount of time, thus increasing profit.

Generally, the use of enzymes is beneficial. In some cases, they can replace harmful chemicals and reduce water and energy consumption in food production. However, enzymes produced by genetically engineered organisms are cause for concern. Not enough is known about the long-term effects of these enzymes on humans and the ecosystem for them to be used across the board.

FDA regulations on enzyme use is a gray area. Enzymes used in the processing of foods do not have to be listed on product labels because they are not considered foods. Also, when enzymes are genetically engineered, the manufacturer is not required to notify the FDA that the enzymes have been modified. The lists of GE enzymes known by the FDA is, by their own admission, “probably incomplete.”

Worldwide, the enzyme market is a $1.3 billion industry. One of the largest enzyme manufacturers are Novo Nordisk, which manufactures GE and non-GE enzymes. The FDA provided us with this partial list of genetically engineered enzymes:

  • Chymosin—used in the production of cheese

  • Novamyl(TM)—used in baked goods to help preserve freshness

  • Alpha amylase—used in the production of white sugar, maltodextrins and nutritive carbohydrate           sweeteners  (corn syrup)

  • Aspartic (proteinase enzyme from R. miehei)—used in the production of cheese

  • Pullulanase—used in the production of high fructose corn syrup

If you want to absolutely avoid genetically engineered enzymes you will have two choices: avoid foods in the following categories, or call the food manufacturers directly and ask them if their enzymes are genetically engineered. They will probably have no idea. Ask them to check and call them back again. Let us know if you get written confirmation.

  • Beers, wines and fruit juices—(Enzymes used: Cereflo, Ceremix, Neutrase, Ultraflo, Termamyl, Fungamyl, AMG, Promozyme, Viscozyme, Finizym, Maturex, Pectinex, Pectinex Ultra SP-L, Pectinex BE-3L, Pectinex AR, Ultrazym, Vinozym, Citrozym, Novoclairzym, Movoferm 12, Glucanex, Bio-Cip Membrane, Peelzym, Olivex/Zietex)

  • Sugar—Enzymes used: Termamyl, Dextranase, Invertase, Alpha Amylase

  • Oils—Enzymes used: Lipozyme IM, Novozym 435, Lecitase, Lipozyme, Novozym 398, Olivex, Zeitex

  • Dairy products—Enzymes used: Lactozym, Palatase, Alcalase, Pancreatic Trypsin Novo (PTN), Flavourzyme, Catazyme, Chymosin

  • Baked goods—Enzymes used: Fungamyl, AMG, Pentopan, Novomyl, Glutenase, Gluzyme

In many cases the enzymes named above are brand names. They may appear under other names as well. Enzymes are usually found in minuscule quantities in the final food product. The toxin found in genetically engineered tryptophan was less than 0.1 percent of the total weight of the product, yet it was enough to kill people. The use of enzymes is pervasive in the food industry. Nothing is known about the long-term effects of genetically engineered enzymes. We include this information so you can make an informed choice about whether you want to eat them or not.

Enzymes produced by genetically modified microorganisms

Novozymes’ enzymes produced by genetically modified microorganisms

Novozymes A/S markets a range of enzymes for various industrial purposes. Many of these enzymes are produced by fermentation of genetically modified microorganisms (GMMs).

There are several advantages of using GMMs for the production of enzymes, including:

  • It is possible to produce enzymes with a higher specificity and purity

  • It is possible to obtain enzymes which would otherwise not be available for economical, occupational health or environmental reasons

  • Due to higher production efficiency there is an additional environmental benefit through reducing energy consumption and waste from the production plants

  • For enzymes used in the food industry particular benefits are for example a better use of raw materials (juice industry), better keeping quality of a final food and thereby less wastage of food (baking industry) and a reduced use of chemicals in the production process (starch industry)

  • For enzymes used in the feed industry particular benefits include a significant reduction in the amount of phosphorus released to the environment from farming

Due to an efficient separation process the final enzyme product does not contain any GMMs.

The enzymes are produced by fermentation of the genetically modified micro organisms (the production strain) which then produces the desired enzyme. The process takes place under well-controlled conditions in closed fermentation tank installations.

After fermentation the enzyme is separated from the production strain, purified and mixed with inert diluents for stabilisation.

The following is a list of Novozymes’ enzymes produced by genetically modified organisms.

