Abstract
Biotechnology of Extremophiles such as Thermus Aquaticus and Deinoccous radiodurans have a plethora of ways to improve human life. This paper reviews the use of said extremophilic enzymes, bacteria and some methodology of the current biotechnology that can take advantage of the extremophiles.
Introduction
Biotechnology is involved with our everyday lives, ranging from crops production, PCR and more. There’s a huge economic incentive to invest in researching extremophiles to be used in said applications. The paper looks at components of Thermus aquaticus and Deinococcus radiodurans and how these microbial enzymes and properties can be used for industry; improving human lives. The enzymes Examined from Thermus Aquaticus are 4-a-glucano-transferase, Taq polymerase, and Klentaq (modified Taq polymerase). For Deinococcus radiodurans its protective capabilities against radiation and the properties of manganese 2+ are also examined.
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Thermus aquaticus
Thermus aquaticus has an enzyme called 4-a-glucano-transferase (TAαGT) which can catalyse the breakdown of glycogen. This can be used in the sweet potato which is an important crop in Asian countries as it contributes significantly to the economy. Increasing the yield of Cycloamylose will be a huge benefit to industrial producers. Using the enzyme can result in a yield of 48.56% showing the highest Cycloamlyose yield reported from starch. This is done by adding the enzyme to E.coli and sequentially adding Isoamylase to the sweet potato from Pseudomonas sp. This is then debranched using TAαGT. This leads to the increase in yield. (Chu Et Al, 2016). Increasing yield means more money for the producers and thus more of an incentive in further research for this biotechnology. Research is also needed to determine if cost outweighs investment into using this enzyme in the sweet potato.
The enzyme can also be improved by altering it using Bacillus stearothermophilus ET1 CGTase E and DE starch binding domains. These were added to the C-section of TAαGT forming TAαGT-E and TAαGT-DE. this is done to enhance starch utilising activity. The only change is that TAαGT-DE is more molar specific to towards amylose and is still able to produce modified amylopectin structure Cycloamylose. (Park, Et Al 2007).
To alter TAaGT enzyme and clone it, the gene was digested and separated on agarose gel 0.8%. Oligonucleotides primers were used based upon T. aquaticus 33923 and was PCR accordingly using a clone as the template. The products were then digested using Ndel and HindIII and inserted into E.coli. To build the modified genes the stop codon needs to be removed in order to be a linker site for the starch binding domains. The gene was further amplified using PCR. The E and DE domain were isolated using forward primers then digested and ligated into the enzyme producing the chimera enzyme. (Park, Et Al 2007).This method in cloning and modifying this enzyme will be beneficial in future research as the biotechnology may be further improved resulting in improved TAaGT enzymes which can be used to increase Cycloamylose production.
Thermus aquaticus is also known for its Taq polymerase which is widely used for PCR, however issues such as microbial DNA from outside sources can contaminate and create a limit to the use of PCR especially during preparation. This can be a huge problem in regard to forensics because contaminates needs to be kept at a minimum due to the lack of DNA present. There is no universally accepted method of preventing contamination of PCR. A reasonable hypothesis to prevent contamination would be during dilution, contaminating DNA will decrease linearly while amplification of target DNA using Tac polymerase will geometrically still increase until target DNA become equal with Taq polymerase. Doing this increases the reliability of qPCR assays for low level bacteria contamination. (Spangler, R., Goddard, N.L., & Thaler, D.S.2009) (Spangler, R., Goddard, N.L., & Thaler, D.S.2009)
Notable points arise with the graph, one being with every dilution background DNA contamination decrease while geometric efficiency stays constant until target DNA comes to equilibrium with Taq Polymerase. (Spangler, R., Goddard, N.L., & Thaler, D.S.2009). if more DNA is needed then diluting would be a problem because the more dilute the solution less Taq polymerase would be present therefore the geometric limit is less. Increase of DNA will still occur but would remain constant once the limit is reached and product inhibition may pose a problem, further research is needed for better ways in preventing contamination from posing an issue and if product inhibition affects diluted solutions to be used in PCR.
Taq polymerase can also be inhibited when blood and soil samples are used. This can create false positive for forensics tests. This may also pose a significantly problem if target DNA is minute and may be damaged due to contamination. The enzyme can be altered by an N-terminal deletion thus changing the enzyme to Klentaq. Barnes,1992 states Klentaq is a better version of the enzyme with improved heat tolerance and stability.
An experiment made by Kermechiev Et Al, 2009 which uses several samples of soil and blood components to test for inhibition against Klentaq10, Taw22 and Wild type Taq. The results are below.
Inhibition in general was seen across all enzymes. Lactoferrin increased activity of all enzymes, the reason for this is unknown. Plasma, Serum IgG and haemoglobin increased activity slightly when used in low concentrations. (Kermechiev Et Al, 2009)
However Wild type Taq was significantly inhibited by whole blood extract and soil extract. This enzyme should not be used when amplifying DNA from soil and blood when there are better alternatives such as Klentaq. Using Klentaq alongside a diluted solution of target DNA may increase overall efficiency and decrease background contamination however, more research is needed to test If there’s a significant difference in reliability between Klentaq and dilution of target DNA compared to Taq polymerase and a non-diluted solution. A recommendation would be conducting independent T-tests and comparing the mean amount of background contamination, the speed in which the geometric limit is reached how diluted the solution should be.
