Urbanek et al. AMB Express (2022) 12:12 https://doi.org/10.1186/s13568-022-01352-7 ORIGINAL ARTICLE Identification of novel extracellular putative chitinase and hydrolase from Geomyces sp. B10I with the biodegradation activity towards polyesters Aneta K. Urbanek1, Miguel Arroyo2, Isabel de la Mata2 and Aleksandra M. Mirończuk1* Abstract Cold-adapted filamentous fungal strain Geomyces sp. B10I has been reported to decompose polyesters such as poly(e-caprolactone) (PCL), poly(butylene succinate) (PBS) and poly(butylene succinate-co-butylene adipate) (PBSA). Here, we identified the enzymes of Geomyces sp. B10I, which appear to be responsible for its biodegradation activity. We compared their amino acid sequences with sequences of well-studied fungal enzymes. Partial purification of an extracellular mixture of the two enzymes, named hydrGB10I and chitGB10I, using ammonium sulfate precipitation and ionic exchange chromatography gave 14.16-fold purity. The amino acid sequence of the proteins obtained from the MALDI-TOF analysis determined the molecular mass of 77.2 kDa and 46.5 kDa, respectively. Conserved domain homology analysis revealed that both proteins belong to the class of hydrolases; hydrGB10I belongs to the glycosyl hydrolase 81 superfamily, while chitGB10I contains the domain of the glycosyl hydrolase 18 superfamily. Phylogenetic analysis suggests a distinct nature of the hydrGB10I and chitGB10I of Geomyces sp. B10I when compared with other fungal polyester-degrading enzymes described to date. Keywords: Geomyces sp. B10I, Polyesters, Chitinase, Hydrolase, Cold-adapted microorganisms © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Introduction Conventional plastics are an integral part of our daily activities and make a significant contribution to vari- ous industries. Unfortunately, the extensive use of plastic causes severe environmental problems. One solution is to develop and commercialize biodegrad- able plastics such as polybutylene succinate (PBS), polybutylene succinate-co-adipate (PBSA), poly- caprolactone (PCL), polylactic acid (PLA) or poly- hydroxybutyrate (PHB), which are decisively more environment-friendly than fossil-based plastics (Nawaz et al. 2015; Jung et al. 2018). Due to their sen- sitivity to the enzymatic attack of microorganisms, the physical and chemical structure of the material and the decomposition conditions, these polyesters can naturally degrade in the environment (Emadian et  al. 2017). Many microorganisms and microbial enzymes have been isolated and described as effective biocata- lysts for the degradation of biodegradable plastics. A particularly large number of hydrolases across many species of microorganisms have been biochemically and structurally characterized (Lee and Huang 2013). Hydrolases represent an important class of enzymes that can increase the rate of degradation of biomass to chemical precursors and can also be effective in removing microplastics from water sources, which is a rapidly growing environmental threat (Johnson et al. 2021). The most frequently mentioned hydrolases Open Access *Correspondence: aleksandra.mironczuk@upwr.edu.pl 1 Department of Biotechnology and Food Microbiology, Faculty of Biotechnology and Food Science, Wroclaw University of Environmental and Life Sciences, Chełmońskiego 37, 51-630 Wrocław, Poland Full list of author information is available at the end of the article http://orcid.org/0000-0003-1604-1635 http://creativecommons.org/licenses/by/4.0/ http://crossmark.crossref.org/dialog/?doi=10.1186/s13568-022-01352-7&domain=pdf Page 2 of 11Urbanek et al. AMB Express (2022) 12:12 revealing activity against polyester-based plastics are lipases, cutinases, esterases, proteases and depolymer- ases (Liu et  al. 2019; Urbanek et  al. 2020). It should be emphasized that among many known enzymes of microbial origin, fungal enzymes with biodegrada- tion activity towards plastic are still little known, and most of them have been reported from Penicillium and Aspergillus species (Kim and Rhee 2003). For instance, Penicillium funiculosum and Penicillium expansum have been reported to produce PHB depolymerase (Brucato and Wong 1991; Gowda and Shivakumar 2015). Aspergillus oryzae RIB40 was able to degrade PBS and PBSA due to secretion of cutinase (Maeda et  al. 2005; Liu et  al. 2009), whereas PHB depolymer- ase from Aspergillus fumigatus 76  T-3 could decom- pose PHB and PES (polyethersulfone). Many other filamentous fungi, belonging to Fusarium (Abe et  al. 2010; Sameshima-Yamashita et  al. 2016; Mao et  al. 2005; Urbanek et al. 2021), Clonostachys, Trichoderma (Urbanek et al. 2017), Geomyces, Sclerotinia, Mortiere- lla (Urbanek et  al. 2021), Alternaria (Abdel-Motaal et al. 2020) or other have been reported as being able to degrade bioplastic without simultaneous enzymatic characterization. It proves that the enzymatic potential of fungi has not been sufficiently discovered yet. In our previous study, we isolated the cold-adapted fungus Geomyces sp. B10I from Antarctic soil sam- ples collected in the vicinity of Arctowski Polish Ant- arctic Station on King George Island, which has been reported to exhibit biodegradation activity towards the polyesters PBSA, PBS and PCL (Urbanek et  al. 2021). Species in this filamentous genus tend to be keratinophilic and psychrophilic. Geomyces are found in a variety of ecosystems and are often the most com- mon group of fungi found in cold environments such as Arctic permafrost, Antarctic soil, and even glacier bank soils at over 3000 m above sea level (Hayes 2012). Since Geomyces sp. is a cold-adapted fungus, likely its enzymes can be active at low temperatures. The