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Introduction:
DNA and RNA are very key biomolecules of all bio-cells. For developing bio-cell to high-biotech, bio-molecular design engineering is an important concern.
Up-gradation of bio-cells often involves bio-molecular redesigning. The redesigning DNA biomolecules for products consists of four major steps. The redesigning of biomolecules may be considered as a non-classical engineering.
The tools required in redesigning are called restriction endonucleases or restriction enzymes. As low volume high value microbial products, restriction enzymes have added important dimensions to the development of bioprocess engineering. A more recent review discussed the sources, production, separation and purification of restriction enzymes in general.
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It emphasized the importance of upstream and downstream processing to obtain pure enzymes for genetic engineering or molecular biology work. Several process engineering variables and state parameters have been found to exert an effect on the maximization of production and many of these bio-molecular design engineering tools.
The restriction enzyme BamH1 is a typical example of this type of tools. A discussion of the bioprocessing and influence of variables, fundamental molecular properties, physiochemical behaviour and application of these non-classical bio-molecular design engineering tools with special reference to BamH1 will, therefore, be useful. In order to maximize production of these tools by bioprocessing, manipulation/optimization of culture conditions is reflected in the behaviour of the cell culture system including its cellular materials. Some important bioprocessing information for production from a biotechnological view point has been provided very recently.
Restriction enzymes of living cells are constitutive in nature. Bacillus amyloliquefaciens synthesize intracellular restriction enzyme Bam H1 during its cultivation. Some information on the behaviour of cellular and cell free native Bam H1 in relation to thermotolerence has been reported recently. Thermotolerence of native restriction enzymes, in general, is extremely poor.
The lower value of activation energy for denaturation of an enzyme represents its lower heat stability. The unusually low value of activation energy for denaturation of native BamH1 (2.63 kcal/mole), besides confirming its extreme heat labile nature, gives quantitative information on its thermal denaturation. Possibility of inhibition of BamH1 by commercial polysaccharides recently reported by Oishi et al. indicated reaction engineering concern of the system.
Perspectives, Results and Discussion in Relation to BamH1:
1. Most suitable in vivo thermotolerence for denovo BamH1:
In cell cultivation of Bacillus amyloliquefaciens H1 at different temperatures one may simulate microbial cells placed into the cultivation medium as two chambers filled with liquids under constant flow and separated by a cell wall with pores of micron sizes and comparable to the mean free path of the molecules where heat is transferred through the system from T1 to T2 (T1 > T2).
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Constitutive molecules of the cells on the T1 side of the cell wall thus have a higher kinetic energy than on the T2 side of the cell wall with higher velocity and frequency of collision of molecule. This causes transportation of hotter fluid from T1 side to T2 side. A steady state in cellular function (not equilibrium) is reached when total number of collisions of molecules per unit time on the T1 side and T2 side of the cell wall are equal.
This optimal steady state is not reached until equal optimal cultivation temperature for growth in interior and exterior of cell is established. At this thermal condition it may be expected that biosynthesis of constitutive macromolecular enzyme proteins like BamH1 will take place at highest level. Above this thermal condition, however, the thermoturbulence or thermo collisions increased within the de novo BamH1 molecule caused by higher temperature and predominant bulk flow transport in comparison with its thermal diffusive flow at lower temperature.
This thermoturbulence might increase the collision within de novo BamH1molecule thereby causing unwinding and cleavage of its H-bonds. The higher the temperature of cultivation above optimal value the severe is the unfolding and inactivation of thermolabile protein BamH1.
2. Engineering perspectives and concern:
The above described concept may be conceivable both in presence or absence of cell wall. In absence of cell wall, however, the average concentration difference is smaller with same energy flow. This means efficiency of the process is lower.
When thermal energy flow stops the transport of molecules from T1 side to T2 side, it may then be passive. Prevalence of the two possible operative principles may be expected in a cellular system. Thus fluid flow and thermodynamic principles are two concerned perspectives in intracellular bio processing.
