Inactivation of SARS-CoV-2 and HCoV-229E in vitro by ColdZyme® a medical device mouth spray against the common cold.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic calls for effective and safe treatments. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing COVID-19 actively replicates in the throat, unlike SARS-CoV, and shows high pharyngeal viral shedding even in patients with mild symptoms of the disease. HCoV-229E is one of four coronaviruses causing the common cold. In this study, the efficacy of ColdZyme® (CZ-MD), a medical device mouth spray, was tested against SARS-CoV-2 and HCoV-229E in vitro. The CZ-MD provides a protective glycerol barrier containing cod trypsin as an ancillary component. Combined, these ingredients can inactivate common cold viruses in the throat and mouth. The CZ-MD is believed to act on the viral surface proteins that would perturb their entry pathway into cells. The efficacy and safety of the CZ-MD have been demonstrated in clinical trials on the common cold. METHOD OF STUDY: The ability of the CZ-MD to inactivate SARS-CoV-2 and HCoV-229E was tested using an in vitro virucidal suspension test (ASTM E1052). RESULTS: CZ-MD inactivated SARS-CoV-2 by 98.3% and HCoV-229E by 99.9%. CONCLUSION: CZ-MD mouth spray can inactivate the respiratory coronaviruses SARS-CoV-2 and HCoV-229E in vitro. Although the in vitro results presented cannot be directly translated into clinical efficacy, the study indicates that CZ-MD might offer a protective barrier against SARS-CoV-2 and a decreased risk of COVID-19 transmission.

Biochemical characterization of a native group III trypsin ZT from Atlantic cod (Gadus morhua).

Atlantic cod trypsin ZT is biochemically characterized for the first time in this report in comparison to a group I trypsin (cod trypsin I). To our knowledge, trypsin ZT is the first thoroughly characterized group III trypsin. A more detailed understanding of trypsin ZT biochemistry may give insight into its physiological role as well as its potential use within the biotechnology sector. Stability is an important factor when it comes to practical applications of enzymes. Compared to trypsin I, trypsin ZT shows differences in pH and heat stability, sensitivity to inhibitors and sub-site substrate specificity as shown by multiplex substrate profiling analysis. Based on the analysis, trypsin ZT cleaved at arginine and lysine as other trypsins. Furthermore, trypsin ZT is better than trypsin I in cleaving peptides containing several consecutive positively charged residues. Lysine- and arginine-rich amino acid sequences are frequently found in human viral proteins. Thus, trypsin ZT may be effective in inactivating human and fish viruses implying a possible role for the enzyme in the natural defence of Atlantic cod. The results from this study can lead to multiple practical applications of trypsin ZT.

Elucidation of different cold-adapted Atlantic cod (Gadus morhua) trypsin X isoenzymes.

Trypsins from Atlantic cod (Gadus morhua), consisting of several isoenzymes, are highly active cold-adapted serine proteases. These trypsins are isolated for biomedical use in an eco-friendly manner from underutilized seafood by-products. Our group has explored the biochemical properties of trypsins and their high potential in biomedicine. For broader utilization of cod trypsins, further characterization of biochemical properties of the individual cod trypsin isoenzymes is of importance. For that purpose, a benzamidine purified trypsin isolate from Atlantic cod was analyzed. Anion exchange chromatography revealed eight peaks containing proteins around 24kDa with tryptic activity. Based on mass spectrometric analysis, one isoenzyme gave the best match to cod trypsin I and six isoenzymes gave the best match to cod trypsin X. Amino terminal sequencing of two of these six trypsin isoenzymes showed identity to cod trypsin X. Three sequence variants of trypsin X were identified by cDNA analysis demonstrating that various forms of this enzyme exist. One trypsin X isoenzyme was selected for further characterization based on abundance and stability. Stepwise increase in catalytic efficiency (kcat/Km) of this trypsin X isoenzyme was obtained with substrates containing one to three amino acid residues. The study demonstrates that the catalytic efficiency of this trypsin X isoenzyme is comparable to that of cod trypsin I, the most abundant and highly active isoenzyme in the benzamidine cod trypsin isolate. Differences in pH stability and sensitivity to inhibitors of the trypsin X isoenzyme compared to cod trypsin I were detected that may be important for practical use.

