Increased production of the important broad-range endolytic protease proteinase K by the fungus Parengyodontium album through adaptation of the growth medium

doi:10.21203/rs.3.rs-7969934/v1

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Increased production of the important broad-range endolytic protease proteinase K by the fungus Parengyodontium album through adaptation of the growth medium

https://doi.org/10.21203/rs.3.rs-7969934/v1

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Proteinase K is a subtilisin-related serine protease produced by the fungus Tritirachium album, now called Parengyodontium album. Proteinase K has a broad substrate spectrum and is used in many molecular biology protocols to efficiently break down unwanted proteins. Proteinase K experienced supply shortages during the COVID-19 pandemic which contributed to delays in testing and a bottleneck in the production of diagnostic kits. Under laboratory and bioprocess conditions proteinase K is secreted by P. album together with a contaminating protease named “aminopeptidase”. The current proteinase K production process uses a specific P. album strain and a culture medium (“reference medium”) containing corn steep liquor, casein peptone and soybean protein as carbon and nitrogen sources. We found that proteinase K was strongly induced upon the addition of other protein-containing components such as feather meal, wheat bran or brown lentils. Based on these components and using statistical design of experiments, we developed novel culture media that led to a significantly higher proteinase K yield (and similar levels of aminopeptidase) when compared to the reference medium (up to 180% increase). This was in line with a strong increase in proteinase K gene expression. In another series of experiments, casein peptone, which was part of the previously employed reference medium, was replaced by soybean protein. This growth medium now enables the animal-free production of proteinase K without reducing the proteinase K yield. The genome of P. album was sequenced (31.4 Mbp; 10,757 genes) and the genes encoding proteinase K and aminopeptidase were identified on chromosomes VII and VIII, respectively.

Proteinase K

aminopeptidase

Parengyodontium album

bioreactor

growth media improvement

In the 1970s a screening program was initiated to identify microorganisms which produce highly active proteases and within the scope of this program the mold Parengyodontium album (former Tritirachium album Limber) was isolated from soil manured with horn chips which contained high levels of keratin [1]. Keratin monomers (40–68 kDa) assemble into bundles to form strong unmineralized filaments and are found in mammals, reptiles, birds and amphibians [2]. Interestingly, there are also indications that keratins occur in plants [3]. Keratin is hard to degrade and the only other biological material known to approximate the toughness of keratinized tissue is chitin. Using an agar plate-based assay P. album was found to secrete a highly active mixture of proteases whereby one protease was dominant [1]. It was found that this dominant protease was one of the few proteases that can break down keratin, hence it was named proteinase K [4]. At present proteinase K (EC 3.4.21.64) is the only protease of commercial interest from P. album [5, 6]. Later P. album was also isolated from seawater, food and cultural heritage sites [7–10]. Proteinase K is a serine protease and a member of the subtilisin-like protease (S8) family [11–13]. It has a broad substrate spectrum and cleaves peptide bonds C-terminally to a series of amino acids within the target proteins. Proteinase K maintains its activity in the presence of high concentrations of SDS (up to 0.5%) or urea (up to 2 M), is active over a broad pH and temperature range and frequently is used in molecular biology protocols such as DNA- or RNA-isolation. Proteinase K digests structural proteins and generally improves the quality of purified DNA or RNA samples [14]. Accordingly, proteinase K is used in sample processing, for example, for PCR-based virus diagnostic tests. Proteinase K is secreted together with a less characterized “aminopeptidase” [1, 15] in the stationary growth phase when the culture medium is depleted of glucose and amino acids, suggesting a catabolite repression of both enzymes [1]. Aminopeptidases catalyze the cleavage of amino acids from the N-terminus. Proteinase K is secreted by P. album in an inactive form, the so-called “preprotein” (384 amino acids). The preprotein consists of a signal peptide of 15 amino acids, a “proprotein” of 90 amino acids and a mature (and active) proteinase K of 279 amino acids (28.9 kDa) (Fig. 1).