Food Applications:

Brand name

Type of enzymes

Main Application

Amylase® AG XXL

Glucoamylase

Juice Industry

Dextrozyme®

Pullulanase / Amyloglucosidase

Starch industry

Finizym® W

Phospholipase

Starch industry

Gluzyme® Mono

Glucose oxidase

Baking industry

Lecitase® Novo

Lipase

Oils and fats industry

Maltogenase®

Maltogenic amylase

Starch industry

Maturex®

Alpha-acetodecarboxylase

Brewing industry

NovoCarne® Tender

Protease

Meat industry

Novoshape®

Pectinesterase

Fruit processing

Novozym® 27080

Carbohydrase / Lipase

Baking industry

NOVOZYM® 27122

Xylanase

Protein Hydrolysis

Novozym® 33081

Polygalacturonase

Juice Industry

Novozym® 46016

Phospholipase

Dairy industry

Novozym® 46019

Cellobiose oxidase

Dairy Industry

Pectinex® XXL

Pectin lyase / Polygalacturonase

Juice Industry

Promozyme® D2

Pullulanase

Starch industry

Saczyme®

Glucoamylase

Alcohol Industry

Toruzyme®

Transferase

Starch industry

Feed Applications:

Brand name

Type of enzymes

Main Application

Bio-Feed® Wheat

Xylanase

Animal feed industry

Bio-feed® Phytase

Phytase

Animal feed industry

Other Applications:

Brand name

Type of enzymes

Main Application

Alcalase®

Subtillisin

Detergent industry

Aquazym® LT-L

Alpha-amylase

Textile industry

BioPrep®

Pectate lyase

Textile industry

Carezyme®

Cellulase

Detergent industry

Clear-Lens® LIPO

Lipase

Personal care industry

DeniLite®

Laccase

Textile industry

DeniMax® 601

Cellulase

Textile Industry

Duramyl®

Alpha-amylase

Detergent industry

Everlase®

Subtillisin

Detergent industry

Extruzyme® Pro

Alpha-amylase

Pet food industry

Greasex®

Lipase

Leather industry

Kannase®

Subtillisin

Detergent industry

Lipex®

Lipase

Detergent industry

Lipolase®

Lipase

Detergent industry

Liquanase®

Subtilisin

Detergent industry

Liquozyme®

Alpha-amylase

Starch and Ethanol industry

Mannaway®

Mannanase

Detergent industry

NovoBate® 100

Trypsin

Leather Industry

Chemical Modification of Enzymes

We know that the proteins synthesized under the control of gene sequences in a cell undergo post translational modification. This leads to stability, structural integrity, altered solubility and viscosity of individual proteins. This may also alter the chemical reactivity.

These alterations can be achieved in vitro and may .sometimes even create entirely new enzyme, by creating new active sites or modifying the old ones. Some of the examples will be described in this section.

Protein Modelling

Utilizing the data generated through X-ray diffraction and NMR studies, models can be constructed with the help of computer graphics. There are computer programmes available (interactive colour graphics programmes) with the help of which a protein structure can be fitted to the electron density map (obtained from X-ray diffraction) by simultaneous display on the screen of computer monitor. Similarly, Van der Waals surfaces for the protein can be displayed and interaction between several molecules simulated.

There are also other interactive molecular graphics which can be used (with the help of programmes) to find out the perturbations (disturbances) in protein structure that will result from specific modifications of amino acid sequences. We know that to some extent the three dimensional structure of a protein can be predicted from the amino acid sequence, but we still have to depend partly on X-ray diffraction patterns for determining the three dimensional structure.

In future when the three dimensional structure can be accurately predicted from amino acid sequence data, this will lead to long term success in protein engineering. The models of proteins, made on the basis of amino acid alterations, can then be tested for the predictions about structure function relationships.

Multienzyme Systems by Gene Fusion ( Bi and Polyfunctional Enzymes)

Multienzyme systems have been artificially synthesized, which can catalyze sequential reactions in many biotechnological production processes. Although, proximity of more than one enzymes can also be achieved by co-immobilization and chemical cross linking, gene fusion appears to have the highest potential in enzyme technology. The gene fusion technology, for preparation of bi-and polyfunctional enzymes, involves joining of structural genes of two or more enzymes. The translational stop singal at the 3′ end of the first gene is removed and ligated in frame to the A TG start codon of the second gene. Alternatively, short linkers (2-10 amino acids) are used. The novel chimaeric gene gives a single polypeptide chain carrying active sites of both genes. This fusion may involve

(i)     two monomeric enzymes

(ii)   a monomeric and a dimeric enzyme or

(iii) two dimeric enzymes.

Rationale of Protein Enzyme Engineering – Although thousands of proteins have been characterized in prokaryotes and eukaryotes, only few became commercially important. This is due to the high cost of isolating and purifying enzymes in sufficient quantities.

Although the cost aspect has been overcome by producing an enzyme in large quantities in bacteria, for its industrial application, an enzyme (outside the cell) should also have some characteristics in addition to those of enzymes in the cells. These characteristics may include the following:
(i) enzyme should be robust with a long life;

(ii) enzyme should be able to use the substrate supplied in the industry even if it differs slightly from that in the cell;
(iii) enzyme should be able to work under conditions (e.g. extremes of pH, temperature and concentration) of the industry even if they differ from those in the cell.

In view of the above, enzyme should be engineered to meet the altered needs. Therefore, efforts have been made to alter the properties of the enzymes. Following is the list of properties that one needs to alter in a predictable manner for protein or enzyme engineering.

