Enzymes in Biotechnology
Enzymes are globular proteins used as biological catalysts, they speed up the rate of chemical reactions. The essential role of the enzyme is to provide an active site, which has a complimentary shape to the molecules that bind there. Substrates are converted to products through the formation of an enzyme-substrate complex. To be effective in a production process the enzyme molecules must be brought into maximum contact with the substrate molecules.
For the purposes of this essay, biotechnology includes Recombinant DNA technology, bio-detergents, Biological fingerprinting and PCR.
In recombinant DNA technology, the DNA from one organism is combined with the DNA from another organism. Often this involves inserting human DNA into the DNA of another organism. When these genetically engineered organisms are cultured, they produce human protein.
When cells make a protein, they first transcribe its gene into a molecule of mRNA. mRNA molecules carry genes, for example, the gene that codes for Insulin. By finding molecules of insulin mRNA they can be used to make artificial insulin genes. To do this, the enzyme reverse-transcriptase is used. This enzyme speeds up the production of cDNA from mRNA.
Once the target gene has been located using a DNA probe, enzymes called restriction endonucleases (or restriction enzymes) control the process of removing it from the chromosome. There are many different restriction enzymes and each ‘cuts’ DNA at a different base sequence.
When the gene has been isolated using the restriction enzyme it is inserted into a vector that has been ‘cut’ with the same restriction enzyme to produce complimentary ‘sticky ends’. For this insertion to be successful, the enzyme ligase must be used to control the process of joining together the sticky ends of the vector and the sticky ends of the gene.
Using this gene technology, bacteria has been produced that can help to reduce pollution by removing existing pollutants and by introducing cleaner industrial processes.
Spillages from oil tankers are a major source of pollution at sea. Although detergents can be used to remove the oil, these detergents can be very harmful to marine life. However, naturally occurring ‘oil-eating’ bacteria are able to oxidise hydrocarbons into carbon dioxide. The genes that enable them to do this are often carried on plasmids. This has enabled the transfer of these genes into marine bacteria. As a result, genetically engineered bacteria can now be used to deal with oil spills.
Another aspect of enzymes in biotechnology that has proven to be particularly useful is in DNA profiling. This is a way of making a pattern from pieces of DNA cut with restriction enzymes. As everybody has different DNA, the pattern, or fingerprint, is unique to each individual.
This works because within the non-coding regions between genes, there are short sequences of bases called core sequences that repeat themselves over and over again. These repeating regions of DNA are called minisatellites. Different individuals have different numbers of repeated core sequences. The greater the number of repeats, the longer the minisatellite. Different people therefore have different sized minisatellites.
The technique for making a DNA fingerprint can be divided into four main steps: Extraction; digestion; separation and hybridisation.
In Digestion, certain restriction enzymes are added to the DNA to cut it. These enzymes recognise specific base sequences and so cut at specific points, close to, but not within the minisatellite region, thus leaving the minisatellite intact. This process produces a number of DNA fragments of different lengths, which are then separated out using electrophoresis to form a unique pattern.
In many instances there is insufficient DNA available for a genetic fingerprint test. This problem is overcome by using the polymerase chain reaction (PCR). PCR involves the repeated replication of DNA in a test-tube, doubling the amount with each replication cycle.
After heating the DNA to separate the double strands, the enzyme DNA polymerase, four different free nucleotides and DNA primers are added. As the mixture cools, the primers join to the DNA at either end of the region that is to be amplified. The enzyme DNA polymerase controls the ‘joining up’ of the free nucleotides to complete the strands. In this way, DNA is replicated, producing two double-stranded molecules.