Chitosan is a polymeric product of deacetylated chitin, which exists in a wide variety of patterns differing in their degree of deacetylation. Our goal is to design chitosan oligomers with a specific pattern of deacetylation. It can then be used for the production of our hydrogels.
We implemented chitin deacetylases originating from the organisms Sinorhizobium meliloti (NodB) and Vibrio cholerae (COD) into our E. coli cells. These enzymes deacetylate chitin individually. NodB targets the first position of the non-reducing end, while COD works similarly on the second unit. By implementing an orthogonal expression system to regulate the patterns, designer chitosan could be adjusted to the respective task. This would allow the expression of each enzyme separately, creating a defined deacetylation pattern.
What are Chitin Deacetylases?
Chitin deacetylases (CDA) mostly occur in marine bacteria, few in insects, and several in fungi .
In fungi, for example, CDAs are involved in cell wall formation, sporulation, and catabolism of chitin oligosaccharides. Many plant fungal pathogens secrete CDAs during plant infection. Plants only detect fungal infections by registering chitin. Fungi “turn invisible” by deacetylating chitin into chitosan and thus, outwit the plant defence system .
The CDAs generate chitosan oligomers from chitin by deacetylating the N-acetylglucosamine units of the substrate . During deacetylation, acetic acid is cleaved off from a glucosamine unit. Some CDAs may even deacetylate chitosan, creating a double deacetylated oligomer .
Chitin deacetylases belong to the carbohydrate esterase family 4. All family members, including NodB protein and chitin deacetylases, share the same primary structure called “NodB homology domain” or “polysaccharide deacetylase domain” .
In medical applications and plant protection, CDAs are used for designing antifungal and antibacterial biofilms .
NodB - Sinorhizobium meliloti
The CDA NodB is isolated from the organism Sinorhizobium meliloti (strain 1021) , which belongs to the family of gram-negative proteobacteria . Rhizobium sp. often form a root endosymbiosis with legumes in nature. Through this, nitrogen assimilation in legumes is provided. For cell signalling the microbial partners and plants exchange diffusible molecules, the so-called nodulation factors (Nod factors) . Belonging to these Nod factors are NodC, NodB and NodA.
For further information to NodC visit the following link to the Chitin Synthase.
The nodB gene is 653 base pairs long and translates into a hydrolase with a molecular weight of approximately 24.4 kDa .
The enzyme works optimally in surroundings with a pH of 9 and temperatures reaching 37 °C . It solely deacetylates the first position of the non-reducing end in a chitin oligomer . Due to regioselectivity , monomers are not deacetylated by NodB, therefore chains of dimers up to hexamers are converted to mono-deacetylated chitosan oligomers .
If NodB is incubated for a long time with the substrate and high enzyme concentrations, the possibility of double-deacetylated oligomers arises. However, the emerging amount is insignificant .
As explained before, CDAs occur in many different organisms and produce chitosan out of chitin to outwit plant defense systems.
NodB deacetylates the first N-acetyl-D-glucosamine unit (GlcNAc) of the non-reducing end . Deacetylation describes hydrolysis of the acetamido group of the GlcNAc units, thus generating acetic acid und D-glucosamine (GlcN) .
Cloning and Expression
We ordered the nodB gene via IDT sequencing and inserted this gene into the pSB1C3 vector through a BioBrick system and verified this via sequencing. The nodB was fused to an Anderson-promoter with defined cleavage sites (BBa_K2380025). The vector includes the RBS BBa_K2380024.
Before cloning the nodB gene on pSB1C3, the gene was cloned in a pUPD-vector containing a T7-promoter. nodB gene was successfully expressed in E. coli BL21 after induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and analysed via SDS-Page. NodB is known to aggregate inclusion bodies within E. coli cells . Inclusion bodies are non-functional, insoluble aggregates that occur during overexpression .
There are many advantages with the occurrence of inclusion bodies. On one hand they yield a very high protein expression level, on the other, they are easy to isolation from cells because of their difference in size and density in comparison to cellular organelles. Inclusion body proteins also degrade slower and resist proteolytic attacks by proteases. Lastly, proteins expressed in inclusion bodies tend to be less contaminated with cellular substances and can be purified with less steps .