The demand for proteinase K rose dramatically during the Corona pandemic (2019 to 2022) when PCR-based virus diagnostics were very common [16] [9]. Production processes for proteinase K using recombinant microorganisms were reported [17, 18]. At Merck (Darmstadt, Germany) Proteinase K is produced using a specific, genetically unmodified P. album strain and we set out to improve this biotechnological process with regard to proteinase K yield. We observed a strong induction of the enzyme when protein-containing components such as lentils, wheat bran or feather meal were added, and now present improved recipes for growth media based on these components.

Chemicals and Materials

All chemicals were purchased from MilliporeSigma (Darmstadt, Germany), if not stated otherwise. Wheat bran was purchased from Herrnmühle (Reichelsheim, Germany). Feather meal was provided by Beckmann & Brehm (Beckeln, Germany). Lentils (Pardina lentils, Lens culinaris) were obtained from Müller’s Mühle GmbH (Hamburg, Germany). Rice husks were obtained from Hopfen und Mehr GmbH (Hamburg, Germany). Horn splinters were purchased from Bauhaus (Belp, Switzerland). The horn splinters were chopped up in a blender and sifted through a kitchen sieve to remove the remaining large splinters to obtain horn meal, which was then added to the nutrient medium. Healing wool was obtained from dm-drogerie markt (Karlsruhe, Germany). Wool pellets were purchased from Franz und Elisabeth Wagner (Murnau-Weindorf, Germany). Insects (whole larvae of Hermetia illucens) were purchased from Amazon (Seattle, USA), whereby its origin and/or composition is unknown. The larvae were shredded in a blender for 10 minutes and then added to the medium. De-gummed silk fibers were sourced from Etsy (New York, USA). Fishmeal was purchased from Amazon, whereby its origin and/or composition and seller is unknown. The artificial substrate for proteinase K, t-butyloxycarbonyl-L-alanyl-L-alanine p-nitroanilide (Boc-Ala-Ala-pNA) (product number: 4006301, CAS number: 50439-35-5), was purchased from Bachem (Bubendorf, Switzerland) as well as the artificial substrate for aminopeptidase H-Leu-pNA (LEUPA; product number: 4001072, CAS number: 4178-93-2). The antifoam agent desmophen 3600 Z (CAS Number: 171904-01-1) was provided from Merck KGaA (Darmstadt, Germany).

Microbial strains and growth conditions

Wild-type P. album (Merck strain No. 2429) was used throughout this study and is available through the “Centraalbureau voor Schimmelcultures at Baarn (Netherlands, CBS no. 747.69)” [19]. P. album was cultivated using Sabouraud dextrose (2%) broth (Merck KGaA) at 26°C. For agar plates, 16 g/L agar-agar was added. Liquid cultures were grown in 250 mL Erlenmeyer flasks (1 L) with baffles in a shaking incubator at 175 rpm and 1 inch deflection. Strain maintenance was performed by freezing stationary phase mycelium (48 h) at -80°C. Pre-cultures for test fermentations were generated by incubation on Sabouraud dextrose (2%) broth at 26°C (72 h). For the main cultures, 144 mL culture medium (recipe see below), 23 mL preculture, the required amount of glucose (208 g/L) and 50 µl desmophen 3600 Z were mixed in a 1 L shake flask and incubated at 26°C, 175 rpm and 1 inch deflection. The original standard medium for fermentation (reference medium) contained 0.367 g/L CaCl₂ x 2 H₂O, 0.011 g/L FeSO₄, 0.283 g/L KH₂PO₄, 0.708 g/L K₂HPO₄, 1 g/L MgSO₄, 0.594 g/L NaCl, 0.0101 g/L ZnSO₄, 3 g/L corn steep liquor dry concentrate (Merck KGaA, product number: 2.76151), 2 g/L peptone from casein (Merck KGaA, product number: 2.80242), 5 g/L Soy Supro 910 (“soybean protein”) (ADM, Chicago, USA; Profam 648 IP) and 10 g/L glucose [19]. The basic medium for the wheat bran/feather meal test series contained 0.367 g/L CaCl₂ x 2 H₂O, 0.011 g/L FeS0₄, 0.283 g/L KH₂PO₄, 0.708 g/L K₂HPO₄, 1 g/L MgSO₄, 0.594 g/L NaCl, 0.0101 g/L ZnSO₄, 3 g/L corn steep liquor dry concentrate and 10 g/L glucose. The optimal amounts of feather meal/wheat bran to be added to this medium were determined using statistical experimental design (MODDE, see Table S1 in the supplement). The basic medium for the improvement of the animal-free medium contained 0.367 g/L CaCl₂ x 2 H₂O, 1 g/L MgSO₄, 0.594 g/L NaCl and 3 g/L corn steep liquor dry concentrate. The optimal amounts of Soy Supro and glucose as well as K₂HPO₄ and KH₂PO₄ were also determined using statistical experimental design (Table S1). K₂HPO₄ and KH₂PO₄ were used in a ratio of 3.2 to 1. The culture medium based on lentils contained 0.367 g/L CaCl₂ x 2 H₂O, 1 g/L MgSO₄, 0.594 g/L NaCl, 3.6/L KH₂PO₄, 7.9 g/L K₂HPO₄ and 3 g/L corn steep liquor. The amounts of lentils, glucose and Soy Supro were determined using statistical experimental design (Table S1).

