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This research could not only provide a novel ?-CGTase but also provide a research basis for changing
the product specificity of ?-CGTase. The enzyme activity was activated by methanol, ethanol,
cyclohexane and dodecanol, but was inhibited by isopropanol and butyl alcohol. The method used to
purify the recombinant CGTase G1 is similar to the procedure used to purify the wild type CGTase
(Sian et al., 2005). The ?-CD cyclization activity of CGTase was measured according to the method
established by Kaneko et al. (1987) with modification. In the longer-range ion pairs, the side-chain
charged group centroids as well as the side chain nitrogen and oxygen atoms are more than 4 A
apart. Second, besides the differences in distance, dissimilarity is noted at the top-left portion of
each diagram in Fig. 3, as indicated by the arrow in CGTase G1. To be able to stand inactivation as
well as to maintain the enzyme activity at high temperature, the secondary and tertiary structures of
Domain A must be stable. Very different CGTase activities are described in the literature. This
applies to the other five CGTases in Fig. 3. Several differences could be pointed out between the six
diagrams shown in Fig. 3. First, variation in distance (verifies by the bold, normal or dash lines) can
be noticed for the other five CGTases. Journal Applied Microbiology, 111, 1129-1137. 8. Martins,
R.F., Delgado, O. and Hatti-Kaul, R. (2003) Sequence Analysis of Cyclodextrin Glycosyltransferase
from the Alkaliphilic Bacillus agaradhaerens. For biomass immobilization the reactivated culture was
added to a column containing sterile charcoal. The Ramachandran plot (Lovell et al., 2003) for the
model generated after three cycles of energy minimization is shown in Fig. 2. There are Fig. 1:
Amino acids alignment of CGTase G1 with some other CGTases with known 3D structure. Journal
of Agricultural and Food Chemistry, 2018, 66(34):9 052-9 060. Leemhuis et al. (2004) applied the
concept of salt bridges in improving thermostability of B. The pH variation was clearly dependent on
the degradation of the carbon source (sodium carbonate). Variations in distance can be observed for
each CGTase. The concentration of total reducing sugars and the pH values were determined and
monitored during the fermentation and are shown in Figure 3. Number of residues in CGTase G1 3D
structure that were in the favoured region was 95.5%, allowed region (4%) and outlier region
(0.4%). Based on the ERRAT plot, VERIFY3D scoring and Ramachandran plot, it is shown that a
good model for CGTase G1 was generated. Preliminary model generated by the Modeler software
has high energy level (9,291.83 kcal mol -1 ). Normally this energy level is considered too high and
due to this reason, a series of energy minimization steps is required to reach a local or more
preferential global minimum level of total energy. In silico, protein structures are more stable in low
energy level. In folded proteins, pairs of neighbouring oppositely charged residues often interact to
form ionic interactions. Due to the contribution of these factors, protein or enzyme can exhibits
enhanced thermodynamic and kinetic stabilities (Kumar et al., 2001). The purpose of this study is to
homology model the structure of CGTase G1 and to study the relationship between ionic interactions
and thermostability. Homology modeling for protein CGTase G1 was executed to understand the
structure of this enzyme and to determine the ionic interactions that exist within the structure. The
bovine bone charcoal is an innovative support material for the immobilization of microorganisms’
producers of enzymes and the use of this microbial support allows its reuse to a significant cost
reduction of the process. The presence of higher quantity of charged residues can increase the
changes of ionic interaction forming. Immobilized cell fermentation was carried in order to determine
the enzyme activity profile during time. The 3D structure of CGTase G1 thus obtained was further
optimized by gradually minimizing energy in three stages. Such statistical works on ionic interactions
were also performed by other researches, for examples in reports by Musafia et al. (1995), Szilagyi
and Zavodszky (2000) and Hendsch (1994). Biochemical Enginerring Jorunal, 41, 79-86. 10. Szejtli,
J. (1988) Cyclodextrin Technology. Comparative protein modeling is based on the reasonable
assumption that two homologous proteins will share very similar structures (Rodriguez et al., 1998).
Because a protein's fold is more evolutionarily conserved than its amino acid sequence, CGTase G1
can be modeled with reasonable accuracy on a very distantly related template, provided that the
relationship between target and template can be discerned through sequence alignment. First the
bone charcoal was characterized and its specific surface area was Figure 3.