(1)      Kinetic properties of enzyme turnover and Michaelis Constant, Km.
(2) Theremostability and the optimum temperature for the enzyme.
(3) Stability and activity of enzyme in nonaqueous solvents.
(4) Substrate and reaction specificity.
(5) Cofactor requirements.
(6) Optimun pH.
(7) Protease resistance.
(8) Allosteric regulation.
(9) Molecular weight and subunit structure.

For a particular class of enzymes, variation in nature may occur for each of the above properties, so that one may like to combine the optimum properties to get the most efficient form of the enzyme.

This aspect of protein engineering will be illustrated using the example of glucose isomerases, which convert glucose into other isomers like fructose and are used to make high fructose corn syrup vital for soft drink industry. It exhibits wide variation in its properties.
Sometimes, it may not be possible to get a combination of optimum properties. For instance, an enzyme with highest activity may not be the most stable. Therefore, a compromise in properties may have to be made, if we have to select an enzyme from the available variability or even if we create variability by mutagenesis.
However, if structure and function relationship of an enzyme is known, the structural features for desirable function may be combined and protein engineering techniques may then be used to create a novel enzyme exhibiting a combination of all desirable functional properties.

Glucose isomerase belongs to a TIM barrel family of enzymes which resemble each other in having a highly characteristic domain called TIM barrel, with active site for catalytic action at one end. This TIM barrel may be found in enzymes that may differ in sequences and may catalyze different reactions.
As earlier discussed, since similarities of structure of protein meant similarities in function, TIM barrel presents a challenge to this concept. However, it is curious tbat some enzymes in this family appear in pairs in their metabolic pathways so that they catalyse two consecutive steps thus showing coupling of their functions.
As an example of two enzymes of TIM barrel family, while ‘triose phosphate isomerase’ is one of the most efficient catalysts, ‘glucose isomerase’ is relatively very inefficient.
Therefore, if ‘glucose isomerase’ enzyme is redesigned to use the highly efficient domain of TIM barrel family, it will be a remarkable achievement for soft drink industry. Efforts in this direction are being made (see later for methods of protein engineering).

Acheivements of Protein Engineering

A number of proteins are known, now, where efforts have been made to know the effects of site specific mutagenesis involving substitution of one or more amino acids. Efforts have also been made to study in detail the function of different regions of a protein. Following are some results of such efforts.

?-lactamase. This enzyme functions in the periplasmic space of bacterial cells. The enzyme hydrolyses and inactivates the beta- lactam ring of penicillin derivatives and helps in transport across the inner membrane. During transport a polypeptide (signal sequence peptide of 23 amino acids) is cleaved off.

Detailed analysis suggested that, transport and processing does not depend on this polypeptide of 23 amino acids alone. An active site involving amino acid serine has also been identified, since its replacement by cysteine leads to reduction in the activity of this enzyme.

Dihydrofolate reductase. In this enzyme, replacement of a single amino acid, aspartic add (ASP) by asparagine (ASN), led to a decrease in specific activity by a thousand fold, suggesting that aspartic acid is very important.(or the active site. Other similar modifications were also examined.

Insulin. It consists of A and B chains linked by C-peptide of 35 amino acids. It was shown that a sequence of 6 amino acids for C-peptide was adequate for the, linking function.

Lactose permease (product of, gene of ‘lac’ operon). This enzyme is involved in transport of lactose and a cysteine to glycine substitution showed that this amino acid was not essential for transport. Further, out of four histidine residues, two at positious 35 and ’39 do’ not play any essential role in transport, while the mutation in any of the other two histidines at positions 208 and 322, lead to loss of transport function.

T4 lysozyme. A mutation of isoleucine to cystine in this enzyme leading to formation of a disulphide bridge led to thermal stability and a 200 fold increase in enzyme activity even at 6T’C.

Human beta interferon. Removal of one of the three cysteine residues’ I led to an improvement in stability of the enzyme.

? repressor. This protein could be engineered to develop a specific site for cro protein, since the alteration led to development of a cro recognition site.I

Acetylcholine receptor. This protein is involved in transport, of acetylcholine through. the membrane. Specific regions of this protein involved in acetylcholine binding and channel formation have been, identified.

Cytochrome C. A phenylalanine residue has been identified to be non-essential for electron transfer but is involved in determining the reduction potential of the protein.

Trypsin. It could be redesigned to have altered substrate specificity.

Subtilisin. Another successful alteration of substrate specificity involved the enzyme subtilisin reported in 1986-87.

Lactate dehydrogenase. A lactate dehydrogenase (LDH) from Bacillus stearothermophilus was modified separately by each of the three substitutiens of amino acids (resulting from mutations; Asp197… Asn; Thr246″‘Gly; Gln102…Arg). The substitution, Gln102″‘Arg, led to change in specificity from lactate to malate, with high efficiency, comparable to that which the native LDH had for lactate.

Lactic protease. Substrate specificity of lactic protease (in E. coli), has been shown to be dramatically modified by replacing active site methionine by alanine (Met19

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