Composition of the newly developed culture media that allow a higher proteinase K yield

The medium containing wheat bran and feather meal as complex protein sources which achieved the best results in terms of proteinase K formation was composed as follows: 0.367 g/L CaCl₂ x 2 H₂O, 0.011 g/L FeS0₄, 0.283 g/L KH₂PO₄, 0.708 g/L K₂HPO₄, 1 g/L MgSO₄, 0.594 g/L NaCl, 0.0101 g/L ZnSO₄, 3 g/L corn steep liquor dry concentrate, 10 g/L glucose, 28 g/L wheat bran and 6.15 g/L feather meal. The production medium not containing animal-derived components (“animal-free” medium) was composed as follows: 0.367 g/L CaCl₂ x 2 H₂O, 1 g/L MgSO₄, 0.594 g/L NaCl, 3 g/L corn steep liquor dry concentrate, 3.6 g/L KH₂PO₄, 7.9 g/L K₂HPO₄, 10 g/L glucose and 7 g/L Soy Supro. The medium containing lentils as a complex protein source and achieving the best results in terms of proteinase K formation was composed as follows: 0.367 g/L CaCl₂ x 2 H₂O, 1 g/L MgSO₄, 0.594 g/L NaCl, 3.6 g/L KH₂PO₄, 7.9 g/L K₂HPO_4, 3 g/L corn steep liquor, 8,4 g/L glucose, 9 g/L lentils and 8,5 g/L Soy Supro.

Proteinase K/aminopeptidase assay

For the determination of proteinase K activity in supernatants of P. album cultures, 10 mg BOC-Ala-Ala-pNA were dissolved in ethanol and diluted in 0.1 M Tris pH 8 to an initial absorbance of 0.666–0.668 at a wavelength of 312 nm to create the “proteinase K reaction solution”. The supernatants were then diluted 1:30 with proteinase K reaction solution pre-warmed to 25°C. An increase of absorbance at 405 nm (release of pNA) was followed for 2 minutes and the activity of the enzyme was calculated according to formula 1.

Formula 1: \(\:\frac{\text{U}}{\text{m}\text{l}}=\frac{{\Delta\:}\text{E}\text{*}\text{t}\text{o}\text{t}\text{a}\text{l}\:\text{v}\text{o}\text{l}\text{u}\text{m}\text{e}\text{*}\text{d}\text{i}\text{l}\text{u}\text{t}\text{i}\text{o}\text{n}}{{\epsilon\:}405\:\left(\text{9,62}\frac{\text{c}\text{m}\text{*}\text{c}\text{m}}{{\mu\:}\text{m}\text{o}\text{l}}\right)\text{*}\text{l}\text{a}\text{y}\text{e}\text{r}\:\text{t}\text{h}\text{i}\text{c}\text{k}\text{n}\text{e}\text{s}\text{s}\text{*}\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\:\text{v}\text{o}\text{l}\text{u}\text{m}\text{e}}\)

To determine aminopeptidase activity in supernatants of P. album cultures, 25.1 mg H-Leu-pNA was dissolved in 1 mL ethanol and 5 mM HCl was added to obtain a final volume of 20 mL (solution A). The P. album culture supernatants were diluted 1:4 with solution A and this mixture was again diluted 1:6.5 with 0,1 M Tris pH 8 (the final assay volume was 3 mL). The absorption increase at 405 nm (release of pNA) was followed for 2 min and the activity was calculated by employing formula 1.