Potential use of Atlantic cod trypsin in biomedicine.

Surface proteins of viruses and bacteria used for cell attachment and invasion are candidates for degradation by proteases. Trypsin from Atlantic cod (Gadus morhua) was previously demonstrated to have efficacy against influenza viruses in vitro and on skin. In this paper, cod trypsin is shown to be 3-12 times more effective in degrading large native proteins than its mesophilic analogue, bovine trypsin. This is in agreement with previous findings where cod trypsin was found to be the most active among twelve different proteases in cleaving various cytokines and pathological proteins. Furthermore, our results show that cod trypsin has high efficacy against herpes simplex virus type 1 (HSV-1) and the respiratory syncytial virus (RSV) in vitro. The results on the antipathogenic properties of cod trypsin are important because rhinovirus, RSV, and influenza are the most predominant pathogenic viruses in upper respiratory tract infections. Results from a clinical study presented in this paper show that a specific formulation containing cod trypsin was preferred for wound healing over other methods used in the study. Apparently, the high digestive ability of the cold-adapted cod trypsin towards large native proteins plays a role in its efficacy against pathogens and its positive effects on wounds.

Characterization of cold-adapted Atlantic cod (Gadus morhua) trypsin I--kinetic parameters, autolysis and thermal stability.

Atlantic cod trypsin I is a highly active cold-adapted protease. This study aimed at further characterization of this enzyme with respect to kinetic parameters, sites of autolysis and stability. For that purpose, trypsin I was purified by anion exchange chromatography. Its purity and identity was verified by SDS-PAGE analysis and mass spectrometry. Concomitantly, another cod trypsin isozyme, trypsin X, previously only described from its cDNA sequence was detected in a separate peak from the ion exchange chromatogram. There was a stepwise increase in the catalytic efficiency (k(cat)/K(m)) of cod trypsin I obtained with substrates containing one to three amino acid residues. As expected, the activity of trypsin I was maintained for longer periods of time at 15 degrees C than at higher temperatures. The residues of the trypsin I molecule most sensitive to autolysis were identified using Edman degradation. Eleven autolytic cleavage sites were detected within the trypsin I molecule. Unfolding experiments demonstrated that autolysis is a contributing factor in the stability of trypsin I. In addition, the data shows that cod trypsin I is less stable towards thermal unfolding than its mesophilic bovine analogue.

Differential protein expression in early Atlantic cod larvae (Gadus morhua) in response to treatment with probiotic bacteria.

Proteome analysis was used to study the effects of probiotic bacteria treatment on the protein expression in early Atlantic cod (Gadus morhua) larvae. The main focus was on proteins connected to immune function, growth and development. The results demonstrated that none of the identified cod larvae proteins showing up-regulation after administration of probiotic bacteria are known to have a function in immunity. In contrast, several proteins exhibiting down-regulation after exposure to the probiotic bacteria may be related to stress and immune responses. This indicates that the probiotic treated larvae were experiencing a lower level of immune-stimulation than the control group. It is therefore tempting to postulate that the probiotic bacteria mixture used in the present study reduced the environmental stress by inhibiting the growth of pathogenic bacteria. Most of the identified up-regulated proteins in the probiotic group relative to the control may be linked to growth and development. The most pronounced up-regulation of proteins was found in several muscle α-actin isoforms indicating improved growth upon probiotic bacteria treatment.

The novel trypsin Y from Atlantic cod (Gadus morhua) - isolation, purification and characterisation.