Immobilized fermentations were carried in a single batch (168 h) and in five sequential batch of 48 h
each. Starch is usually the substrate used in the production of CGTase. Glu 229, Ser 429 and Pro 663
(red colour) are those energetically unfavorable residues only three amino acid- Glu229, Ser429 and
Pro663 that fall into energetically unfavorable region in the Ramachandran plot. They were
correlated with the CGTase activity ( Figure 5 ). The total numbers of ionic pairs in the CGTases
protein structure are summarized in Table 3 (row No. 6). CGTase G1 and CGTase Bacillus
stearothermophiles have total interactions of 78 and 85, respectively. Surprisingly, CGTase G1 has 74
negatively charged residues and this number is greater than the two other thermostable CGTases. The
fourth Arg found in CGTase G1 biggest networking was Arg579 and its connecting Asp194 and
Asp628, acting as a conduit between Domain B and E. Journal of Inclusion Phenomena and
Macrocyclic Chemistry, 78, 465-470. 13. Bergamasco, R.C., Zanin, G.M. and Moraes, F.F. (2005)
Sulfluramid Volatility Reduction by Beta-Cyclodextrin. For biomass immobilization the reactivated
culture was added to a column containing sterile charcoal. While the ERRAT plot justified the
geometrical errors, VERIFY 3D program validates the structure by a statistical approach that
measures the compatibility of an amino acid sequence with the model structure. TS1-1 and
characterization of the recombinant enzyme. Preliminary model generated by the Modeler software
has high energy level (9,291.83 kcal mol -1 ). Normally this energy level is considered too high and
due to this reason, a series of energy minimization steps is required to reach a local or more
preferential global minimum level of total energy. Leemhuis et al. (2004) applied the concept of salt
bridges in improving thermostability of B. Journal of Fermentation Bioengineering, 85, 416-421.
(98)80086-X 21. Postma, J., Nijhuis, E.H. and Someus, E. (2010) Selection of Phosphorus
Solubilizing Bacteria with Biocontrol Potential for Growth in Phosphorus Rich Animal Bone
Charcoal. Blasting to NCBI database was done using the BLAST program and automated sequence
alignment between CGTase G1 and the template sequence was carried out. Acknowledgements The
authors thank the Coordination of Improvement of Higher Education Personnel (CAPES) for the
financial support. G1 was originally isolated locally from soil at a rubber estate. Science Alert works
with a wide variety of publishers, including academic societies, universities, and commercial
publishers. Nevertheless, all the earlier data revealed that there are differences between the
thermophilic and mesophilic CGTases. The liquid culture was maintained in an incubator at 130 rpm
and 37?C for 24 h. 2.2. Preparation of Matrix and Immobilization Bovine bone charcoal samples
were provided by the company “Bonechar Activated Charcoal from Brazil”. CGTase from Bacillus
stearothermophiles (1CYG) was chosen as template because it turned out to have the highest E value
6.3 e -227 and has the closest sequence identity of 62% and similarity of 78% to CGTase G1.
Verification of the models in each stage of minimization processes were determined using the
ERRAT and Verify3D programs (Colovos and Yeates, 1993). These results demonstrate that aeration,
cell immobilization, proper choice of immobilization matrix and the type of reactor used are
important features in the CGTase production processes. Conflicts of Interest The authors have no
conflicts of interest to declare. The CGTase G1 can withstand higher temperature in a longer period
of time. Biochemical Engineering Journal, 46, 278-285. 49. Kuo, C.C., Lin, C.A., Chen, J.Y., Lin,
M.T. and Duan, K.J. (2009) Production of Cyclodextrin Glucanotransferase from an Alkalophilic
Bacillus sp. Kian Mau Goh, Nor Muhammad Mahadi, Osman Hassan, Raja Noor Zaliha Raja Abdul
Rahman and Rosli Md. Accounts Chem. Res., 37: 123-130. CrossRef PubMed Direct Link. These
amino acids were located near the TIM-barrel N-terminal of the CGTase. Probably because of this,
CGTase G1 has better tolerance ability to heat.