Protein purification from the supernatants of P. album cultures

Suspended particles and cells were removed from supernatants of P. album cultures by centrifugation at room temperature (15 min, 20,000 x g). The resulting supernatants were dialyzed (Serva, Heidelberg, Germany) against an equilibration buffer (pH 5.5) containing 0.02 M acetic acid and 0.5 mM CaCl₂ until a conductivity lower than 1.9 mS/cm was reached. The protein solution was loaded to an Eshmuno® CPX column (MilliporeSigma). The column was washed (3 column volumes) using equilibration buffer. Aminopeptidase eluted from the column when the equilibration buffer containing 30 mM NaCl was used, whereas proteinase K eluted from the column using equilibration buffer containing 70 mM NaCl.

Preparation of dry mass of mycelial samples from P. album

P. album cultures were centrifuged at 20,000 x g for 5 minutes at room temperature. The supernatants were removed and the precipitates were dried at 60°C for 7 days. Insoluble components of the growth medium which were present when e.g. wheat bran was added could not be separated from the mycelium and thus these particles added to the specified dry weight.

Statistical experimental design (MODDE)

The MODDE 13 software (Sartorius, Gottingen, Germany) was used to create the design matrices. The measured values were incorporated into MODDE 13 and the model coefficients were determined using the autotune function. Data points with a standard deviation of more than 2 were excluded. The analysis tool integrated in the software was used to calculate the optimal media composition to achieve the highest proteinase K activity in the supernatants.

SDS-PAGE analysis of culture supernatants

Samples of the supernatants (36 µL) were incubated with 1 µL 0.2 M phenylmethansulfonylfluorid dissolved in 2-propanol for 24 h at 20°C. Subsequently, 4 µL of NuPAGE™ sample reducing agent 10x (Thermo Fisher Scientific, Darmstadt, Germany) were added and incubated for 1 h at 30°C. Next, 13.3 µl of NuPAGE™ LDS sample buffer (Thermo Fisher Scientific) were added and samples were incubated for 5 min at 95°C. After centrifugation (1 min, 13,000 x g at room temperature) 20 µl were loaded to 1.0 mm Novex™ Tris-Glycine Plus Midi Protein Gels (4 to 20%) (Thermo Fisher Scientific) and the gels were run at 200 V for 30 min. The proteins within the gels were stained using “PageBlue™ Protein Staining Solution” (Thermo Fisher scientific).

Transcription analysis using quantitative PCR

The mRNA was isolated from mycelial samples of P. album according to the protocol included with the RNeasy Plant Mini Kit for RNA extraction (Qiagen, Hilden, Germany). The reverse transcription reaction was performed employing the Transcriptor First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany). The quantitative polymerase chain reaction (qPCR) was performed using a LightCycler 480 II instrument (Roche). The configuration of the instrument and evaluation of the data was conducted as described previously [20]. The oligonucleotides for amplification of the constitutively expressed gene encoding actin were “primer actin B FW” (5´ GAC GTC CGA AAG GAT CTG TAT G 3´) and “primer actin B RV” (5´ AAG AAG GAG CAA GAG TGG TAA TC 3´). Expression of the proteinase K gene was monitored using the oligonucleotides “primer proteinase K FW” (5´CAC GTC TAC AAG AAC GTC TTC A 3´) and “primer proteinase K RV” (5´ AGC ATC CTG CTC AAT GTA CTC 3´). The oligonucleotide sequences for the actin gene and the proteinase K gene were derived from the genome sequence of P. album (unpublished) which was sequenced by CeBiTech (Bielefeld, Germany). Proteinase K gene expression was normalized to gene expression of P. album actin B [21].

