*Communication* **Rational Design of a Calcium-Independent Trypsin Variant**

**Andreas H. Simon † , Sandra Liebscher † , Ariunkhur Kattner † , Christof Kattner and Frank Bordusa \***

> Institute of Biochemistry and Biotechnology, Charles Tanford Protein Centre (CTP), Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 3a, 06120 Halle, Germany **\*** Correspondence: frank.bordusa@biochemtech.uni-halle.de; Tel.: +49-345-5524800

† These authors contributed equally to this work.

**Abstract:** Trypsin is a long-known serine protease widely used in biochemical, analytical, biotechnological, or biocatalytic applications. The high biotechnological potential is based on its high catalytic activity, substrate specificity, and catalytic robustness in non-physiological reaction conditions. The latter is mainly due to its stable protein fold, to which six intramolecular disulfide bridges make a significant contribution. Although trypsin does not depend on cofactors, it essentially requires the binding of calcium ions to its calcium-binding site to obtain complete enzymatic activity and stability. This behavior is inevitably associated with a limitation of the enzyme's applicability. To make trypsin intrinsically calcium-independent, we removed the native calcium-binding site and replaced it with another disulfide bridge. The resulting stabilized apo-trypsin (aTn) retains full catalytic activity as proven by enzyme kinetics. Studies using Ellmann's reagent further prove that the two inserted cysteines at positions Glu70 and Glu80 are in their oxidized state, creating the desired functional disulfide bond. Furthermore, aTn is independent of calcium ions, possesses increased thermal and functional stability, and significantly reduced autolysis compared to wildtype trypsin. Finally, we confirmed our experimental data by solving the X-ray crystal structure of aTn.

**Keywords:** protease; trypsin; calcium-binding; enzyme engineering

## **1. Introduction**

In nature, the serine protease trypsin (EC 3.4.21.4) is involved in the digestive hydrolysis of proteins into peptides in many vertebrates and has been used widely in various biotechnological processes. The enzyme cleaves peptide bonds on the carboxyl side of lysine and arginine. Catalytic activity is mediated by three highly conserved residues corresponding to the catalytic triad H57, D102, and S195 (chymotrypsinogen numbering) [1,2]. Like many other proteases, trypsin is synthesized as an inactive zymogen and converted into the active enzyme by limited proteolytical cleavage between Arg15 and Ile16 [3]. This cleavage induces conformational changes in the enzyme by forming a salt bridge between Ile16 and Asp194. This interaction stabilizes the substrate-binding site and the oxyanion hole, solidifying the transition state negative charge of the scissile peptide bond carbonyl oxygen [4,5]. The stability of the active state of trypsin is commonly mediated by three disulfide bonds (C42-C58, C168-C182, and C191-C220), which mainly contribute to the high catalytic activity, substrate specificity as well as catalytic robustness in non-physiological reaction conditions [6]. Furthermore, the enzyme has no need for cofactors, which is crucial for many applications.

Nevertheless, trypsin is highly dependent on Ca2+-ions [7]. There are two calciumbinding sites within the enzyme. The first one is localized in the zymogenic peptide and necessary for activation processes via enterokinase. The other calcium-binding site is localized in the calcium-binding loop (CBL) and is necessary for the enzyme's activity. The absence of calcium ions leads to a considerable increase in autodigestion [8]. Furthermore, a temperature-dependent trypsin activation by calcium ions can be observed [7]. This

**Citation:** Simon, A.H.; Liebscher, S.; Kattner, A.; Kattner, C.; Bordusa, F. Rational Design of a Calcium-Independent Trypsin Variant. *Catalysts* **2022**, *12*, 990. https://doi.org/10.3390/ catal12090990

Academic Editors: Chia-Hung Kuo, Chun-Yung Huang, Chwen-Jen Shieh and Cheng-Di Dong

Received: 1 August 2022 Accepted: 29 August 2022 Published: 1 September 2022

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Ca2+-induced thermal stability is also described for other proteases like thermolysin or subtilisins [9,10].

One of the most striking uses of trypsin is the detachment of adherent cells from culture flasks in animal cell cultures. Since exogenous calcium causes cell differentiation and inhibition of proliferation, trypsin is used with EDTA for this application [11]. Since trypsin activity and stability are Ca2+ dependent, the use of EDTA makes it necessary to increase the amount of enzyme, which could alter the physiology, protein expression, and metabolism of cultured cells [12].

Besides cell culture, trypsin is also the most used enzyme in proteomics. Its cleavage carboxyterminal of Arg and Lys results in a positive charge at the peptide C-terminus, being advantageous for MS analysis [13]. Furthermore, some MS applications would profit from a Ca2+-free buffer system due to the prevention of insoluble salts resulting in a possible decrease of ion signals due to buffer-induced ionization suppression [14].

The insertion of artificial cysteines forming cross-linkage within the protein of interest to increase stability is valuable in protein chemistry [15–17]. In the case of subtilisin, the deletion of the calcium-binding loop leads to a drastic stability reduction, which can be restored by inserting a disulfide bridge within the enzyme [18]. This work demonstrates that reduced stability in subtilisin can be compensated by inserting a disulfide bridge. In the present work we were able to show that in trypsin the calcium binding site can even be directly replaced by a disulfide bridge, and thus a more stable, calcium-independent trypsin variant was generated.

#### **2. Results and Discussion**