Final pH of the batches was never below 8.8, which is very close to the ideal pH for B. Table 1
resumes the energy level and structure quality for the models in different steps. As a conclusion, it
was presumed that the triad Asp181-Arg185-Asp175 plays an important factor in the networking
that caused the half life of CGTase G1 to be slightly higher compared to other CGTase originally
produced by mesophilic strains, typically B. Cyclodextrins have various applications in the food,
cosmetic, pharmaceutical and agrochemical industries. Number of residues in CGTase G1 3D
structure that were in the favoured region was 95.5%, allowed region (4%) and outlier region
(0.4%). Based on the ERRAT plot, VERIFY3D scoring and Ramachandran plot, it is shown that a
good model for CGTase G1 was generated. Enzyme production was not linked to carbon
consumption, since the increase in CGTase activity was noticed after a rapid carbon uptake. Journal
Applied Microbiology, 111, 1129-1137. 8. Martins, R.F., Delgado, O. and Hatti-Kaul, R. (2003)
Sequence Analysis of Cyclodextrin Glycosyltransferase from the Alkaliphilic Bacillus
agaradhaerens. The pH variation was clearly dependent on the degradation of the carbon source
(sodium carbonate). The presences of ionic interactions inside the CGTase G1 structure were.
Blasting to NCBI database was done using the BLAST program and automated sequence alignment
between CGTase G1 and the template sequence was carried out. Protein Eng. Des. and Sel., 13: 179-
191. PubMed Direct Link Kumar, S. and R. Nussinov, 2001. How do thermophilic proteins deal with
heat. The catalytic residues of CGTase G1 are differentiated by magenta color. Leemhuis et al.
(2004) applied the concept of salt bridges in improving thermostability of B. Biochemical
Engineering Journal, 46, 278-285. 49. Kuo, C.C., Lin, C.A., Chen, J.Y., Lin, M.T. and Duan, K.J.
(2009) Production of Cyclodextrin Glucanotransferase from an Alkalophilic Bacillus sp. Enzyme
Microb. Technol., 39: 74-84. CrossRef Direct Link Ramazzotti, M., C. Parrini, M. Stefani, G. Manao
and D. Degl'innocenti, 2006. Springer, Dordrecht. 11. Bekers, O., Uijtendaal, E.V., Beijnen, J.H.,
Bult, A. and Underberg, W.J.M. (1991) Cyclodextrins in the Pharmaceutical Field. If the cross
domains interactions (between Domain B and other domains) were taken into consideration, the
thermostable CGTases still have higher ionic pairs than the normal CGTases. In Table 3 (row No. 10),
the number in brackets refers to the numbers of interaction between Domain B with other domains in
the protein structure. The CGTase is considered a multifunctional enzyme, which catalyzes four
reactions, among which cyclization is considered the most important. The bolded-line indicates the
salt-bridges, while the normal-line represents N-O pairs. Acknowledgements The authors thank the
Coordination of Improvement of Higher Education Personnel (CAPES) for the financial support.
The results of enzymatic activity achieved show the potential of CGTase production in a short time
with Bacillus firmus strain 37 immobilized in bovine bone charcoal matrix and using air
supplementation in the production medium. This indicates that aeration is a very important parameter
in CGTase production. Probably because of this, CGTase G1 has better tolerance ability to heat.
Introduction CGTase ( ? -1,4-glucan-4-glycosyltransferase) is a bacterial enzyme which has a
molecular mass of around 70 - 78 kD and whose amino acid sequence shows structural similarity to ?
-amylase enzyme. Comparing the values of enzyme activity with free and immobilized cells, it was
found that with the process carried with immobilized B. References 1. Cucolo, G.R., Alves-Prado,
H.F., Gomes, E. and Silva, R. (2006) Optimisation of the Production of CGTase from Bacillus sp
Subgroup Alcalophilus E16 by the Submerged Fermentation of Cassava Starch. Generally, it can be
observed from Table 3 (Row No. 1) that thermostable CGTases exhibited slightly shorter amino acid
sequence than of the mesophilic CGTases. The fourth Arg found in CGTase G1 biggest networking
was Arg579 and its connecting Asp194 and Asp628, acting as a conduit between Domain B and E.
CGTase activity (circle), pH (triangle) and total reducing sugars concentration (square) versus time
for enzyme production with immobilized Bacillus firmus strain 37 on bone charcoal.