Proteinase K activity in supernatants of liquid P. album cultures changes strongly upon the addition of different proteinaceous components

Culture media used in microbial bioprocesses contain mixtures of defined, less defined, or undefined additives. Our presently used proteinase K fermentation medium (“reference medium”) contained salts, corn steep liquor dry concentrate, casein peptone, Soy Supro (soybean meal) and glucose and we set out to further improve this recipe. The addition of several undefined complex protein-containing substances was tested for their effects on the production and secretion of proteinase K. The protein-containing compounds were added to a liquid culture of a wild-type P. album strain and proteinase K activities were determined in the supernatants of the cultures. The tested protein-containing components replaced the main carbon and nitrogen sources casein peptone and Soy Supro of the reference medium with an equal amount of the new nitrogen source. When the carbon and nitrogen source corn steep liquor dry concentrate was increased in the reference medium, proteinase K activity in the supernatant of the test cultures (1 L shake flasks) was not enhanced, so we did not vary this component in our experiments. Since most of the complex proteinaceous components we added were not fully soluble, the resulting dry masses of the fermentation samples increased as the corresponding particles were not fully degraded by the fungus. First, we investigated whether keratin containing compounds like avian feathers, mammalian wool (healing wool or wool pellets), mammalian horn splinters or mammalian horn meal would lead to higher proteinase K activities when compared to just using the reference medium (Fig. 2). Wheat bran and rice husks were also tested, as it was reported that these components would promote the production of proteases by other organisms [22] and which possibly also contain keratin-like proteins [3]. Insect meal, fishmeal and silk contain high levels of protein and thus were tested although only silk and fishmeal were reported to contain keratin [23]. The volume activity of secreted proteinase K was monitored in the supernatants of the cultures in addition to the volume activity of a second protease activity present in P. album, aminopeptidase, to find out whether addition of the complex substrate would change the relative amounts of the two proteases during fermentation (Fig. 2). The addition of horn meal did neither change proteinase K activity nor aminopeptidase activities in the supernatant. The addition of horn splinters resulted in lower proteinase K and aminopeptidase activities. This was most likely due to the smaller surface area of these particles, reducing degradation of this carbon and nitrogen source by the released proteases. The healing wool turned into felt balls during cultivation and the fungi almost exclusively grew inside those balls. This probably led to a reduced amount of biomass and explains the comparably low volume activities of proteinase K and aminopeptidase in the supernatants. Organic compressed wool (“wool pellets“) is used as a fertilizer and represents a comparably cheap renewable medium component and thus was tested as a carbon and nitrogen source. The wool pellets did not turn into felt balls, however, P. album growth was comparably poor which was probably also due to the reduced surface area of the substrate. Proteinase K activity was also comparably low. Feather meal led to an increase of proteinase K activity in the supernatant, interestingly, however, this was not the case for the contaminating aminopeptidase. Feather meal was the only β-keratin (the main structural elements are β-pleated sheets) [24] tested and it was pretreated with heat and pressure to increase its bioavailability [25]. The addition of wheat bran led to an increase in both the amount of secreted proteinase K and aminopeptidase, either due to an increase in biomass or to a stronger induction of these enzymes. It is important to note, that the increase in dry weight may also have been caused by the addition of not degradable particles that were naturally present in wheat bran. Addition of rice husks resulted in poor growth of P. album and, accordingly, we only found low levels of secreted proteinase K and aminopeptidase. Notably, rice husks often are contaminated with fungicides, which also could be an explanation for the reduced growth. Silk also turned into felt balls with a small surface to volume ratio which may explain its bad performance. Towards the end of the fermentation these balls disintegrated due to the protease activity, but the proteinase K levels in the supernatant were nevertheless low. Insect meal and fish meal were well dispersed and apparently stimulated growth of P. album, however, protease production was low and we concluded that the enzyme was not induced by these components.