use of cold-adapted enzymes may reduce the need of heating, increase the quality, sustainability and cost-effective- ness of technological processes (Santiago et  al. 2016). Moreover, these excellent biocatalysts may play a role in the biodegradation of plastic. Here, we partially purified and characterized two novel enzymes produced by Geomyces sp. B10I. Based on the basic local alignment search tool (BLAST) (Alts- chul et al. 1990), both proteins were found to belong to the hydrolase class, and more specifically to the hydro- lase and chitinase families. As hydrolases are common enzymes with biodegradable activity, both enzymes are perceived to degrade polyesters and have been named hydrGB10I and chitGB10I, respectively. Materials and methods Fungal strain and culture media The cold-adapted strain of filamentous fungi Geomyces sp. B10I with PBSA/PBS/PCL-degrading activity depos- ited in Collection of Industrial Microorganisms (IAFB) (Warsaw, Poland) under number KPP 3680 was used in this study. For the enzyme production 2xYT medium composed of tryptone 1.6%, yeast extract 1.0%, NaCl 0.5% and Tween 80 0.1% was used. Ten flasks contain- ing 100 mL of 2xYT liquid medium were inoculated with 2–3 loops (20–30 µg) of mycelium taken from the plate culture and incubated at 21  °C, 140  rpm for 72–96  h. Minimal mineral (MM) medium containing 0.1% emulsi- fied polyesters (PBSA, PBS or PCL) was prepared for the examination of BP-degrading enzymatic activity as pre- viously described (Urbanek et  al. 2021). Cell-free broth, fractions after stepwise precipitation with ammonium sulfate and fractions collected after IEC were preserved at 4 or − 20 °C. Materials Polybutylene succinate (PBS) and polybutylene succi- nate-co-adipate (PBSA) under the trade names Bionolle 1020MD and Bionolle 3020MD, respectively, were pur- chased from Showa Denko K.K. (Japan). Polycaprol- actone (PCL) was obtained from TRESNO (Poland). Chemicals used to prepare the culture media and car- tridges for ion-exchange chromatography Bio-Scale Mini UNOsphere Q (strong anion exchanger) 5 mL and 1 mL, Bio-Scale Mini UNOsphere S (strong cation exchanger) 5 mL and 1 mL (GE Healthcare, Sweden) were purchased from Sigma-Aldrich (Germany). 0.5% biopolymer emul- sions were prepared as previously described (Urbanek et al. 2021) and used in plate and turbidimetric assays to determine the biodegradation activity at each step of the purification procedure. Partial protein purification steps After aerobic cultivation, the crude enzyme solution was obtained by centrifugation (10,000×g for 20  min, 4  °C) and paper filtration. The crude enzyme solution (350 mL) was initially concentrated with polyethylene glycol (PEG) to 158 mL. Next, ammonium sulfate (AS) was added to the solution in four steps to 20, 40, 60 and 80% satura- tion at 4 °C with stirring, respectively (Chua et al. 2013). The mixtures were centrifuged at 17,000×g at 4  °C for 20 min. Obtained pellets were dissolved in 20 mM Tris– HCl buffer (pH 8) and were treated as enzyme solutions marked as 20, 40, 60 and 80% AS, respectively. Subse- quently, plate assays indicating biodegradation activ- ity were performed. 80  µL of enzyme solutions were applied into wells in MM medium containing emulsified bioplastics. The halo zones formed during incubation Page 3 of 11Urbanek et al. AMB Express (2022) 12:12 corresponded to biodegradation activity of fractions. The enzyme solution with the highest activity was dialyzed, clarified and loaded onto Bio-Scale Mini UNOsphere Q 5  mL (GE Healthcare, Sweden) or Bio-Scale Mini UNOsphere S 5 mL (GE Healthcare, Sweden) cartridges using a Bio-Rad Duo Logic LP chromatography sys- tem (Bio-Rad, USA). The cartridges were previously equilibrated with 20  mM Tris–HCl buffer (pH 8.0) and high-degassed buffer according to the manufacturer’s instructions. Elution was performed first isocratically with the same buffer and then with a linear gradient from 0 to 1 M NaCl (0–100% buffer B). The flow rate was main- tained at 1  mL/min. The active fractions were pooled and dialyzed overnight against 20  mM Tris–HCl buffer (pH 8.0). The enzyme solution was then applied to Bio- Scale Mini UNOsphere Q 5 mL (GE Healthcare, Sweden) equilibrated with 20 mM Tris–HCl buffer (pH 9.0). The column was washed with the same buffer, and the active fractions were eluted isocratically and with a 0–1 M NaCl linear gradient. The active fractions were pooled again, dialyzed against 20  mM Tris–HCl buffer (pH 9.0) and then loaded onto Bio-Scale Mini UNOsphere Q 1  mL (GE Healthcare, Sweden) equilibrated with 20 mM Tris– HCl buffer (pH 9.0). The active fractions were eluted isocratically. All fractions obtained at every purification step were analyzed for enzymatic activity in a plate assays (described in “Biodegradation activity” section). The pro- tein concentration was determined according to Bradford (Bradford 1976) using BSA as standard. The molecular mass of the purified proteins was determined by SDS- PAGE using 12.5% polyacrylamide gel (Laemmli 1970). Unstained SDS-PAGE Broad Range Standard (Bio-Rad) was used as a molecular weight marker. Biodegradation activity Wells were aseptically cut in solid MM medium supple- mented with 0.1% bioplastic emulsions. 