Enzyme stabilization by selection and genetic engineering
Enzyme stabilization by selection
Biocatalysts are inherently labile; therefore their operational stability is very importance for any
bioprocess. An industrial disadvantage of the most commercially used biocatalysts- enzymes and
enzyme complexes - is their relatively low stability.
Selection of industrial enzymes from any sources is depends on the following factors: a.
specificity b. pH c. thermostability d. activation or inhibition e. availability and cost
Therefore, microorganisms which require extreme physicochemical conditions for their growth
and proliferation - and enzymes derived from them - bear a potential for overcoming this
situation.
The particular, enzymes derived from thermophilic microorganisms show an increased thermal
stability compared to those of mesophilic microorganisms. They are called thermostable if they
have a maximum reaction temperature above that of the optimum growth temperature for the
microorganism. This definition is invalid in view of the fact that enzymes from microorganisms
growing at temperatures above 90 OC are "thermostable" regardless of whether their optimum
activity temperature is below or above the optimum growth temperature. It becomes obvious that
thermostability of an enzyme is a relative term.
Stability of the enzyme is a function of the stabilizing forces which include hydrogen bonding,
hydrophobic bonding, ionic interactions, metal binding, and/or disulfide linkages . Such
stabilizing effects contribute to the long-term stability of an enzyme. Thermostability is also
connected with a higher resistance to most chemical denaturants. A suitable method for
characterizing the enzyme stability is the scanning calorimetry.
Thermophilic microorganisms are a source of more stable enzymes. Mixed populations of
bacteria which seemed to occur in the ocean near the Galapagos Island in a depth of 2500 metres
were generally an optimum growth temperature is 250OC at a pressure of 250 atm .
There is reliable verification of the existence of microorganisms at temperatures approaching the
boiling point of water given in the (Table 1 )
Table 1. Optimum growth temperature ( Topt ) of thermophilic bacteria
Species
Topt ( OC)
Bacillus acidocaldarius
Bacillus stearothermophilus
Caldarobacterium hydrogenophilum
60 - 65
55 - 70
74 - 76
Clostridium thermohydrosulfuricum
Methanobacterium thermolithotrophicum
Pyrococcus furiosus
Pyrodictium occultum
Sulfolobus acidocaldarius
Thermoproteus tenax
Thermotoga maritime
Thermus aquaticus
67 - 70
65 - 70
100
105
70 - 75
88
80
70
These bacteria occur in natural and/or artificial habitats. As an example the original isolations of
Thermus aquaticus were from hot spring algal mats. Incubation in aerobic liquid medium at 70
up to 75OC led to the formation of visible turbidity, often with clumps.
Brock and Freeze isolated one strain of Thermus aquaticus from a hot water tap in Indiana.
Brock and Yoder found another strain of Thermus aquaticus in a creek receiving thermal
pollution. Ramely and Hixson as well as Brock and Boylen and Heinritz et al. obtained strains of
Thermus aquaticus in a creek receiving thermal pollution and domestic hot water reservoirs,
respectively. It thus seems likely that Thermus is capable of growing in manmade habitats of
high temperature.
Table 2 shows the specific growth rate and the specific yield coefficient of selected thermophilic
bacteria utilizing glucose as the carbon and energy sources at temperatures up to 80OC.
Table 2. Specific growth rate (μ) specific yield coefficient ( Y x/ s), and optimum growth
temperature (& topt ) of selected thermophilic microorganisms utilizing glucose.
From the table 2 we find that the specific growth rate is in the range of the values of mesophilic
bacteria. On the other hand, the specific yield coefficient is obviously lower compared to
mesophiles.
The specific growth rate and/or the specific yield coefficient of thermophilic bacteria could be
improved by
- continuous instead of batch cultivation preventing inhibition of bacterial growth and
proliferation due to carbon substrate and/or metabolites
- increasing system pressure in fermenters.