The reaction product was identified by high performance liquid chromatography-mass spectrometry
(HPLC-MS), which indicated that the enzyme was a ?-CGTase. Introduction CGTase ( ? -1,4-
glucan-4-glycosyltransferase) is a bacterial enzyme which has a molecular mass of around 70 - 78 kD
and whose amino acid sequence shows structural similarity to ? -amylase enzyme. The residue pairs
that are involved in the ionic interactions were preliminary predicted using the WHAT- IF web
interface program (Rodriguez et al., 1998). The residues numbers involved in the interactions were
extracted and the coordinates of the charged atoms were subsequently identified using the Yasara
View software (Krieger and Vriend, 2002). This indicates that CGTase production is not directly
associated to biomass production, in repeated batches with B. From statistical analysis, Kumar and
Nussinov (2002b) mentioned that normally thermostable proteins have slightly more pairs of
interactions compared to the mesophilic proteins of the same kind. Immobilized cell fermentation
was carried in order to determine the enzyme activity profile during time. Table 1 resumes the energy
level and structure quality for the models in different steps. Enzyme Microb. Technol., 39: 74-84.
CrossRef Direct Link Ramazzotti, M., C. Parrini, M. Stefani, G. Manao and D. Degl'innocenti, 2006.
Biotechnology Letters, 25, 1555-1562. 9. Mazzer, C., Ferreira, L.R., Rodella, J.R.T., Moriwaki, C.
and Matioli, G. (2008) Cyclodextrin Production by Bacillus firmus Strain 37 Immobilized on
Inorganic Matrices and Alginate Gel. Twenty of the networking residues are actually located in
Domain A and B of CGTase G1. Advances in Biotechnological Processes, 6, 31-71. 5. Matioli, G.,
Zanin, G.M. and Moares, F.F. (2002) Influence of Substrate and Product Concentrations on the
Production of Cyclodextrins by CGTase of Bacillus firmus, Strain no 37. Moreover, our results
showed that a proper combination of that features allows to obtain high concentrations of the
enzyme, and this, along with the reuse of the microbial support, conducts to a significant cost
reduction of the process. New Science Press, London. Rahman, K., R.M. Illias, O. Hassan, N.A.N.
Mahmood and N.A.A. Rashid, 2006. Molecular cloning of a cyclodextrin glucanotransferase gene
from alkalophilic Bacillus sp. The structures of CGTase G1 generated at different optimization steps
were also evaluated using the VERIFY 3D program in DS Modeling package. In CGTase G1, the
corresponding residues are Asp181 and Arg185 ( Fig. 3 ). Surprisingly, these two residues were
located very closely in the 3D structure and have a strong salt bridge interaction with centroid
distance of 3.49 A (results not shown). Abbreviation used: CGTase from Bacillus stearothermophiles
(1CYG, template used in modeling CGTase G1), CGTase from Bacillus irculans strain 8 (1CGT),
CGTase from alkalophilic Bacillus sp. 1011 (1PAM), CGTase from Thermoanaerobacterium
thermosulfurigenes strain EM1 (1CIU) and CGTase from Bacillus circulans 251 (1CXI). ?-helices
were indicated by red-bars while ? -sheets were shown as blue-arrows Fig. 2: Full Ramachandran
plot of the main-chain conformation angles of final CGTase G1 model. In the metabolism of CGTase
microorganism producers, starch is converted to CD, producing energy. B1001 by site-directed
mutagenesis. J. Biosci. Bioeng., 89: 206-209. CrossRef PubMed Zhou, H.X., 2004. Loops, linkages,
rings, catenanes, cages and crowders: Entropy-based strategies for stabilizing proteins. The two
arrows and dashed-line oval indicate the other three main variations found in the structures The new
pair of ionic interaction introduced is actually the forth variation mentioned above for the biggest
ionic interaction networking in CGTases. Journal of Molecular Catalysis B: Enzymatic, 49, 1-7. 4.
Bender, H. (1986) Production, Characterization and Application of CDs. The molecular weight of
the recombinant enzyme was about 80 kDa, the optimum temperature was 50 ?, and the optimum pH
was 10.0. There was no detectable ?-cyclodextrin production. ACKNOWLEDGMENTS The author
would like to thank Mohd Ghanee for technical advices. It was important to identify the phases of
cell growth, since the results will be considered in the immobilization of the microorganism in bone
coal, that is, cell immobilization should be made in the period in which B. Second, the flexibility of
these amino acids was higher. By taking CGTase G1 as an example, 50 out of a total 74 negatively
charged amino acid (Asp and Glu) were involved in the ionic interactions. As an overall picture,
almost all residues in the networking were conserved. The results of CGTase activities are shown in
Figure 2. The observed low decrease in pH is also interesting because since enzymes are proteins, the
ionic characters of the amino and carboxylic groups are affected by changes in pH. Ionic networking
comparison: Protein stabilization by ionic networking is much greater than the contribution by
individual interactions (Kumar et al., 2000). Location of networking is believed to be important as
well.