A design of experiments analysis identifies a highly effective combination of feather meal and wheat bran and further improved proteinase K production using P. album

The experiments of the previous section revealed that the addition of feather meal and wheat bran increased the proteinase K activity in cultures of P. album (Fig. 2). Accordingly, we tested various combinations of both components with the aim of further enhancing proteinase K production. To reduce the number of experiments a DOE simulation was carried out. The influence of feather meal and wheat bran on proteinase K production was initially simulated using a two-level factorial design and the medium was further improved using a central composite design (Table S1). Our calculations to determine an optimal dosage range for feather meal revealed that the addition of 3–13 g/L feather meal should be most beneficial for proteinase K production (Fig. 3A). The combinations shown in Fig. 3A were tested in shake flask cultures and proteinase K activities were determined in the supernatants. The corresponding data were used to generate a prediction (Fig. 3B). Wheat bran was tested in a dosage range between 8–30 g/L. As we anticipated that the presence of too much solid matter in the medium would negatively affect downstream processing, an artificial limit of 30 g/L wheat bran was set. It has long been known that the ratio between nitrogen and carbon sources in the culture media is important for the protease productivity of fungi and that elevated nitrogen levels reduce the amount of secreted proteinase K [26]. Due to the fact that the exact proportion of available C and N sources in wheat bran was not known, we could not deduce an ideal ratio for proteinase K production and, accordingly, these data points (Table S1) have been adjusted employing the DOE software MODDE. The model parameters indicated a high probability of success (Table S2). The model suggested an optimal combination of 28 g/L wheat bran and 6 g/L feather meal (Fig. 3B). Notably, the optimal amounts of wheat bran and feather meal for the production of another (contaminating) P. album protease, “aminopeptidase”, were found at the limits specified for wheat bran and feather meal (Fig. S1). P. album was grown in shake flasks containing 28 g/L wheat bran and 6 g/L feather meal (see Fig. 3B) replacing 2 g/L casein peptone and 5 g/L Soy Supro and keeping 3 g/L corn steep liquor and all the other components of the reference medium. We found relatively low proteinase K activities in the culture supernatants during the first 50 h of cultivation. After 50 h, however, we measured a strong increase in proteinase K activity (Fig. 4). At the end the culture medium contained almost twice as much proteinase K activity (0.539 ± 0.033 U/mL) when compared to the reference, close to the value predicted by the model (0.557 U/mL) (Fig. 3B). The space-time-yield of the novel process within 96 h cultivation time increased to 180% compared to the reference. The process was successfully upscaled to a total volume of 4 L in a glass bioreactor (volume activity of proteinase K 0.458 U/mL after 78h) which could be sterilized in an autoclave. Unfortunately, when scaling up the process from 4 L glass bioreactors to 50 L steel bioreactors, the addition of wheat bran and feather meal proved to be problematic with regard to the sterilization process. Wheat bran and feather meal caused massive foaming during sterilization on site. The foam clogged the exhaust air filter, causing large amounts of wheat bran and feather meal to stick to the top of the baffle plates. The sterilization process was less efficient due to the insulating effect of the wheat bran and feather meal accumulations and we frequently encountered microbial contaminations. However, we are convinced that the addition of wheat bran and feather meal is very useful when employing a modified bioreactor with a more advanced sterilization option.

Improvement of the reference medium with regard to proteinase K activity and conversion to an animal-free medium