100 µL cell-free extracts or fractions collected during IEC were applied to each well. The plates were incubated for up to 24 h at 40  °C. After incubation, the emerging clean zones were observed as a result of enzymatic hydrolysis. The visible halo zone confirmed the presence of enzymes with bio- degradation activity in the applied samples. Moreover, the activity of the enzyme solutions was determined by measuring the decrease in turbidity of homogeneous PBSA, PBS and PCL suspensions accord- ing to García-Hidalgo et al. (2012) with slight modifica- tions (García-Hidalgo et al. 2012). Briefly, a total of 3 mL of the standard reaction mixture containing 50  mM Tris–HCl buffer (pH 8.0), 2  mM CaCl2 and 200  μg/mL of sonicated homogeneous suspension of polyesters (1 mg/mL) in deionized water was pre-incubated at 40 °C for 5 min. Then, the reaction was started by adding the enzyme solution and the decrease in turbidity was meas- ured every 5 min at 630 nm during the 40 min incubation period at 40  °C. Controls without enzymes were car- ried out in parallel to ascertain possible non-enzymatic hydrolysis of the polyesters. All activity assays were per- formed in duplicate. One unit (U) of enzyme activity was defined as the amount of enzyme which catalyzes a decrease of 0.01 absorbance units per minute at 630 nm under the assay conditions (García-Hidalgo et  al. 2012). Protein concentration was determined as previously described (Bradford 1976) and the specific activity (U/mg protein) was calculated. Matrix‑assisted laser desorption‑time‑of‑flight (MALDI‑TOF) analysis Single bands obtained in SDS-PAGE were cut from the gel and analyzed by MALDI-TOF mass spectros- copy using single fragmentation (only one mass spec- troscopy—MS—analysis) and double fragmentation of given peptides (MS/MS) in 4800 Proteomics Analyzer (AB SCIEX). The MASCOT database with similarity search against SwissProt database (https:// www. expasy. org/ resou rces/ unipr otkb- swiss- prot) with a taxonomic restriction to fungi was used to identify proteins based on mass spectroscopy data (Wojtusik et al. 2018). Phylogenetic analyses Subsequently, the obtained protein sequences were compared with known proteins with proved biodegra- dation activity within the BLASTp tool (Altschul et  al. 1990). Multiple sequence alignment was built within ClustalOmega (https:// www. ebi. ac. uk/ Tools/ msa/ clust alo/) (Madeira et  al. 2019) and next edited and visual- ised within Jalview (Waterhouse et al. 2009). The analy- sis was conducted to reveal any similarities of hydrGB10I and chitGB10I to the other known hydrolases involved in biodegradation such as depolymerases and cutinases. HydrGB10I was also compared to other members of the glycoside hydrolase family 81, and chitGB10I to chi- tinases. Protein sequences restricted to fungal proteins were retrieved from NCBI (National Centre for Biotech- nology Information) (https:// www. ncbi. nlm. nih. gov), UniProt (https:// www. unipr ot. org) and PMBD (Plas- tics Microbial Biodegradation Database) (http:// pmbd. genome- mining. cn/ home) databases. Evolutionary phylogenetic analyses were performed within MEGA-X software using the neighbour-joining method (Kumar et al. 2018). Both the accession numbers and the names of the selected sequences are given under the alignment results presented in Additional file 1: Figs. S4, S5. The percentage identity of hydrGB10I and chit- GB10I to other sequences is coloured blue. The most https://www.expasy.org/resources/uniprotkb-swiss-prot https://www.expasy.org/resources/uniprotkb-swiss-prot https://www.ebi.ac.uk/Tools/msa/clustalo/ https://www.ebi.ac.uk/Tools/msa/clustalo/ https://www.ncbi.nlm.nih.gov https://www.uniprot.org http://pmbd.genome-mining.cn/home http://pmbd.genome-mining.cn/home Page 4 of 11Urbanek et al. AMB Express (2022) 12:12 conserved residues in each group are the most intense in colour, and the least conserved are the palest. PSSA (protein sequence-structure analysis) and homol- ogy modelling were performed within RaptorX (Källberg et al. 2012). The three-dimensional structures of the iden- tified proteins were visualized by PyMOL (Schrödinger 2010). SWISS-MODEL (https:// swiss model. expasy. org), accessible via the Expasy webserver (Waterhouse et  al. 2018) was also used to model protein structure homol- ogy modelling to obtain more precise information such as GMQE (Global Model Quality Estimate) value. Amino acid sequences Accession numbers for amino acid sequences of proteins in GenBank are KFY49210.1 and KFY57494.1. Results Inoculum and enzyme production Cold-adapted fungus Geomyces sp. B10I with the ability to degrade bioplastic (Additional file 1: Fig. S1) was cul- tivated aerobically in 2xYT medium supplemented with Tween 80. The addition of Tween 80 was necessary for the extracellular secretion of enzymes. It was observed that the biodegradation activity exhibited by the fungus was closely related to the release of spores (Additional file 1: Fig. S1). Thus, the culture was stopped when spore release was observed under the microscope. Cell-free broth containing the enzymes was obtained by cen- trifugation and filtration and was used as crude enzyme solution. Partial protein purification steps Three hundred and fifty  millilitre of cell-free broth was initially concentrated to 158 mL. Next, the proteins were precipitated by the stepwise addition of ammonium sul- fate (AS). Pellets obtained after centrifugation were dis- solved in 20  mM Tris–HCl buffer and tested in a plate assays for hydrolytic activity. The largest halo zone was observed for 60% AS saturation (Fig. 1) and this fraction was selected for the purification process. The dialyzed and clarified fraction (11 mL) was loaded onto Bio-Scale Mini UNOsphere Q 5  mL, strong anion exchanger (GE Healthcare, Sweden). The enzyme with biodegrada- tion activity was eluted both isocratically with 20  mM Tris–HCl buffer (pH 8.0) and with linear NaCl gradient (0–1.0 M) in the same buffer. The pooled, dialyzed and adjusted to pH 9.0 active fractions (Fig.  2A and Additional file  