Purification and Refolding
To purify the enzyme, a C-terminal-directed mutagenesis was performed to add a His-taq. The nodB gene was again expressed in E. coli BL21. The cells were disrupted via sonication and in a second sonication step the inclusion bodies were solubilized in a highly concentrated guanidine hydrochloride buffer containing β-mercaptoethanol. High concentrations of chaotropic denaturants such as guanidine hydrochloride and reducing agents (β-mercaptoethanol) provide the solubilization of inclusion bodies . To prevent non-native disulfide bond formations β-mercaptoethanol was used. To refold the bound protein, the 6 M guanidine-hydrochloride buffer was exchanged to a 6 M urea buffer. During slow removal of the denaturant urea, the protein was refolded into the native state .
Affinity purification of His-tagged recombinant proteins and subsequent on-column refolding, gives us the feasibility to purify and refold NodB in a single chromatographic step . The protein was purified via an ÄKTA in combination with a 1 mL HisTrap column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).
Enzyme Reaction and Assay
A reaction volume was set to 1 ml with 1 mM chitin pentamers (AG Prof. Dr. Bruno Moerschbacher, WWU Münster, Institut für Biologie und Biotechnologie der Pflanzen) and 2,5 µM purified NodB eluted in NH4HCO3 adjusted to pH 9. Four reactions were initiated and incubated overnight at 37 °C. After approximately 20 hours the reactions were heat-inactivated at 80 °C for 20 minutes to denature remaining enzymes.
NodB is a carbohydrate esterase which deacetylates the first N-acetyl-D-glucosamine unit (GlcNAc) of the non-reducing substrate end . Deacetylation describes hydrolysis of the acetamido group in the GlcNAc-units, thus generating acetic acid und D-glucosamine (GlcN) .
Acetic acid was indirectly detected via an acetic acid assay kit (Acetate Kinase Manual Format, Megazyme, Bray, Ireland). NADH consumption is measured at 340 nm wavelength. Thus, the amount of acetic acid is stoichiometric with the amount of NAD+ of the last reaction step.
Results and Discussion
Expression and Purification
The nodB gene was successfully expressed in E. coli BL21 after induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The expression temperature was reduced to 30 °C.
Analysis utilizing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed single bands for the NodB enzyme (approximately 24,4 kDa). Results are shown in figure 6.
After tagging the protein with an His-tag, we performed purification and refolding through an ÄKTA pure system and verified the success through another SDS-Page.
Enzyme Reaction and Assay
The following figures show data in which the background of chitin is already detracted from the measured results. Since the blank showed auto-hydrolysis of ATP, resulting in ADP, they were subtracted as well.
The measurement of four replicate enzyme reactions shows an indirect detection of acetic acid via NAD+ absorbance. This proves that NodB has generated acetic acid during deacetylation of chitin into chitosan.
Approximately 10 µg acetic acid in 0.2 x sample volume, 6 µg in 0.15 x sample volume and 4 µg in 0.1 sample volume were detected (figure 7). The 60 % standard solution resulted in 26 µg acetic acid, about 6.5 times more acetic acid as in our samples.
4 µg acetic acid were detected in the 0.1 x sample volume. In the 0.15 x sample volume the amount of acetic acid was 1.5 times higher and in the 0.2 x sample volume it was 2.5 times higher (figure 8).
About 1 mg of chitin was used as substrate. If chitin is completely deacetylated by NodB into chitosan about 60 µg acetic acid are expected to be produced. In our results about 50 µg acetic acid were produced. Consequently, approximately 83 % chitin has been deacetylated. This extrapolated result is shown in figure 9.
We verified the expression of NodB in E. coli BL21 with a SDS-Page. After tagging the protein with a His-tag, we performed purification and refolding through an ÄKTA pure system, and verified the success through another SDS-Page.
To test whether NodB works properly, we used the acetic acid assay kit (Acetate Kinase Manual Format, Megazyme, Bray, Ireland). NodB deacetylates chitin to create chitosan. This chemical step releases acetic acid. As already explained in the methods, the amount of acetic acid is indirectly measured via amount of NAD+. Thus, the amount of acetic acid is stoichiometric with the amount of NAD+ in the last reaction step. NADH consumption is measured at 340 nm.
As the graphs show, this verification was successful and indicates that NodB was refolded properly and is present in its active form. We are thankful for the advise Prof. Dr. Bruno Moerschbacher (also see integrated Human Practices) gave us concerning NodB purification and the recommendation to use the acetic acid assay kit from Megazyme. After successfully purifying NodB, we would appreciate having the opportunity to share our results with his research group and eventually optimize our purification process. The many reaction steps of the kit result in somewhat inaccurate data. Additionally, we would need to try different acetic acid assay kits to evaluate the best one for our purpose. The measurement of background from NodB interaction with the assay reactions is another control, which is needed to be measured to complete our calculation.