Animal-free culture media do not use animal components such as blood serum or extracts from animals. These animal-free media are often used in research, biotechnology and food production to avoid ethical concerns or bottlenecks in the availability of complex media components made from animal tissue. Our previous experiments had shown that a specific combination of carbon source and nitrogen sources was necessary to achieve a high proteinase K level. It was also observed that P. album only produced proteinase K efficiently at a pH value between 7–8 [26]. Therefore, we first adjusted the pH of the reference medium from 5.8 to 6.9, and then used the software MODDE 13 to predict optimal combinations of KH₂PO₄ and K₂HPO₄ (creating the buffer system), glucose, and soy bean meal (Table S3). Moreover, casein peptone was omitted with the objective to create an “animal-free” medium. The shake flask experiments using the different combinations of KH₂PO₄/K₂HPO₄, glucose, and soy bean meal revealed that most of the MODDE 13 tested combinations resulted in a similar proteinase K activity, however, two combinations were clearly superior (Fig. S2). From these data a model was generated. Tentatively, an optimal proteinase K activity at 11 g/L glucose, 7.2 g/L Soy Supro, and 15 g/L KH₂PO₄/K₂HPO₄ was predicted (Fig. S3). This combination was experimentally tested in three independent shake flask experiments (Fig. 5) and compared to the center point in the DOE designs space (Fig. S2; 0.3 U/mL). Since the resulting activity of the MODDE prediction (Fig. S3; 0.26 U/mL) was lower than the center point (Fig. S2), the model was considered unreliable (see Table S4), and we concentrated on the experimental data (Fig. S2) to develop an animal free medium. The center point of the designs space (glucose 10 g/L, Soy Supro 7 g/L and phosphate buffer 11.5 g/L) resulted in a proteinase K activity of 0.274 ± 0.007 U/mL, while another tested medium composition containing 13 g/L glucose, 8.5 g/L Soy Supro, and 15 g/L phosphate buffer also produced a high proteinase K activity of 0.284 ± 0.006 U/mL (Fig. S2). Since the activities of both points were within the margin of error and the center point had a significantly lower concentration of the carbon sources glucose and Soy Supro this medium was chosen for the next experiments. This medium (called “animal-free medium” in the following) led to a continuous, almost linear increase in activity after 56 h, while the reference medium resulted in a reduced increase in activity after 56 h (Fig. 5). This novel animal-free medium can therefore replace the reference medium without any loss of volume activity and was also successfully used in an upscaling experiment. Still, the animal-free medium clearly produced less proteinase K activity (0.299 ± 0.012 U/mL) when compared to the medium containing feather meal and wheat bran (0.539 ± 0.033 U/mL) (see Fig. 4 of the preceding section).

The addition of lentils strongly enhances proteinase K activities in the supernatants of P. album cultures

The addition of wheat bran and feather meal strongly induced proteinase K activity (see Fig. 4), however, caused massive problems during sterilization. Therefore, we tested another readily available proteinaceous component, brown lentils (Lens culinaris spp., cultivar Pardina) [27], with regard to the induction of proteinase K. Again, a DOE model suggested a variety of combinations of all possible carbon and nitrogen sources present in the medium and these combinations were tested experimentally in shake flasks (Fig. 6). The measured proteinase K activities were used to generate a MODDE 13 prediction (Table S5). This time, the data points showed a better distribution of activities, and the resulting model was also more reliable (Table S6). As a result, an optimal composition of the nutrient medium was predicted to contain 8.4 g/L glucose, 8.5 g/L soy protein, and 9 g/L lentils (Fig. S4). The different proteinaceous media components of the reference medium were replaced by lentils. The combination of lentils and Soy Supro indeed led to a strong increase in proteinase K secretion (Fig. 7). After 96 h of cultivation, a proteinase K activity of 0.485 ± 0.017 U/mL was measured. The curve pattern was similar to that of the wheat bran and feather meal medium indicating a strong induction of proteinase K by lentils or degradation products thereof. In the lentil-based medium, proteinase K secretion was slightly lower at the start of fermentation but then increased significantly. A final comparison of the reference medium to the animal-free medium, the wheat bran and feather meal containing medium and the lentil-based medium is shown in Fig. 7. Exemplarily, an SDS-PAGE analysis of supernatants of P. album cultures (reference medium, animal-free medium and lentil-based medium) is shown in Fig. S5. As foaming was not observed during sterilization employing the lentil-based medium this medium represents a much better option for our production process at Merck. Proteinase K was purified from the supernatants of a 50 L bioreactor containing the lentil-based medium and the reference medium. The standard protocol for protein purification yielded proteinase K preparations with a similar purity, suggesting that downstream processing in a large-scale production process should be possible in a similar manner as before.

