Professor Huang Mingtao of South China University of Technology, et al.: Research progress of Clostridium histolyticus collagenase

Collagen is the most abundant type of protein in mammals, accounting for more than 30% of the total protein content in the body, and is widely distributed in
the skin, arteries, tendons, cartilage and most extracellular matrix.
Collagen contains a large number of repetitive "Gly-X-Y" amino acid sequences (Gly is glycine; X and Y are any amino acids, of which X is mainly proline and Y is mainly hydroxyproline), which can form a spiral structure with three peptide chains regularly wound, thereby giving collagen high mechanical strength and stability
.
This property of collagen plays an important role in supporting tissue structure and protecting body organs, but because its structure is stable and difficult to hydrolyze, protein utilization is low
.

Xiao Han, Liu Xiuhao, Huang Mingtao* of the School of Food Science and Engineering, South China University of Technology, based on the latest research progress at home and abroad, summarized the structural characteristics, hydrolysis mechanism, production status and practical application of Clostridium histolyticum collagenase, and looked forward to their future research directions, in order to provide a reference for the further development and utilization of microbial collagenase
.

1.
Structural characteristics of microbial collagenase

Microbial collagenases typically have multiple domains (Figure 2), namely propeptide, activating domain, peptidase domain, PKD, and CBD
.
According to their functions, the activation domain and peptidase domain can be classified as catalytic functional modules, and PKD and CBD can be classified as collagen recruitment modules
.
The difference in collagen collection modules is also an important manifestation
of the structural differences of collagenases derived from different strains.

CBD in the collagen recruitment module specifically identifies the three-stranded helix structure that binds collagen, which is an important structure for collagenase hydrolysis of insoluble natural collagen, which tends to bind to the looser part
of the collagen triple-stranded helix.
PKD does not bind closely to collagen, but it can further strengthen the binding ability
of CBD to fibrous collagen.
As shown in Figure 3, ColG contains two tandem CBD and one PKD, and the presence of Ca2+ can make the junction between the two CBDs in ColG change from a α-helix to a β-fold, enhance the binding of CBD to collagen, and also make it not easily hydrolyzed by other proteases, improving the stability
of full-length collagenase.
ColH contains one CBD and two PKDs, CBD can slide along collagen fibers to find sites that are easier to hydrolyze, and the presence of two PKDs makes it more affinity for collagen
.
According to this structural feature, the two can synergize to hydrolyze collagen, that is, ColG first anchors the most vulnerable areas of collagen fibers to swell the fibrils, and ColH binds to these swollen areas for hydrolysis

2.
Hydrolysis mechanism of collagenase

In 2011, Eckhard et al.
first analyzed the overall crystal structure of collagenase ColG, and speculated the mechanism of hydrolysis of collagen and microfibrillar collagen (composed of 5 three-stranded helixes) by heterologous expression of each domain of the enzyme, which was roughly divided into the following three steps, as shown in
Figure 4.

First, the peptidase domain of collagenase binds to the collagen triple helix (Figure 4A); At this time, collagenase is open, the activation domain does not interact with the substrate, and collagen has not yet been hydrolyzed; Collagenase then closes and the activation domain interacts with the triple-stranded helix (Figure 4B).

During hydrolysis, collagenase gradually opens into a semi-open conformation, unwinding and sequentially degrading the three α-chains (Figure 4C
).
After the three strands are thoroughly hydrolyzed, collagenase returns to its initial open state for the next round of hydrolysis
.
For the more complex microfibrillary collagen formed by 5 three-stranded helix winding, the hydrolysis steps are also roughly the same, but only one three-stranded helix collagen is hydrolyzed at a time, and the rest is discharged outside the enzyme to wait for the next hydrolysis (Figure 4D~F).

3.
Collagenase activity detection method

The enzyme activity detection methods of collagenase mainly include plate method, color rendering method and fluorescence method
.

The plate method can be used for preliminary screening of strains
with collagen hydrolytic activity.
The specific method is to culture the strain with collagenase activity on the gelatin plate, treat the plate with acid mercury reagent, and the gelatin denatured and precipitated without degradation, and the hydrolysis circle around the colony can be observed, and the strength of the strain's ability to decompose collagen can be preliminarily judged by the size of the ratio of the hydrolysis circle to the colony diameter
.
This method is easy to operate and can be used to preliminarily identify the hydrolytic activity of collagenase, but the size of the hydrolysis circle varies according to the observation time, and the sensitivity is low, and it is mostly used for qualitative detection
.

Among the chromogenic methods, ninhydrin chromogenic method is a conventional collagenase enzyme activity detection method, which characterizes the hydrolytic activity
of collagenase by the content of free amino acids released from the enzymatic hydrolysis substrate into the system.
This method typically uses soluble collagen, insoluble collagen, and denatured collagen gelatin as substrates to define enzyme activity U
in terms of glycine or leucine production equivalent.

In addition to natural or denatured collagen as substrates, some synthetic specific substrates are also commonly used for the detection of collagenase activity, such as Azocoll, FALGPA(N-(3-[2-furyl]acryloyl)- L-leucylglycyl-L-prolyl-L-alanine) and Pz-PLGPA (PZ-L-prolyl-L-leucyl-glycyl-L-prolyl-D-arginine), among others
.
Azocoll, a collagen linked to an azo dye, is released by the dye after hydrolysis by collagenase, and enzyme activity
can be calculated by measuring absorbance at a wavelength of 520 nm.
FALGPA and Pz-PLGPA are synthetic collagenase oligopeptide substrates that are not easily hydrolyzed
by other proteases.

In addition to these common substrates, Saikumari et al.
synthesized a new fluorescent substrate that can be specifically cleaved by Clostridium histolyticum collagenase without being affected by other proteases, and the substrate is hydrolyzed and excited by 336 nm excitation light, and the emission fluorescence at 490 nm wavelength is significantly enhanced and the response is fast and sensitive
。 Go et al.
used the characteristics of phthalaldehyde (OPA) combined with free amino acids to produce fluorescence, and added a mass concentration of 1 mg/mL OPA (0.
1 mol/L PBS, pH 7.
4) to the reaction solution after collagenase and substrate, and detected the fluorescence intensity
at 460 nm wavelength after being excited by 360 nm excitation light.
These methods are fast and sensitive, allowing large amounts of data to be obtained in a short period of time, but fluorescent reagents are often more expensive and expensive to detect
.

4.
Current status of collagenase production