At some point in the future, mass spectrometry is another point that has to be tackled as well. Retrospectively, hoping to achieve even better purification results, we would like to try to express nodB in colder conditions for about 40 hours . The expression at lower temperatures enables us to express nodB without the need of refolding from inclusion bodies.
COD -Vibrio Cholerae
In order to bring more variability in our produced chitosans, we decided to implement a second chitin deacetylase. Because of its bacterial origin, we picked COD isolated from the gram-negative organism Vibrio cholerae O1 biovar El Tor str. N16961 . It is not part of its pathogenic activity. During our conversation with Professor Moerschbacher, he informed us that the expression of COD in E. coli has been successfully performed by himself and his group. In addition, it was already proven that both NodB and COD could be expressed in the same organism .
The cod gene is 1296 base pairs long and translates into a hydrolase with a molecular weight of approximately 45.5 kDa .
The enzyme works optimally in surroundings with a pH of 8 and temperatures reaching 45 °C  .
As mentioned before, deacetylases target different units in a chitin molecule. Which unit is deacetylated depends on the chosen enzyme. In the case of COD, the second position from the non-reducing end is deacetylated   . If both enzymes – COD and NodB – are active at once we are able to create a deacetylation pattern involving the first two units.
In contrast to NodB, COD does not deacetylate chitosan twice, after long incubation periods .
CODs catalytic part is its N-terminal domain, while the other two domains make up carbohydrate-binding molecules. The catalytic domain correlates to a carbohydrate esterase domain (CDA) .
COD deacetylates the second N-acetyl-D-glucosamine unit (GlcNAc) of the non-reducing end , thus generating acetic acid and D-glucosamine (GlcN).
We ordered the cod gene via IDT sequencing and inserted this gene into the pSB1C3 vector through a BioBrick system and verified this via sequencing. The cod was fused to a T7-promoter (BBa_I719005). The vector includes the RBS BBa_K2380024.
We induced the BL21 cells, containing the pSB1C3 vector with the T7 promoter system, with IPTG to start expression. In order to validate the successful expression, we performed a SDS-Page.
Results and Discussion
The cod gene was successfully expressed in E. coli BL21 after induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The expression temperature was reduced to 30 °C.
Analysis utilizing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), shown in figure 14, revealed single bands for the COD enzyme (approximately 45.5 kDa).
Further approach would include adding His-tags and purification via affinity chromatography. To test the enzyme activity the amount of released acetic acid during enzymatic reaction can be measured, using an acetic acid assay kit, or via thin-layer chromatography.
Considering the importance of using two CDAs, an orthogonal expression system could be implemented to create multiple patterns of deacetylation.
Since chitin oligomers are deacetylated at specific positions by different deacetylases, the usage of different enzymes makes it possible to create chitosans with defined patterns and special properties. While our project only involves two deacetylases at this time, others could be introduced as well, giving us the opportunity to create varying patterns. To accomplish this future goal, we would require an orthogonal expression system. Unregulated parallel usage of several CDAs only results in chitosan oligomers that are deacetylated by all enzymes. If they could be individually induced, each combination of CDAs may be expressed and different chitosan oligomers could be constructed for various applications. To achieve an effective regulation, we have been searching for a suitable system. During this, we have stumbled upon a solution in a modern research issue – the T7-split-RNA-Polymerases.
The general concept is to split T7-RNAPs into a C-terminal and N-terminal domain. The biological activity of the split T7-RNAPs is restored when both fragments are in direct proximity to each other. The assembly of the split fragments can be made dependent on the dimerization of fusion proteins. Only when both domains are expressed the proteins can dimerize, reassembling the RNAP. If inducible dimerization systems are chosen as fusion proteins the dimerization must additionally be induced, for instance by small molecules or light.
Mutants of the T7-RNAP carrying amino acid substitutions in the region that mainly determine the polymerase’s promoter specificity address altered T7 promoter (PT7) sequences. This would allow the expression of the genes of interest orthogonally, when placing several orthogonal variants of T7 promoter in front of the gene. These promoters can be placed upstream of the genes of the different CDAs. Via chemical or light induction the dimerizations of fused dimer systems mediate the reassembly of various split T7-RNAPs, that are orthogonally addressing variants of PT7. Thus, a orthogonal regulatory system is established.
We have not been able to introduce this system into our project and the iGEM Registry of Standard Biological Parts yet, but we are planning to facilitate such a system in our next years project.
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