One of the possible ways to stabilize the conformation is to have a higher quantity of ionic
interactions. Applied Microbiology and Biotechnology, 2005, 66(5):475-485. Then, the centroid
distance between the opposite charged residues were computed manually (using Microsoft Excel) by
taking the average of the coordinates of the heavy (non hydrogen) atoms in the side chain charged
group. Agricultural and Biological Chemistry, 40, 753-757. 26. Silva, L., Bieli, B., Junior, O.,
Matioli, G., Zanin, G. and Moraes, F. (2018) Bovine Bone Charcoal as Support Material for
Immobilization of Bacillus firmus Strain 37 and Production of Cyclomaltodextrin
Glucanotransferase by Batch Fermentation in a Fluidized Bed. It was also noticed a direct
relationship between the CGTase activity and biomass concentration of B. The locations of the
residues that form the biggest networking in CGTase G1 is shown in Fig. 4. These 22 amino acids
are in green colour. Besides that, earlier studies (Leemhuis et al., 2004) suggested that Domain B is
also important for the stability of CGTase. However, the predominance of each CD depends on pH,
temperature, its microbial origin, i.e. the microorganism producing the enzyme and also the reaction
time. PubMed Direct Link Martins, R.F. and R. Hatti-Kaul, 2002. A new cyclodextrin
glycosyltransferase from an alkaliphilic Bacillus agaradhaerens isolate: Purification and
characterisation. The reaction product was identified by high performance liquid chromatography-
mass spectrometry (HPLC-MS), which indicated that the enzyme was a ?-CGTase. In CGTase T.
thermosulfurigenes EM1, the consensus residues Asp184-Arg188- Glu183 were located too far away
from the main networking body resulting in separation among the two networks. The 3D structure of
CGTase G1 thus obtained was further optimized by gradual energy minimization in three stages.
ERRAT program was designed to detect local errors within the geometry in the 3D structure using a
nine residue sliding window approach. ACKNOWLEDGMENTS The author would like to thank
Mohd Ghanee for technical advices. The method used to purify the recombinant CGTase G1 is
similar to the procedure used to purify the wild type CGTase (Sian et al., 2005). The ?-CD
cyclization activity of CGTase was measured according to the method established by Kaneko et al.
(1987) with modification. Biotechnology Letters, 25, 1555-1562. 9. Mazzer, C., Ferreira, L.R.,
Rodella, J.R.T., Moriwaki, C. and Matioli, G. (2008) Cyclodextrin Production by Bacillus firmus
Strain 37 Immobilized on Inorganic Matrices and Alginate Gel. One of the possible reasons is
because of richer ionic interactions was found in CGTase G1 than other mesophilic CGTases.
Semina: Exact Technology Science, 28, 143-160. 20. Liu, Y.K., Seki, M., Tanaka, H., Furusaki, S.
(1998) Characteristics of Loofa (Luffa cylindrica) Sponge as a Carrier for Plant Cell Immobilization.
In Figure 2, it was observed that CGTase enzyme activity rapidly increased with time until 48 h,
maintaining relatively constant to 84 h. The lines indicate the ionic interaction between the amino
acids. The calculation was based on the reported method by Kumar and Nussinov (2002a). CGTase
activity (circle) and specific activity of CGTase (diamond) against time enzyme production with
Bacillus firmus strain 37 immobilized on bone charcoal. The ionic interactions in the mesophilic and
thermophilic CGTase homologous were compared. Bioprocess and Biosystems Engineering, 2015,
38(7):1 291-1 301. Apart from that, Arg often forms a triad Asp-Arg-Asp in the ionic networking for
proteins. Second, the flexibility of these amino acids was higher. CGTase from Bacillus
stearothermophiles (1CYG) was chosen as template because it turned out to have the highest E value
6.3 e -227 and has the closest sequence identity of 62% and similarity of 78% to CGTase G1. The
pH variation was clearly dependent on the degradation of the carbon source (sodium carbonate).
Interestingly, CGTase G1 has the shortest Table 2: Thermostability behavior of selected CGTases
Table 3: Summary of ionic interactions in various CGTases. Ionic networking comparison: Protein
stabilization by ionic networking is much greater than the contribution by individual interactions
(Kumar et al., 2000). Location of networking is believed to be important as well.