Transcription analysis of the proteinase K gene in P. album confirms a strong induction of the enzyme

The results described above revealed that different culture media led to higher proteinase K activities in the supernatants of P. album cultures when compared to the previously used reference culture medium. One culture medium contained feather meal and wheat bran, the second was modified in terms of pH value and buffer strength and the third contained lentils as a nitrogen and carbon source. The latter two culture media were animal-free. Since the addition of feather meal and wheat bran led to excessive foaming during the sterilization process, scaling up to production scale was not possible. Fortunately, the addition of lentils also led to an increase in the volume activity of proteinase K but did not cause excessive foaming during the sterilization process. An important question was whether proteinase K activity increased solely due to the increase in biomass or whether the enzyme was indeed induced more strongly upon addition of e. g. lentil protein. Total RNA was isolated from the mycelium of P. album sampled during growth at two different time points, one in the exponential growth phase and one during the highest increase of proteinase K activity during fermentation (Figure S6) and the transcript level of the proteinase K gene was determined using quantitative reverse transcriptase PCR. Expression of the gene encoding proteinase K was normalized to expression of a constitutively expressed gene actin B of P. album [28]. To our knowledge this is the first report on monitoring gene expression in P. album. The data in Table 1 clearly show that proteinase K was strongly induced (up to a factor of about 871,000) in all cultures when exponential and stationary growth phases were compared. When the data were normalized to the reference medium we found that the change in mRNA levels corresponded to the measured activity levels of secreted proteinase K. In case of the animal free medium we found no increase in proteinase K gene expression and thus the higher final proteinase K yield is solely due to the longer production period. This also explains the differences when comparing mRNA levels and secreted proteinase K activities when comparing the wheat bran and feather meal medium to the lentil based medium (compare Table 1 and Fig. 7).

Table 1
Expression of the gene encoding proteinase K of *Parengyodontium album* depending on the growth medium. Expression change over time was calculated using the 2-^ΔΔCT method [21]. mRNA change of 375,338 means that expression of the proteinase K gene was 375,338 times higher in the later stages of cultivation compared to the start (see Fig. S6).
	mRNA change upon induction	Fold mRNA change compared to the reference medium
Reference medium	358,953	1
Animal free medium	375,338	1.05
Lentil-based medium	870,880	2.43
Feather meal and wheat bran-based medium	728,485	2.03

This study is the first to systematically explore the production of the important broad-range endolytic protease proteinase K by the fungus Parengyodontium album and of a second (contaminating) extracellular protease, aminopeptidase. Both proteases hydrolyze extracellular proteins into free peptides and amino acids, thereby supplying the fungus with carbon- and nitrogen-containing organic compounds. Since keratins are a widespread source of protein for fungi in nature, we assumed that the addition of selected keratin-containing compounds would stimulate formation of proteinase K by P. album. The overall goal was to improve the current production process based on corn steep liquor, casein peptone, and soy protein, as these protein sources do not contain keratin and we assumed that they would not induce the production of proteinase K to the same extent. Our results revealed that different culture media led to higher proteinase K activities (up to 180%) in the supernatants of P. album cultures when compared to the previously used culture medium. One culture medium contained feather meal and wheat bran, the second was modified in terms of pH value and buffer strength and the third contained lentils as a nitrogen and carbon source. The latter two culture media were animal-free, which was another goal of our study. Feather meal led to an increase of proteinase K activity in the supernatant, interestingly, however, this was not the case for the contaminating aminopeptidase. Gene expression studies confirmed the observed induction of proteinase K upon changing the growth medium. The genome of P. album was sequenced and the locations of the genes encoding proteinase K and aminopeptidase were identified.

Acknowledgements

We thank Prof. Harald Kolmar from the Technical University of Darmstadt (Germany) for helpful discussions.

Funding

The work was in part funded by the state of Baden-Württemberg (Germany).

Conflict of interest

The authors declare that they have no conflicts of interest associated with this publication.

Author Contribution

MM, KW and JS wrote the main manuscript text.MM, JS, KHP, HM and KW designed the study.JS conducted the experiments.All authors reviewed the manuscript.

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Increased production of the important broad-range endolytic protease proteinase K by the fungus Parengyodontium album through adaptation of the growth medium

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Abstract

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Introduction

Materials and Methods

Chemicals and Materials

Microbial strains and growth conditions

Composition of the newly developed culture media that allow a higher proteinase K yield

Proteinase K/aminopeptidase assay

Statistical experimental design (MODDE)

SDS-PAGE analysis of culture supernatants

Transcription analysis using quantitative PCR

Results and discussion

Conclusions

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Conflict of interest

Author Contribution

References

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