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Latest revision as of 23:37, 1 November 2017

Chitin deacetylase NodB

Chitin deacetylase (CDA) NodB from Sinorhizobium meliloti. The enzyme is a hydrolase and deacetylates chitin to chitosan. It solely deacetylates the first position of the non-reducing end in a chitin oligomer [2]. We used it for the production of chitosan with a defined deacetylation pattern. The CDA NodB forms inclusion bodies in E.coli [4].

Biology and Usage

Enzyme properties

A chitin deacetylase isolated from the gram-negative proteobacteria [1] Sinorhizobium meliloti (strain 1021) [3]. The gene itself is 653 base pairs long and is translated into a hydrolase with a molecular weight of approximately 24,4 kDa [2]. The enzyme solely deacetylates the first position of the non-reducing end in a chitin oligomer, setting the acetylated amino-group free via hydrolysis. Expressed in E. coli the enzyme accumulates within the cell in insoluble inclusion bodies and must be purified and refolded to gain functionality. The enzyme's optimal working conditions are in surroundings of pH 9 and temperatures ranging around 37 degrees Celsius.

Application

Chemically or biologically modified chitosan oligomers allow a multitude of applications in modern day projects ranging from agriculture to applied medicine. Due to its stability, easy handling and inexpensive raw material chitosan can be produced in large scales and used as a substrate for further modifications. Due to its antibacterial features usage in clinical environments is also possible.

Purification

During enzyme expression it is important to consider that the protein must either be purified and refolded before usage or be used with a weak promoter to allow for a significant amount of soluble NodB within the cell. Otherwise it will, as already explained, accumulate in inclusion bodies and therefore be unusable [4].

Mechanism

CDAs occur in many different organisms and produce chitosan out of chitin to outwit plant defence systems. NodB deacetylates the first N-acetyl-D-glucosamine unit (GlcNAc) of the non-reducing end [2]. Deacetylation describes hydrolysis of the acetamido group of the GlcNAc units, thus generating acetic acid und D-glucosamine (GlcN) [1].

Methods

Cloning and Expression

The nodB gene was synthesized by IDT and inserted into BioBrick vector pSB1C3 vector and verified via Sanger sequencing (Eurofins Genomics). 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 [4]. Inclusion bodies are non-functional, insoluble aggregates that occur during overexpression [4].
Advantages appearing with inclusion bodies are versatile. 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 contaminants. 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 [2].
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 [5]. 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 [5]. 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 [6]. The protein was purified via an ÄKTA in combination with a 1 mL HisTrap column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The activity of the purified enzyme was evaluated via acetic acid detection (side product of deacetylation). 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 ([http://2017.igem.org/Team:TU_Darmstadt/project/chitin_deacetylase more information]).

Results

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 featuring the protein with an His-tag, we performed purification and refolding with an ÄKTA pure chromatography system using 1 mL HisTrap columns (GE Healthcare) and verified the purification success through another SDS-Page.

Figure 6. SDS Pages of NodB. On the left: SDS-PAGE of chitin deacetylase NodB protein. The arrow marks the position of NodB. From left to right: E. coli BL21 transformed with pSB1C3-nodB, E. coli BL21 transformed with pUPD-nodB after being induced with IPTG for 3h, 6h and 24h, non transformed BL21 parallel to induced cultures and 24h after, E. coli BL21 transformed with pSB1C3-nodB with and without (#) regulatory elements parallel to induced cultures. Usage of PageRuler Prestained Protein Ladder 10 to 180 kDa from Thermo Fischer Scientific.
On the right: SDS-PAGE of purified chitin deacetylase NodB. The arrow marks the position of NodB. Fractions E1-3 show the first flow-through, while Fractions E4-8 are from Elution. Fractions E4-7 show a signal at the appropriate size of NodB. Usage of PageRuler Prestained Protein Ladder 10 to 180 kDa from Thermo Fisher Scientific.

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 NADH consumption at 340 nm wavelength. The amount of acetic acid is stoichiometric with the amount of NAD⁺ in the last reaction step. 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.

Conclusion

We verified the expression of NodB in E. coli BL21 with a SDS-Page. After featuring the protein with a His-tag, we performed purification and refolding of NodB on an ÄKTA pure chromatography 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. The amount of acetic acid is indirectly measured via the amount of NADH consumption at 340 nm wavelength. Thus, the amount of acetic acid is stoichiometric with the amount of NAD⁺ in the last reaction step. 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 [3]. The expression at lower temperatures enables us to express nodB without the need of refolding from inclusion bodies.

References

[1] Muriel Gargaud (Editor-in-Chief), Ricardo Amils, Jose ́ Cernicharo Quintanilla, Henderson James (Jim) Cleaves II, William M. Irvine, Daniele L. Pinti and Michel Viso (Eds.) (2011); Encyclopedia of Astrobiology, Springer-Verlag Berlin Heidelberg, DOI: 10.1007/978-3-642-11274-4, Pages
[2] Hamer, S.N. et al. Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases (2015). Sci. Rep. 5, 8716; DOI:10.1038/srep08716
[3] Alex Bateman (PI), Cathy Wu, and Ioannis Xenarios; UniProtKB - P02963 (NODB_RHIME); http://www.uniprot.org/uniprot/P02963; last visited: 10/19/2017
[4] Rémi Chambon, Stéphanie Pradeau, Sébastien Fort, Sylvain Cottaz, Sylvie Armand (2011); High yield production of Rhizobium NodB chitin deacetylase and its use for in vitro synthesis of lipo-chitinoligosaccharide precursors; Carbohydrate Research 442, 25-30; DOI: 10.1016/j.carres.2017.02.007
[5] Mohan Singh, S., Kumar Panda, A. (2015) Review: Solubilization and Refolding of Bacterial Inclusion Body Proteins; Journal of bioscience and bioengineering Vol. 99, No. 4, 303–310. DOI: 10.1263/jbb.99.303
[6] GE Healthcare Bio-Sciences AB, Uppsala, Sweden (2007), Rapid and efficient purification and refolding of a (histidine) 6-tagged recombinant protein produced in E. coli as inclusion bodies; Application note 18-1134-37 AC; https://www.gelifesciences.co.jp/catalog/pdf/18113437ac.pdf; last visited: 10/09/2017 <p/> Sequence and Features