Advantage of More stable enzymes from thermophilic microorganisms
Thermostable enzymes have several advantages over their counterparts from mesophilic
microorganisms
- higher thermal stability and resistance to most of the chemical denatu- higher storage stability,
- increased reaction rate and comparable catalytic activity,
- lower viscosity of reaction mixture and improved mass-transfer,
- lower danger of contamination in microbial enzyme production as well as
Fig 1: Thermal stability of a thermostable β-galactosidase of Bacillus stearothermophilus TP32
compared to the mesophilic β-galactosidase of Escherichia coli
Fig 2: Resistance of the thermostable (β-galactosidase of Bacillus stearothermophilus TP 32 and
the mesophilic β–galactosidase of Escherichia coli to ethanol and propan-2-ol under incubation
of the enzyme for 60 min at 50OC
Application of thermostable enzymes
Because of these facts thermostable carbohydratases, proteases, and oxidoreductases were
introduced recently into starch processing, hydrolysis of cellulose and lactose, brewing, baking,
food processing, waste water treatment, biosensors and/or other applications.
Thermostable and highly specific enzymes, e.g. DNA polymerases and RNA polymerases open
up new dimensions in molecular biology and genetic engineering. As an example the DNA
polymerase of Thermus aquaticus made possible the polymerase chain reaction ( PCR )
technology.
Enzyme stabilization by genetic engineering
Genetic engineering and protein engineering are modern techniques already in use for the
commercial production of biocatalysts of improved stability, not only to high temperatures, but
also to extremes of pH, oxidizing agents and organic solvents.
Cloning and expression in suitable hosts is being used routinely by major enzyme production
companies to produce improved biocatalysts..
Protein engineering is also being used to obtain improved biocatalysts, the case of alkaline
protease being a paradigm. Already in the market, thermostable proteases capable to withstand
harsh washing conditions (high pH, high concentration of strong oxidants) are products of
protein engineering produced by point amino acid substitutions in the most labile region of the
molecules
In the production of syrups from cornstarch, thermostability of a-amylase is severely reduced
below pH 6, which poses the inconvenience of pH adjustment before and after starch
liquefaction. A thermostable a-amylase from Bacillus licheniformis, active at low pH and low
Ca++concentration has been recently patented.
A thermostable glucose isomerase is a major challenge in the production of high-fructose corn
syrup. Equilibrium is favored at high temperature, so that at 110 °C 55 % HFCS could be
produced at the enzyme reactor stage, without the cumbersome process of sugar fractionation
now used.
It was shown that specific substitution of a surface arginine residue for lysine, obtained by sitedirected mutagenesis, produced a substantial thermal stabilization in the glucose isomerase from
Actinoplanes missouriensis.
Protein engineering is a powerful tool for the design of robust biocatalysts and probably most
future biocatalysts will be produced by engineered organisms.
Genetic gngineering follows the convenience of cloning termophilic genes into more suitable
mesophilic hosts. Those systems will be highly productive and the enzymes produced will retain
its original thermostability. In fact, in a number of cases thermophilic genes have been cloned
and expressed in mesophilic hosts, producing enzymes highly active and stable at high
temperatures. Some examples are in Table below and other bacterial hosts pose some problems
in expressing genes from archibacteria, because of misreading of intervened genes.
E.coli and other bacterial hosts pose some problems in expressing genes from archibacteria,
because of misreading of intervened genes. This is not the case with eubacterial genes, being
therefore better candidates for cloning into bacterial hosts
Following Industrial thermostable enzymes, commercial enzymes from thermophiles and
termophilic genes cloned in mesophilic hosts given in the table below.
In some cases, remarkable similarities are observed between thermophilic enzymes and their
mesophilic counterparts, homology being as high as 85% (Vieille and Zekus, 1996).
Thermostability is the result of differences in specific aminoacid sequences and it has been
ascribed to a more rigid configuration and to the high number of hydrophobic interactions.
By examining primary sequences of termophilic enzymes and mesophilic counterparts the nonconserved regions, as those possibly linked to the thermostable phenotype, can be identified.
This opens up the possibility of using protein engineering techniques (Imanaka et al., 1988) to
produce point mutations in the mesophilic structural gene, which will result in the corresponding
aminoacid substitution in the primary structure of the encoded protein (Daniel, 1996). Good
results have been obtained in several cases when replacing aminoacids for those corresponding
to the thermophilic protein. Most effective zones for substitution will be the more flexible for
being the more labile (Vieille and Zekus, 1996). However, homology between mesophilic
enzymes and their thermophilic counterparts are usually between 30 and 50 % and no general
strategy for converting mesophilic into thermophilic enzymes have emerged yet, making
thermophiles or the genes derived from them the preferred source for thermostable enzymes in
the foreseeable future (Adams and Kelly, 1998)