(i) Intracellular Micro flow Concern:
From standard fluid flow concept the bulk flow in the cell may be given by Fb = v.c. where v is the fluid velocity and c is the concentration fluid of the nutrient molecules. However, under the influence of thermal gradient, the diffusive flow (Fd) in the cell may exist and computable from Fd = K1 ∆c in which K, is defined as the ratio of diffusivity (D) to the thickness of the resistance layer (x, cell wall thickness).
Under thermal influence it could be shown under certain conditions that Fb and Fd have equal influence (i.e., Fb = Fd) and thus in vivo thermolability of de novo BamH1 is of great concern from thermodynamic view point also. In order to illustrate this is the relative importance of the flow components Fb and Fd need be characterised from their ratio. This ratio of Fb to Fd is defined as Peclet number (Npe). Thus,
For fluid flow under working temperature across cell wall of the organism if one considers an individual cell as a continuous micro bioreactor placed within a physical bioreactor and assume following reasonable values.
Experimental support of in vivo thermolability of BamH1:
Thermal inactivation profile of native BamH1 has been observed in vitro system by incubating it at temperatures ranging from 25 to 50°C for varying target of time ranging from 50 to 240 mins. Ten microliter of BamH1 having 5 units/pl activities were subjected to heat treatment. In each experiment the residual BamH1 activity after heat treatment was determined by serial dilution method using DNA as substrate.
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Results of serial dilution method are reported. Knowing the thermolability behavior of BamH1 in vivo system it was aimed to assess the nature of in vivo thermolability of BamH1 during cell cultivation at different temperatures keeping other conditions same in all batch runs.
The experimental results support the perspective of fluid flow as discussed in previous section. It can be seen from this figure that till most suitable cultivation temperature has been provided specific BamH1 production has increased. It indicated that binding constitutive molecules of BamH1 take place most suitable by favouring in vivo bio-reaction system.
Above this temperature because of bulk fluid flow mediated higher quantum of thermal energy penetration the units of molecules for constitution of BamH1 presumably got unfolding initiation and attacked by other protein molecules in vivo. It rendered hindrance in de novo enzyme thereby decreasing the activity depending on the thermal quantum and followed similar pattern as specific growth rate profile.
(ii) Thermodynamic Perspective:
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Considering Kuhn’s concept one may assume that temporary inactivation of restriction enzyme occur, due to separation of broken chain by unwinding of large number of turns (N) of its DNA helix. The separated chains have a higher degree of freedom than similar chain segments bonded within double helix of DNA. Consequently unfolding of (BamH1 like protein) DNA molecule is accompanied by an increase in entropy or decrease of free energy. This concern is of special significance in computation of decrease in free energy in relation to ‘a’ which is the ratio of the half life of the enzyme to total thermal exposure time.
(iii) Cell Culture Engineering Parameter in Relation to Unfolding Initiation Time:
When N>>1 the initiation time of unfolding of the restriction enzyme in relation to maximum specific growth rate (µm) is of great importance in addition to ‘a’ as defined above. The variation in the value of µm as a function of temperature as in Fig. 2.11 is useful in determining the number of turns unfolded from its initiation time.
(iv) Oxygen Mass Transfer Concern:
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Bacillus amyloliquefaciens H1 is an aerobic organism. During its cultivation for BamH1 synthesis in the cell, the influence of oxygen mass transfer by the aeration-agitation unit operation indicated significant effect on BamH1 specific activity.
The profiles of the influence of cell cultivation variables on production of the restriction enzyme BamH1 have been published. The BamH1 yield in relation to the control variables has been shown. From the results of the influence of cultivation variables it appeared that using a Marubishi bioreacter in bioprocessing for BamH1 production, the most suitable values of the parameters are temperature 37°C, pH 7.0, aeration-agitation 2 vvm-500 rpm with corresponding KLa 260-290 h‑1.