1: S2A) were applied onto the same column at pH 9.0. Here, the active enzymes were eluted isocratically (Fig. 2B and Additional file 1: S2B). Fractions were pooled again and applied onto Bio-Scale Mini UNOsphere Q 1  mL pH 9.0 and again eluted isocratically (Fig.  3C and Additional file  1: S3C). The peaks corresponding to the collected fractions with biodegradation activity in the plate assays are marked as red squares in the chromatograms (Additional file  1: Fig. S2). An application of active fraction onto cartridge packed with strong cation exchanger gave no result in purification (data not shown). During the protein puri- fication steps, fractions containing enzymatically active proteins, i.e., cell-free broth, 60% AS fraction, pooled fraction from isocratic elution and fractions eluted in gradient were analysed by SDS-PAGE. Despite the fact, that fraction no. 6 collected after the third round of IEC did not bind to the resin, showed only two bands in SDS- PAGE (lane 6, Fig.  3) and exhibited hydrolytic activity in the plate assay towards PBSA (well 6, Fig.  2C). Simi- lar effect was observed in a plate assays towards PBS and Fig. 1 Comparison of enzymatic activity in a plate assay of cell-free broth (sup), concentrated cell-free broth (sup conc.) and fractions precipitated with (NH4)2SO4 (20, 40, 60 and 80% AS). The largest halo zone was observed in the fractions saturated with AS up to 60%. All fractions were applied on MM medium supplemented with A 0.1% PBSA; B 0.1% PBS; C 0.1% PCL https://swissmodel.expasy.org Page 5 of 11Urbanek et al. AMB Express (2022) 12:12 PCL (data not shown). This fraction contained 2.05  mg of partially purified proteins calculated by the Bradford measurement. The specific biodegradation activity of the partially purified enzymes was measured by evaluating their abil- ity to hydrolyze the PBSA emulsion in a turbidimetric assay. Although the activity of partially purified enzymes was confirmed by the plate assay, no reduction in the tur- bidity of homogeneous PBSA was observed. The yield and purification factors in the ammonium sulfate precip- itation step were 31.31% and 14.16-fold, respectively, and the specific activity for PBSA was 91.89 U/mg (Table 1). Next, the enzymatic activity towards different types of polyesters was compared using a turbidimetric assay. The results suggest that 40  °C is the optimal tempera- ture for enzyme activity. The activity expressed in U/mL was twice as high as the activity in the other temperature variants for all polyesters. Moreover, the highest activity Fig. 2 Biodegradation activity towards 0.1% PBSA in a plate assay exhibited by fractions collected throughout chromatographic steps: A pH 8.0, Bio-Scale Mini UNOsphere Q 5 mL; B pH 9.0, Bio-Scale Mini UNOsphere Q 5 mL; C pH 9.0, Bio-Scale Mini UNOsphere Q 1 mL. The numbers represent the consecutively collected factions; the letter P represents permeate Fig. 3 Electrophoretic profile of proteins. Lane 1—molecular weight standard (Unstained SDS-PAGE Broad Range Standard, Bio-Rad); lane 2— free-cell supernatant; lane 3—60% AS fraction; lane 4—pooled fractions (wash + gradient) exhibiting the activity after the first round of IEC; lane 5—pooled wash fractions after the second round of IEC; lane 6—pooled fractions after the third round of IEC; lane 7—fraction 6 after the third round of IEC showing only two bands and biodegradation activity. PBSA1—hydrGB10I, PBSA2—chitGB10I Page 6 of 11Urbanek et al. AMB Express (2022) 12:12 was indicated for PBS and the lowest for PCL (Table 2). It should be noted that a decrease in absorbance was also observed at 30 and 20 °C. Activities at 20 °C were half the value of the activity at 40  °C. Negative controls (buffer instead of enzyme mixture) indicated no decreases in turbidity. Matrix‑assisted laser desorption‑time‑of‑flight (MALDI‑TOF) analysis The fractions collected during the purification process showing biodegradation activity in the plate assay were separated by SDS-PAGE. Flow-through fraction after the 3rd round of IEC showed activity (well 6) and revealed only two bands (Fig. 4, lane 6). Bands named PBSA1 (hydrGB10I) and PBSA2 (chit- GB10I) were separated from the gel (two repetitions of each band) and analyzed by MALDI-TOF. The results consisted of the combined peptide fingerprint analy- sis and peptide fragmentation of the bands. Additional file 1: Fig. S3 shows the peptide fingerprint result of par- tially purified proteins. Analysis revealed that hydrGB10I contained 714 aa with a predicted molecular mass of 77.248 kDa and a calculated pI 6.75, while chitGB10Icon- tained 428 aa with a molecular mass of 46.482 kDa and pI Table 1 Purification of native enzymes from the cell-free broth of Geomyces sp. B10I. The activity was measured in a turbidimetric assay with PBSA as a substrate for enzyme activity. Unsuccessfully, it was not possible to determine the activity of purified enzymes after the IEC procedure. The assays were performed in duplicate Volume (mL) Protein (µg/mL) Activity (U/mL) Total protein (mg) Total activity (U) Specific activity (U/mg) Purification factor Yield (%) Free-cell broth 350.0 77.31 ± 0.71 4.93 ± 0.01 27.06 ± 0.25 1.725.5 ± 4.95 63.77 ± 0.77 1 100 Concentrated free-cell broth 158.0 139.16 ± 2.56 10.76 ± 0.14 21.99 ± 0.4 1.700.08 ± 22.34 77.32 ± 0.40 1.21 98.52 60% (NH4)2SO4 11.0 52.47 ± 2.67 54.82 ± 2.8 0.66 ± 0.03 592.02 ± 15.24 903.22 ± 22.71 14.16 34.31 Table 2 Biodegradation activity of free-cell broth after concentration and precipitation with (NH4)2SO4 towards polyesters: PBSA, PBS and PCL at different temperatures expressed in U/mL measured in turbidimetric assay. The experiment was