This report describes the isolation and partial characterization of the novel group III trypsin Y from the pyloric caeca of Atlantic cod. Other Atlantic cod trypsins have been used as food processing aids with good results. Trypsin Y was purified by p-aminobenzamidine affinity chromatography and characterized by SDS-PAGE and western blot analysis, as well as by activity measurements towards synthetic substrates. Identification of trypsin Y was done with polyclonal antibodies raised towards the recombinant form of the enzyme and by MALDI-TOF mass spectrometry. Trypsin Y is the only group III trypsin isolated from its native source and characterized by biochemical methods. In accordance with the r-trypsin Y, the native enzyme shows dual substrate specificity, i.e. towards trypsin and chymotrypsin specific substrates. This, along with the high cold-adapted character of trypsin Y, may be valuable for its use as a processing aid for sensitive products such as seafood.

Proteome analysis of abundant proteins in two age groups of early Atlantic cod (Gadus morhua) larvae.

The protein expression profiles of two different age groups of Atlantic cod larvae, at 6 days post hatch (dph) and 24 dph, were compared using 2-dimensional electrophoresis and MALDI-TOF mass spectrometry analysis. The life stage of the cod larvae under study covers the first feeding period, generally characterized by high mortality in cod hatcheries. Despite visible morphological and functional changes in larvae from 6 to 24 dph, the pattern of abundant proteins is largely conserved. Interestingly, more than half of the proteins identified in the present study corresponded to different isoforms of the same proteins. The keratins showed the most pronounced developmental stage specific expression pattern. Type II keratins were more dominant in younger larvae and type I keratins in the older larval group. Four isoforms of the fast skeletal muscle alpha-actin, 3 isoforms of beta-2 tubulin and 2 isoforms of alpha-actin were detected. The different isoforms may be either encoded by different genes or generated by post-translational modifications (PTMs) of the same gene product. The PTMs are largely overlooked by studies based on mRNA detection. Therefore, the proteome approach to understanding the cellular mechanisms underlying fish development is important.

Expression and purification of a cold-adapted group III trypsin in Escherichia coli.

The recently classified group III trypsins include members like Atlantic cod (Gadus morhua) trypsin Y as well as seven analogues from other cold-adapted fish species. The eight group III trypsins have been characterized from their cDNAs and deduced amino acid sequences but none of the enzymes have been isolated from their native sources. This study describes the successful expression and purification of a recombinant HP-thioredoxin-trypsin Y fusion protein in the His-Patch ThioFusion Escherichia coli expression system and its purification by chromatographic methods. The recombinant form of trypsin Y was previously expressed in Pichia pastoris making it the first biochemically characterized group III trypsin. It has dual substrate specificity towards trypsin and chymotrypsin substrates and demonstrates an increasing activity at temperatures between 2 and 21 degrees C with a complete inactivation at 30 degrees C. The aim of the study was to facilitate further studies of recombinant trypsin Y by finding an expression system yielding higher amounts of the enzyme than possible in our hands in the P. pastoris system. Also, commercial production of trypsin Y will require an efficient and inexpensive expression system like the His-Patch ThioFusion E. coli expression system described here as the enzyme is produced in very low amounts in the Atlantic cod.

Development of a qRT-PCR assay to determine the relative mRNA expression of two different trypsins in Atlantic cod (Gadus morhua).

A fluorescence resonance energy transfer (FRET) based quantitative RT-PCR method (qRT PCR) was developed in this study for measuring the mRNA expression of trypsins Y and I in the Atlantic cod. Atlantic cod beta-actin was used as the reference gene and standard curves were created for quantification of the mRNA expression levels. For yet unknown reasons, the Atlantic cod (Gadus morhua) produces several trypsins with different characteristics. Trypsin I is the most common and best characterized of these but trypsin Y is a recently discovered enzyme. The recombinant form of trypsin Y was found to have unique characteristics relative to trypsin I. The native form of trypsin Y has proven difficult to isolate from the cod and activity assays do not distinguish between the activities of trypsin I and trypsin Y. The results show that trypsin Y mRNA is expressed in a very low copy number relative to that of trypsin I (ratio of 1:1340), which may explain the difficulty of isolating the native form of trypsin Y.