These results, however, indicated that pH and temperature in the used range did not cause significant change in the specific BamH1 production. However, the existence of optimum pH and temperature for a maximum yield of BamH1 could be seen. Variation of aeration and agitation could show an appreciable increase in specific BamH1 yield upto 500 rpm and 2 vvm aeration. Above KLa 280 h-1 the BamH1 yield decreased.
Interpretation of the observations:
Increase in BamH1 concentration with the increase in aeration-agitation upto certain level (1 vvm, 500rpm, KLa 270 h-1 in B.E. Marubishi Bioreactor, 71) was probably associated with induction of this restriction enzyme in the organism by oxygen. Possibility of induction of microbial enzyme in presence of oxygen has been reported in the literature.
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However, evidence of such induction for constitutive restriction enzyme is scanty. It is, however, stated that superoxide dismutase (SOD) activity is present in practically all aerobes like Bacillus subtilis, B. stearothermophilus, B. propillae, B. megaterium etc. It is likely, therefore, that it may also be present in B. amyloliquefaciens suggesting the existence of operation of SOD theory of induction-repression in the cell.
Exposure of this organism upto certain level as stated above induces BamH1 synthesis mechanism but at elevated oxygen concentration in the cell caused to form more H2O2 in vivo which perhaps led to the synthesis of more SOD or catalase if insufficient is present to cope with increased O2 generation.
Exposure of B. subtilis to elevated oxygen concentration increases its catalase activity but not SOD and this organism is equally sensitive to high oxygen level whether grown previously in air or 100% pure oxygen. It seems, therefore, below the critical level of oxygen as mentioned above the SOD/catalase level in the cell falls below the level at which BamH1 synthesis is induced.
However, on its exposure to above this critical level of oxygen SOD/catalase activity in the cell is promptly resynthesised and cause repression of BamH1 synthesis thereby showing decline in its activity. Until definite evidence is available it may also be due to in vivo oxidant mediated DNA damage. When cells are subjected to high extracellular oxygen stress leading to an intracellular micro-molar concentration of nascent H202, temporary DNA strand cleavage may also occur. It is because the oxygen stress leads to activation of some specific DNA cleaving mechanism in monoionic or diionic metal-dependent endonuclease.
(v) Basic Enzyme Reaction Engineering Concern:
The restriction enzyme BamH1 has been exploited extensively both for R&D and industrial purposes. In producing important bio-chemical’s/ chemicals through the use of redesigned microorganism BamH1 played a significant role. However, because of the problems associated with stabilizing and making BamH1 reaction inhibition free it has become a task and challenge to the enzyme engineers to stabilize restriction enzymes, in general, at room temperature by some economic process. In order to develop such economic means extensive analysis of the system from reaction engineering perspective has become essential.
A preliminary attempt in this regard was made at BERC, IIT Delhi by observing thermotolerence of native and cross linked BamH1. Reactions were carried out in a volume of 10µ1 in 10 mM potassium phosphate buffer (pH 7.0) containing 20pg BamH1 protein at a level of 5 units/ml and cross linking reagents (DMA, DMS and DTBP) at levels ranging from 0.01 to 0.2% (WW).
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The temperature of reaction ranged from 25 to 50°C for varying length of time using substrate as stated in earlier literature. The experimental data of the time profile of percent residual activity of native and cross linked BamH1 with temperature as parameter has been reported. Analysis of, these data could show a relationship between Kd and time of exposure at various temperatures (Fig. 2.12). The apparent activation energies required for deactivation of native BamH1 could be computed from these data. This plot in form of a time profile (Fig. 2.12) indicated to involve two stages of deactivation in the temperature range.
Properties of the Bio-molecular Cleavage Tools:
1. General properties:
The bio-molecular cleavage tools like restriction endonucleases are strain specific and site specific enzymes which cleave double stranded DNA. Because of their unique nature of controllable, predictable, infrequent and site specific cleavage, restriction endonucleases have been proved to be extremely important tools in dissecting, analyzing, and inserting newer genetic information by cloning at molecular level. Based on their cleavage requirements and properties, these tools have been grouped under four types as type I, II, III and IV.