performed in duplicate 20 °C 30 °C 40 °C PBSA PBS PCL PBSA PBS PCL PBSA PBS PCL Concentrated free-cell broth 44.72 ± 0.34 80.96 ± 0.68 16.8 ± 0.23 40.48 ± 3.62 89.44 ± 2.04 8.64 ± 0.23 101.6 ± 5.09 124.44 ± 1.19 39.28 ± 4.41 60% (NH4)2SO4 165.6 ± 1.13 205.6 ± 0.57 60.88 ± 0.34 221.4 ± 3.62 309.4 ± 8.77 50.88 ± 1.58 369.6 ± 7.07 466.6 ± 11.60 65.76 ± 2.49 Fig. 4 Model of A hydrGB10I (hydrolase) and B chitGB10I (chitinase) visualized within the PyMOL based on results of homology modelling obtained from RaptorX and COACH servers. Models present the three-dimensional structure of proteins. The secondary structure elements are coloured in blue (α-helices), red (β-sheets) and pink (loops). The surface structure is coloured in pink Page 7 of 11Urbanek et al. AMB Express (2022) 12:12 of 8.13. The obtained amino acid sequences (Additional file  1: Fig. S4) were compared with known sequences within the BLASTp tool. The search indicated that both hydrGB10Iand chitGB10I proteins showed identical values (100% identity, 0.0 e-value and 100% coverage) to the proteins characterized as hypothetical proteins from Pseudogymnoascus sp. VKM F-4515 with acces- sion numbers KFY49210.1 and KFY57494.1, respectively. Both proteins contained conserved domains of glyco- side hydrolases. The conserved domain of hydrGB10I belonged to the glycosyl hydrolase 81 superfamily. In turn, protein chitGB10I contained a domain of glycosyl hydrolase family 18 (GH18_chitinase). We found that 10 chitGB10I residues: Tyr47, Phe75, Asp171, Asp173, Glu175, Met241, Tyr243, Asp244, Tyr297, Trp382 appeared in the sequence of the glycosyl hydrolase 18 family as an active site. This active site was found in chitinase C from Bacillus circulans, chitinase B from Saccharophagus degradans 2–40 or chitinase from Bur- kholderia dolosa AU0158. The analysed proteins were named hydrGB10I and chitGB10I for PBSA1 and PBSA2 bands, respectively. The structure homology conducted within the Swiss-Model server (https:// swiss model. expasy. org/) suggested that both proteins are mono- mers. The modelling results showed that the sequence of hydrGB10I was 30.13% identical with 0.96 coverage of the template structure of the glycoside hydrolase fam- ily 81 endo-[beta]-1,3-glucanase, whereas the sequence of chitGB10I was 59.44% identical with 0.92 coverage to the crystal structure of a chitinase CrChi1 from the fun- gus Clonostachys rosea. The GMQE value for PBSA1 was 0.70 and for PBSA2 0.78, which represents high reliabil- ity in the target–template alignment. In addition, a total of 42 and 781 templates were found to match the target PBSA1 and PBSA2 sequences, respectively. The three- dimensional structure of proteins was visualized within the PyMOL program and was presented in Fig. 4. The analysis showed that the sequences have some conserved amino acids. The residues identified to be more than 60% conserved in the structure of hydrGB10I are Arg21, Pro100, Gln111, Asp129, Asn139, Pro148, Tyr151, Val192, Tyr208, Gly626, His628 and Gly667, whereas in chitGB10I sequence are Ala10, Ala12, Gly55, Ala61, Trp102, Gly169, Trp174, Leu197, Asn224, Trp249, Ala257, Gly293 and Gly333. The phylogenetic trees were conducted within the Mega-X software using the neigh- bour-joining method and are presented in Figs. 5,  6. We found that hydrGB10I is the most similar to glycoside hydrolase family (the bootstrap value = 100) and dis- tinct from other fungal enzymes with the biodegradation activity, whereas chitGB10I shows similarity to other chi- tinases and is the closest to LC-cutinase that exhibited the activity. Discussion Extremophilic microorganisms are gaining more and more importance in biotechnological research and indus- trial applications. Psychrophiles have developed unique adaptive strategies to maintain their metabolic activity in the cold conditions which resulted in a stable membranes and a cell walls, unique compounds (e.g., exo-polysac- charides), proteins (the cold-shock protein, antifreeze/ ice-nucleating protein) and genes (Arora and Panosyan 2019; Bhatia et al. 2021). Most importantly, they produce cold-active enzymes that catalyze biochemical reactions at low temperatures making them an attractive resource for biotechnological applications. Cold-active enzymes are primarily an alternative for the application of chemi- cals, lower the required temperature of a reaction and can prevent undesirable chemical reactions (Santiago et  al. 2016). They are used in detergents, the paper and food industry or for bioremediation purposes (Arora and Panosyan 2019). Fig. 5 Neighbour-joining tree of hydrGB10I (Geomyces sp. B10I) and other enzymes with the biodegradation activity. The bootstrap consensus tree inferred from 1000 replicates and the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches https://swissmodel.expasy.org/ https://swissmodel.expasy.org/ Page 8 of 11Urbanek et al. AMB Express (2022) 12:12 Cold-adapted fungus Geomyces sp. B10I has been reported to exhibit biodegradation activity against PBSA, PBS and PCL (Urbanek et  al. 2021). In this study, we searched for enzymes that appear to be responsible for the biodegradation activity of Geomyces sp. B10I. As a result, two enzymes were partially purified, next identi- fied and named hydrGB10I and chitGB10I. It should be emphasized that the complete purification of proteins may sometimes be complicated to perform. Problems usually arise from unknown properties and difficulties in selecting the appropriate resin binding conditions. The literature shows incomplete purification processes as in the case of PCL depolymerase from Alcaligenes faecalis. PCL depolymerase could not be purified to