Deinococcus radiodurans
Deinococcus radiodurans is a polyextremophile as it can survive many extreme environments such as: radiation and cold. Daly Et Al, 2010 shows that Deinococcus radiodurans cell extract prevent protein oxidation from high levels of ionizing radiation. It is extremely important to consider protein protection because they are the first targets of oxidation from radiation, so being able to prevent or treat protein oxidation can be a first step in alleviating or preventing damage. (Gebicki & Du, 2004).
Radiation protection is done by Applying ex Vivo Deinococcus radiodurans ultrafiltrate on cells. Daly Et All, 2010 ultra-centrifuged and ultra-filtrated, D. Radiodurans (DR), radiation sensitive Pseudomonas putida (PP) , E. col (EC) and Thermus thermophilus (TT). When these ultrafiltrates was mixed with E. coli proteins and bombarded with radiation. significant protein damaged was present which can be detected by carbonyl using a western blot analysis; however, DR ultrafiltrate was extremely protective and less carbonyl was detected.
(Daly, M.J. 2012).The dose of radiation which is needed to kill 90% of an organism and how many double stranded breaks occur D.radiodurans and Bdelloid rofftier have shown a higher number of DSBs but still a higher survival with more radiation, this could mean radiation tolerant organism have enhanced DNA repair. Further research is needed on the mechanism of D.radiodurans DNA repair so it can be used for bioremediation at nuclear sites.
D. Radiodurans Also have high amounts of manganese which are roughly 15-150 times greater than radiation sensitive bacteria. (Daly Et All, 2004). Due to the high amount of manganese present in DR, Daly Et All, 2010 tested manganese protection with Bamhl (endonucleases) and states that the presence of manganese is paramount due to Mg2+, Ca2+, Fe2+, Ni2+, Cu2+ and Zn2+ having no protective effect when combined with other molecules. When concentrations of Mn2+ was lowered radiation protection was lost. Applications of this can include altering bacteria and adding manganese to E.coli to be used in bioremediation.
The radiation protective capabilities can be used to protect mice from radiation in comparison to untreated mice. They show that Mn2+- decapeptide complexes (MDP) based on DR protected female mice from radiation syndrome comparatively to non-treated mice. (Gupta Et All, 2016). Initially they tested the toxicity of MDP in vivo to mice and this showed MDP is non-toxic. This was administrated at a dose of 300mg Dp1/kg in a volume of 200uL in 2 doses orally or injected once. This is done daily. The experiment was conducted for 30 days and all mice were euthanized, and blood samples were collected. In terms of protection, MDP was administrated before and after radiation at 300mg DP1/kg caused 100% survival
Further research is needed especially with higher mammalians to determine if side effects are present and if successful. Experimentation can be conducted for humans who work at radiation sites so there would minimal ethical issues because protection from the radiation would already be in place for the worker and MDP will aid in the protection.
Conclusion
Biotechnology of the extremophiles can be extremely advantages, in both commercial and medical settings. Thermus Aquaticus have two enzymes TAqGT and Taq polymerase which we can take advantage of. TAaGT is especially useful for the starch industry and can potentially increase profits due to increase in yield of Cycloamylose. This enzyme can also be modified and potentially improved using Bacillus stearothermophilus starch binding domain ET1 CGtase DE and E, then cloned using PCR. These modifications allow the enzyme to be more molar specific to amylose and be still able to form Cycloamylose. Having the ability to clone this enzyme can allow more modifications to occur and potentially better improvements to happen further increasing yield to the sweet potato and maybe other crops. (Chu Et Al, 2016), (Park, Et Al 2007).
Biotechnology can also take advantage of existing enzymes such as Taq polymerase. Its function is to clone DNA (Via PCR) but this can be further improved with a N-Terminal deletion turning the enzyme to Klentaq. Klentaq is said to be more stable and heat tolerant than Taq polymerase. PCR is done geometrically, and contamination could affect PCR, this can be a huge issue if limited DNA is present as this could affect cloning of said DNA. Diluting the solution in which DNA is present can linearly decrease contamination while keeping the increase of DNA geometrically consistent however the geometric limit is smaller due to less enzyme being present. This is only an issue if a lot of DNA is needed. An independent student t test is needed to test contamination and reliability of both Klentaq and Taq polymerase with and without dilution of the DNA in the solution. (Spangler, R., Goddard, N.L., & Thaler, D.S.2009), (Barnes,1992), (Kermechiev Et Al, 2009).
Deinoccous radiodurans contain a high amount of manganese 2+ and its ultrafiltrate can be used to protect proteins from radiation damage. In the above experiment the ultrafiltrate protected restriction endonucleases for 66 days from radiation and protected E.coli proteins without forming a significant amount of carbonyl. Deinoccous radiodurans may also have enhanced DNA repair capabilities than other organism but more research is needed. (Daly Et Al, 2010), (Gebicki & Du, 2004). (Daly, M.J. 2012), (Daly Et All, 2004).
MDP which is manganese 2+ decapeptide based upon the bacteria manganese properties allows the researchers to protect mice from radiation with a 100% survival rate with radiation protected mice. MDP is also non-toxic to the mice. More research is needed with higher mammalian so human application can be considered and potentially experimented on. (Gupta Et All, 2016).
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