The type I or deoxyribonuclease I is the most commonly known endonuclease. This type has very little specificity and cleaves phosphodiester linkages in a DNA molecule indiscriminately. However, the type II or restriction enzymes would never do that but would attack a DNA molecule only at the specific sequence.
Like type I the deoxyribonuclease type III cleave away from the recognition sequence and are not of much use to recombinant DNA technology. EcoRI, BamH1 etc. are examples of type II restriction endonucleases produced by culturing microorganism (s). Their general properties have been discussed in the literature and in a more recent book.
2. Stability considerations:
The interaction of cross-linkers with BamH1 to impart resistance to thermal unfolding has been considered as a suitable strategy in stabilizing BamH1. The most suitable cross-linking reagent will be that which causes minimum inactivation of BamH1. Also the concentration of this suitable reagent at which it will cause maximum stabilization should be taken into account from an economic viewpoint.
On the basis of this consideration the effect of different concentrations of three crosslinking reagents, namely glutaraldehyde and dimethyl adipimidate (DMA), dimethyl suberimidate (DMS) and dimethyl 3, 3-dithiobispropionimidate (DTBP) on the activity of BamH1 has been observed in the BERC Laboratory.
The preparation of these cross-linked BamH1 compounds has been described. From the experimental results it was seen that the activation energies (E) required for native and DMA. DMS and DTBP cross-linked preparations were 2.63, 5.21, 6.55 and 9.2 kcal/mol, respectively. This increase in E values of cross-linked preparations over native BamH1 indicated that stabilization against thermal inactivation was achieved by cross-linking with a suitable reagent.
Forefront Application Areas:
Some of the forefront application areas with special reference to BamH1 have been discussed in a recent literature.
Other possible application areas of restriction enzymes are discussed below:
1. Agriculture:
Characterization and modification of genetic makeup of plants has been made possible with the help of recombinant DNA technology. Many plant proteins are deficient in amino acids that are essential for human health. Genes coding for the synthesis of a protein rich in essential amino acids could be introduced to improve the quality of plant protein.
It might also be possible to transfer genes for resistance to pests, herbicides, microbial toxins and for nitrogen fixation from Rhizobium strains which cause nodulation on many different leguminous plants such as peanut, soybean, mungbean and a few others. Rhizobium also forms nodules on non-leguminous plants called Troma.
Many nitrogen fixing trees such as Alnus, possess nodule formed by an Actinomycete frankia. These bacteria may be for nodule formation on nonleguminous plants. Nitrogen fixing capability could also be conferred on many free living bacteria that are abundant in soil and plant rhizospheres. Recently, cloning and expression of whole ‘nif’ genes of Klebsiella oxytoca in E. coli is reported.
Resistance to pests and disease to plants is often conferred by a single gene. Resistance based on a single major gene or few such genes becomes ineffective against new races of pathogens and pests a few years of its introduction into a crop-plant. Therefore, novel strategies are needed for obtaining crop resistance to pests and pathogens.
2. Industry:
A large number of industrial and academic R&D laboratories are working on gene technology for several industrially important microorganisms. Animal genes responsible for insulin, interferon and human growth hormone have been successfully cloned in E. coli in an effort towards their commercialization.
Production of human insulin by engineered E. coli cells from laboratory scale (10L) is known. Costly media constituents for fermentation might be replaced by inexpensive naturally occurring abundant substrates such as cellulose. Genes for efficient utilization of cellulose and other substrates can be isolated and cloned into organisms of commercial interest. With development of such genetically engineered microbes that utilize inexpensive media constituents more and more natural products will be produced by fermentation biotransformation replacing their expensive chemical synthesis or isolation from natural sources.
3. Medical sciences:
Gene therapy is well known in more recent years. A number of approaches were described envisaging the use of E. coli for production of antigenic material, extra-cellular secretion of the antigenic protein, production of antigen inserted in the surface of bacteria or intracellular production of immunogenic protein.