homogeneity due to inactivation during gel filtration. The enzyme did not bind to any other resin when IEC and HIC (hydro- phobic interaction chromatography) was performed. Nonetheless, partial purification resulted in a 7.3-fold purity with a yield of 90.4% (Oda et al. 1997). In our study, partial purification resulted in 14.16-fold purity and SDS- PAGE analysis revealed the presence of two proteins in the active fraction collected during the IEC. The amino acid sequences of both enzymes obtained from MALDI-TOF analysis were identical to hypothetical proteins of Pseudogymnoascus sp. VKM F-4515 (Leush- kin et  al. 2015). Up to date, this is the first report pre- senting the possible enzymatic function of these proteins and showing the cold-adapted fungus Geomyces sp. B10I (Pseudogymnoascus sp.) as a potential source of enzymes involved in the biodegradation of polyesters. Both proteins contain conserved domains of glycoside hydrolases. Glycoside hydrolases (GHs) (EC 3.2.1.x.) are the key enzymes of carbohydrate metabolism. This extremely common group of enzymes hydrolyzes the glycosidic bond between carbohydrates or between the carbohydrate and non-carbohydrate moiety (Henrissat 1991). GHs are produced by a variety of microorganisms, including bacteria and fungi, where these enzymes are involved in cell wall recycling during carbon starvation, thereby generating energy and building blocks that can be used for maintenance and sporulation (van Munster et al. 2015). Furthermore, a conserved domain of the gly- cosyl hydrolase 81 superfamily (Ala15 through Gly713) was found in the hydrGB10I amino acid sequence. GH81 (EC 3.2.1.39) is a family of eukaryotic β-1,3-glucanases with endo-β-1,3-glucanase activity. Mouyna et al. (2016) proved that members of the GH81 and GH16 families play an important role in the morphogenesis of Asper- gillus fumigatus. Endo β-1,3-glucanases are essential for the proper assembly of the conidial cell wall and thus for the efficient release of spores (Mouyna et  al. 2016). In turn, analysis of the deduced amino acid sequence of chitGB10I revealed possession of the GH18 family cata- lytic domain (Asn44 through Asp387). The GH18 family, commonly known as chitinases (EC 3.2.1.14), is involved in carbohydrate metabolism along with chitin and poly- saccharides degradation. Chitinases catalyze the random endohydrolysis of the 1,4-beta-linkages of N-acetylglu- cosamine in chitin and chitodextrins. Moreover, some chitinases can also hydrolyze related polymers. The size of chitinases varies from 20 kDa to about 90 kDa (Bhat- tacharya et  al. 2007), which means that chitGB10I is of medium size (46.5 kDa). Fungal chitinases are not as well classified as bacterial chitinases and are identified by their similarity to a family 18 of bacteria and plants. It is sup- posed, that one of the roles of fungal chitinases is in the hyphal growth, branching and spore germination (Takaya et  al. 1998; Sándor et  al. 1998). For instance, multiple chitinase activity was observed in germinating spores in Mucor rouxii cells (Pedraza-Reyes and Lopez-Romero Fig. 6 Neighbour-joining tree of chitGB10I (Geomyces sp. B10I) and other enzymes with the biodegradation activity. The bootstrap consensus tree inferred from 1000 replicates and the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test is shown next to the branches Page 9 of 11Urbanek et al. AMB Express (2022) 12:12 1991). ChitGB10I, like other GH18 chitinases, con- tains a characteristic DxDxE amino acid sequence motif (171Asp-172Ile-173Asp-174Trp-175Glu), in which glutamate is a catalytic residue essential for activity (Malecki et al. 2020). As we observed the activity of Geomyces sp. B10I towards polyesters during sporulation, it is possible that both enzymes hydrGB10I and chitGB10I are responsible for the degradation abilities of this fungus. Unfortunately, there is still little information available on the biochemical properties of fungal plastic-degrading enzymes or their structural characteristics compared to bacterial polyester-degrading enzymes. The obtained sequences of hydrGB10I and chitGB10I showed dissimi- larities in comparison with other polyester-degrading fungal enzymes reported to date. The enzyme names, accession numbers, and fungal source used for the align- ment are listed below in Figs. 5,  6 and below Additional file 1: Figs. S5 and S6. ChitGB10I was found to be simi- lar to Streptomyces thermoviolaceus chitinase (Chua et  al. 2013). To date, there is no more information on other chitinases showing biodegradation activity. In turn, hydrGB10I was more related to cutinases than to depoly- merases, as is shown by the phylogenetic tree (Fig. 6). Although the Geomyces sp. B10I is a cold-adapted fun- gus, the enzyme mixture tested in turbidimetric assays exhibited the highest biodegradation activity at 40  °C. This result shows that the optimal temperature for enzyme activity may differ from the growth temperature of the microorganism (Santiago et al. 2016). For instance, the optimal activity of pectinase from Geomyces sp. F09- T3-2 was indicated at 30 °C whereas the optimal temper- ature for fungal growth was 15 °C (Poveda et al. 2018). In turn, Mao et al. (2015) cultivated Geomyces pannorum at 20 °C while its α-amylase exhibited an optimal activity at 40 °C (Mao et al. 2015). The research of Engqvist (2018) suggests that the difference in growth temperatures and enzyme optima are caused by extrinsic factors such as increased action by chaperones, higher protein turnover rates, molecular crowding, or other undiscovered mecha- nisms (Engqvist 2018). However, taking into account the purpose of our research, it should