Atlantic cod trypsins: from basic research to practical applications.

Atlantic cod trypsin I is an appropriate representative of the traditionally classified cold-adapted group I trypsins, and the recombinant form of cod trypsin Y is the only biochemically characterized member of the novel group III trypsins. Trypsin Y is adapted to lower temperatures than all other presently known trypsins. This review describes the basic characteristics of and practical uses for trypsins of Atlantic cod, as well as those of other organisms. Overexpression of the recombinant forms of cod trypsins I and Y in microorganisms is explained as well as the advantages of using site-directed mutagenesis to increase their stability toward autolysis and thermal inactivation. Trypsins appear to play a key role in the nutrition and development of marine fish. We discuss the potential use of cod trypsins as biomarkers to evaluate the nutritional status of cod larvae and describe the industrial applications of cod trypsin I and other trypsins.

Recombinant cold-adapted trypsin I from Atlantic cod-expression, purification, and identification.

Atlantic cod trypsin I is a cold-adapted proteolytic enzyme exhibiting approximately 20 times higher catalytic efficiency (kcat/KM) than its mesophilic bovine counterpart for the simple amide substrate BAPNA. In general, cold-adapted proteolytic enzymes are sensitive to autolytic degradation, thermal inactivation as well as molecular aggregation, even at temperatures as low as 18-25 degrees C which may explain the problems observed with their expression, activation, and purification. Prior to the data presented here, there have been no reports in the literature on the expression of psychrophilic or cold-adapted proteolytic enzymes from fish. Nevertheless, numerous cold-adapted proteolytic microbial enzymes have been successfully expressed in bacteria and yeast. This report describes successful expression, activation, and purification of the recombinant cod trypsin I in the His-Patch ThioFusion Escherichia coli expression system. The E. coli pThioHis expression vector used in the study enabled the formation of a fusion protein between a highly soluble fraction of HP-thioredoxin contained in the vector and the N-terminal end of the precursor form of cod trypsin I. The HP-thioredoxin part of the fusion protein binds to a metal-chelating ProBond column, which facilitated its purification. The cod trypsin I part of the purified fusion protein was released by proteolytic cleavage, resulting in concomitant activation of the recombinant enzyme. The recombinant cod trypsin I was purified to homogeneity on a trypsin-specific benzamidine affinity column. The identity of the recombinant enzyme was demonstrated by electrophoresis and chromatography.

Cold-adapted and mesophilic brachyurins.

Two different types of brachyurins, termed I and II, have been described in the literature. Within type I there are two subtypes, Ia and Ib. The prototype for the type I brachyurins is Fiddler crab collagenase I. Its cold-adapted analogue from Antarctic krill, termed euphaulysin, shares many of its characteristics. Both enzymes are distinguished by their broad substrate specificity as well as the ability to cleave collagen. The precursor form of euphaulysin has been expressed in Pichia pastoris and processed to its fully active form using cod trypsin. A molecular model of euphaulysin, based on the known crystal structure of crab collagenase I, indicates that the core structure of these enzymes is almost identical. As a cold-adapted enzyme, euphaulysin has a higher catalytic efficiency than crab collagenase I. It is also more sensitive to thermal inactivation and autolysis. Furthermore, euphaulysin has an increased length of several surface loops compared to crab collagenase I. Extended surface loops have been suggested to play a role in the cold activity of some bacterial enzymes. Sensitivity to autolysis is an important factor which contributes to the thermal instability of euphaulysin. Substitution of a highly exposed residue in the 'autolysis loop' of euphaulysin resulted in an increased stability of the enzyme towards thermal inactivation without altering its catalytic efficiency.