Recombinant DNA technology is being used to develop vaccines against deadly viral diseases. Segments of viral genome may be used to produce appropriate viral antigen in alternative hosts like Saccharomyces cerevisiae or Bacillus subtilis. The core antigen gene is expressed in E. coli and when injected into rabbits, the antigen produced by E. coli induces antibody that reacts with human serum core antigen.
Genes that code for surface protein of foot and mouth disease virus can be identified employing rDNA technology. Such genes can be introduced into appropriate host to produce protein that may be an effective vaccine. A DNA sequence from foul plaque virus was inserted into an E. coli plasmid. The recombinant plasmid directed the synthesis of a protein that is 0.75% of total cell protein and reacts with antisera to FOV hemaglutinin. Hopes are also raised to cure hereditary diseases like Haemophilia and colour blindness. Antibiotics producing genes can be cloned in multi-copy plasmids in suitable microbes leading to economical production of medicines.
4. Pollution control:
Metals aromatics, hydrocarbons and other rotting organic wastes which cause pollution can be effectively controlled using genetically manipulated microbes. Many bacteria harbour plasmids that carry genes for transformation of metals, aromatics, and other hydrocarbons.
Since they are located on plasmids, such genes can be isolated and introduced into organism like Azotobacter. Azotobacter can fix nitrogen and utilize the excessive carbon present in the municipal sewage. The microbially treated sewage can be used as fertilizer. Genes responsible for dehalogenase activity can be introduced into organisms that grow in sewage polluted with halogenated chemicals.
5. Nucleic acid research:
In addition to the above mentioned applied uses, restriction enzymes are also extensively used to reveal the mysterious facts contained in nucleic acids.
6. Miscellaneous:
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(i) Complementation Test:
Small pieces of genomes of industrially important organisms can be ligated to appropriate vectors that replicate in such organisms as well as in E. coli which allows application of complementation test for understanding regulatory mechanisms. Such vectors that replicate in E. coli as well as in some other organisms such B. subtilis or yeast are already being used to shuttle genes between two different host organisms.
(ii) In Vitro Localized Mutagensis:
Introducing small deletions or insertions with the help of restriction enzymes, in vitro mutagensis may be caused in vitro mutagenesis offers the possibility of mutating isolated purified DNA sequences to obtain enzymes with altered modified properties.
(iii) Genome Organisation:
Physical mapping of chromosome or gene.
Application of a series of restriction enzymes helps in physical mapping of gene or chromosome. Availability of cloned probes will speed up chromosome mapping. Using labelled probes of complimentary RNA prepared from cloned sea urchin genes, five human histone genes have been localized to the leg arm of chromosome VII.
(iv) Gene Structure:
Gene structure principally refers to DNA sequencing through which gene structure can be described in terms of intervening sequences, overlapping genes and location of promoter. As restriction enzymes are capable of generating limited specific cuts, they can be used as the first step in DNA sequencing.
(v) Origin of DNA Replication:
The origin and direction of replication of several viral chromosomes and plasmids has been revealed using restriction enzymes, particularly EcoRI.
(vi) Protein-DNA Interaction:
The type II restriction endonucleases appear to be excellent models for the study of sequence- specific protein DNA interactions whose mechanism has not been elucidated clearly.
Future Scope:
Due to their unique nature of controllable, predictable, infrequent and site specific cleavage of DNA, restriction enzymes are proved to be extremely important tools in dissecting, analysing and reconstructing genetic information at the molecular level. To make their use economical, convenient and most effective, their enzymology has to be investigated more thoroughly to reveal their behaviour in terms of kinetics and dynamics to enhance new stability.
Considerable emphasis on optimization of culture conditions and harvest period are also needed to increase their productivity. Though, several restriction enzymes are being overproduced by genetically redesigned strains, the optimization of their productivities still remains to be achieved to get maximum recovery and in large scale. A great potential for R&D efforts, therefore, remains as future scope in this field.