be highlighted that the biodegradation activity was also detected at 20 and 30 °C. 50% of the activity at 40 °C was preserved at 20 °C, which is of great importance if the enzymes are to be used at ambient or room temperature. Thus the indicated bio- degradation activity at 20 and 30 °C is a promising result in the search for enzymes adapted to lower temperatures than thermophilic. These enzymes could be an attrac- tive targets for the biodegradation of bioplastic as well as other industrial applications. Cold-adapted enzymes have a few advantages over mesophilic orthologs in that they operate at low temperature, could reduce the energy cost in the reaction and attenuate side-reactions and they could be easily inactivated by heat (Nandanwar et  al. 2020). The information presented in this study is useful in the context of future research on the biodegradability of conventional plastics. The biodegradation activity of hydrGB10I and chitGB10I on fossil and aromatic plastics should be investigated. The production of these enzymes and their large-scale practical application should also be taken into account. Since Geomyces sp. is a fungus with- out GRAS status, it cannot be used directly as an enzyme producer. Moreover, purification of chitGB10I and hydrGB10I is a complicated process when enzymes are produced by the filamentous fungus. Therefore, research should focus on cloning sequences to other microorgan- isms for the independent secretion of enzymes and their easier purification. Abbreviations PCL: Poly(e-caprolactone); PBS: Poly(butylene succinate); PBSA: Poly(butylene succ inate-co-butylene adipate); IEC: Ion exchange chromatography; MALDI-TOF: Matrix assisted laser desorption ionization-time of flight mass spectrometry; PHB: Polyhydroxybutyrate; PES: Polyethersulfone; PLA: Polylactic acid; PDLLA: Poly-DL-lactide; PLLA: Poly-L-lactide; BLAST: Basic local alignment search tool; MM: Minimal medium; PEG: Polyethylene glycol; BP: Biodegrad- able plastic; BSA: Bovine serum albumin; SDS-PAGE: Sodium dodecyl-sulfate polyacrylamide gel electrophoresis; NCBI: National Centre for Biotechnology Information; PSSA: Protein sequence-structure analysis; GMQE: Global model quality estimate; AS: Ammonium sulfate; HIC: Hydrophobic interaction chro- matography; GH: Glycoside hydrolase; GRAS: Generally recognized as safe. Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s13568- 022- 01352-7. Additional file 1: Figure S1. Geomyces sp. B10I and its biodegradation activity. A Microscopic morphology at total magnification 25.2 ×: growing hyphae; B spore release; C biodegradation activity towards 0.1% PBSA in a plate assay; D Biodegradation activity of free-cell supernatant collected at different growth stages: red square represents free-cell supernatant obtained during hyphae growth, whereas black square from spore release stage; numbers represent the number of the flask. Figure S2. Chromato- graphic profiles of purification: A pH 8.0; B pH 9.0; C pH 9.0. The peaks with biodegradable activity are marked as a red square in the chromatograms. The flow rate was maintained at 1 ml/min for each purification step. Figure S3. Peptide fingerprinting MS result for bands signed as PBSA1 (A) and PBSA2 (B). Molecular weight of the analysed proteins was 77248 Da and 46482 Da, respectively. The search within BLAST revealed that both proteins had shown essentially identical properties (100% identity) to proteins characterized as a hypothetical proteins from Pseudogymnoascus sp. VKM F-4515 with accession numbers KFY49210.1 and KFY57494.1, respectively. Figure S4. Amino acid sequences of (A) hydrGB10I and (B) chitGB10I. Figure S5. Multiple sequence alignment of hydrGB10I with other enzymes exhibiting biodegradable activity. Conserved columns in each group are coloured in blue. Alignment conservation, quality, con- sensus and occupancy are displayed below the alignments. EAL84505.1— PHB depolymerase (Aspergillus fumigatus Af293); AAB05922.1—cutinase (Fusarium petroliphilum); BAN42607.1—cutinase-like enzyme (Cryptococ- cus sp. BPD1A); B2NHN2—PHB depolymerase (Talaromyces funiculosus); QED41487.1—serine hydrolase (Pestalotiopsis microspore); PVH79176.1— glycoside hydrolase family 81 protein (Cadophora sp. DSE1049); KAF8866935.1—family 81 glycosyl hydrolase (Acephala macrosclerotio- rum); A0A370T8Q3—PHB depolymerase (Venustampulla echinocandica); https://doi.org/10.1186/s13568-022-01352-7 https://doi.org/10.1186/s13568-022-01352-7 Page 10 of 11Urbanek et al. AMB Express (2022) 12:12 P52956—Cutinase 1 (Aspergillus oryzae RIB40). Figure S6. Multiple sequence alignment of chitGB10I with other enzymes exhibiting biode- gradable activity. Conserved columns in each group are coloured in blue. Alignment conservation, quality, consensus and occupancy are displayed below the alignments. BAA88834.1—Chi25 (Streptomyces thermoviola- ceus); ABV57861.1—chitinase CrCH1 (Clonostachys rosea); AMP42675.1— chitinase (Rhizomucor miehei); X0BTD8—cutinase (Fusarium oxysporum f. sp. raphani 54005); B2NHN2—PHB depolymerase (Talaromyces funiculo- sus); BAM28634.1—cutinase C (Aspergillus oryzae); BAN42607.1—cutinase- like enzyme (Cryptococcus sp. BPD1A), EAL84505.1—PHB depolymerase (Aspergillus fumigatus Af293); G9BY57.1—LC-cutinase. Figure S7. The scheme of purification procedure. Acknowledgements Not applicable. Authors’ contributions Conceptualization, IDLM and AMM; methodology, AKU, IDLM and MA; inves- tigation, AKU; writing—original draft preparation, AKU; writing—review and editing, AMM; visualization, AKU; supervision, IDLM, AMM. All authors have read and approved the final manuscript. Funding This study was performed as part of the Ph.D. research program project enti- tled ‘Isolation and purification of enzymes produced by fungal strains capable of degradation of bioplastic’ (PPN/IWA/2018/1/00050/U/00001) and was financially supported by the Polish National Agency for Academic Exchange. This study was financed by the National Science Centre, Poland, Project UMO-2017/27/B/NZ9/02218. Availability of data and materials The authors promise the availability of data and materials. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details 1 Department of Biotechnology and Food Microbiology, Faculty of Biotechnol- ogy and Food Science, Wroclaw University of Environmental and Life Sciences, Chełmońskiego 37, 51-630 Wrocław, Poland. 2 Department of Biochemistry and Molecular Biology, Faculty of Biology, Universidad Complutense de Madrid, C. de José Antonio Novais, 12, 28040 Madrid, Spain. Received: 19 October 2021 Accepted: 22 January 2022 References Abdel-Motaal FF, Mahmoud WN, El-Zayad SA (2020) Eco-friendly bio- degradation of poly (ε-caprolactone) (PCL) by the fungus Alternaria alternata-ST01. Plant Cell Biotechnol Mol Biol 20:1447–1455 Abe M, Kobayashi K, Honma N, Nakasaki K (2010) Microbial degradation of poly(butylene succinate) by Fusarium solani in soil environments. Polym Degrad Stab 95:138–143. https:// doi. org/ 10. 1016/j. polym degra dstab. 2009. 11. 042 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local align- ment search tool. J Mol Biol 215:403–410 Arora NK, Panosyan H (2019) Extremophiles: applications and roles in environ- mental sustainability. Environ Sustain 2:217–218. https:// doi. org/ 10. 1007/ s42398- 019- 00082-0 Bhatia RK, Ullah S, Hoque MZ, Ahmad I, Yang YH, Bhatt AK, Bhatia SK (2021) Psychrophiles: a source of cold-adapted enzymes for energy efficient biotechnological industrial processes. J Environ Chem Eng 9(1):104607. https:// doi. org/ 10. 1016/j. jece. 2020. 104607 Bhattacharya D, Nagpure A, Gupta RK (2007) Bacterial chitinases: properties and potential. Crit Rev Biotechnol 27:21–28. https:// doi. org/ 10. 1080/ 07388 55060 11682 23 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 Brucato CL, Wong SS (1991) Extracellular poly(3-hydroxybutyrate) depolymer- ase from Penicillium funiculosum: general characteristics and active site studies. Arch Biochem Biophys 290:497–502. https:// doi. org/ 10. 1016/ 0003- 9861(91) 90572-z Chua TK, Tseng M, Yang MK (2013) Degradation of poly(ε-caprolactone) by thermophilic Streptomyces thermoviolaceus subsp. thermoviolaceus 76T–2. AMB Express. https:// doi. org/ 10. 1186/ 2191- 0855-3-8 Emadian SM, Onay TT, Demirel B (2017) Biodegradation of bioplastics in natu- ral environments. Waste Manag 59:526–536. https:// doi. org/ 10. 1016/j. wasman. 2016. 10. 006 Engqvist MKM (2018) Correlating enzyme annotations with a large set of microbial growth temperatures reveals metabolic adaptations to growth at diverse temperatures. BMC Microbiol 18:177. https:// doi. org/ 10. 1186/ s12866- 018- 1320-7 García-Hidalgo J, Hormigo D, Prieto MA, Arroyo M, de la Mata I (2012) Extracel- lular production of Streptomyces exfoliatus poly(3-hydroxybutyrate) depolymerase in Rhodococcus sp. T104: determination of optimal biocatalyst conditions. Appl Microbiol Biotechnol 93:1975–1988. https:// doi. org/ 10. 1007/ s00253- 011- 3527-5 Gowda USV, Shivakumar S (2015) Poly(-β-hydroxybutyrate) (PHB) depolymer- ase PHAZ Pen from Penicillium expansum: purification, characterization and kinetic studies. 3 Biotech 5(6):901–909. https:// doi. org/ 10. 1007/ s13205- 015- 0287-4 Hayes MA (2012) The geomyces fungi: ecology and distribution. Bioscience 62(9):819–823. https:// doi. org/ 10. 1525/ bio. 2012. 62.9.7 Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280:309–316. https:// doi. org/ 10. 1042/ bj280 0309 Johnson AM, Barlow DE, Kelly AL, Varaljay VA, Crookes-Goodson WJ, Biffinger JC (2021) Current progress towards understanding the biodegradation of synthetic condensation polymers with active hydrolases. Polym Int 70(7):977–983. https:// doi. org/ 10. 1002/ pi. 6131 Jung HW, Yang MK, Su RC (2018) Purification, characterization, and gene clon- ing of an Aspergillus fumigatus polyhydroxybutyrate depolymerase used for degradation of polyhydroxybutyrate, polyethylene succinate, and polybutylene succinate. Polym Degrad Stab 154:186–194. https:// doi. org/ 10. 1016/j. polym degra dstab. 2018. 06. 002 Källberg M, Wang H, Wang S, Peng J, Wang Z, Lu H, Xu J (2012) Template-based protein structure modeling using the RaptorX web server. Nat Protoc 7:1511–1522. https:// doi. org/ 10. 1038/ nprot. 2012. 085 Kim DY, Rhee YH (2003) Biodegradation of microbial and synthetic polyesters by fungi. Appl Microbiol Biotechnol 61:300–308. https:// doi. org/ 10. 1007/ s00253- 002- 1205-3 Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549. https:// doi. org/ 10. 1093/ molbev/ msy096 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. https:// doi. org/ 10. 1038/ 22768 0a0 Lee TK, Huang KC (2013) The role of hydrolases in bacterial cell-wall growth. Curr Opin Microbiol 16:760–766. https:// doi. org/ 10. 1016/j. mib. 2013. 08. 005 Leushkin EV, Logacheva MD, Penin AA, Sutormin RA, Gerasimov ES, Kochkina GA, Ivanushkina NE, Vasilenko OV, Kondrashov AS, Ozerskaya SM (2015) Comparative genome analysis of Pseudogymnoascus spp. reveals primar- ily clonal evolution with small genome fragments exchanged between lineages. BMC Genom. https:// doi. org/ 10. 1186/ s12864- 015- 1570-9 https://doi.org/10.1016/j.polymdegradstab.2009.11.042 https://doi.org/10.1016/j.polymdegradstab.2009.11.042 https://doi.org/10.1007/s42398-019-00082-0 https://doi.org/10.1007/s42398-019-00082-0 https://doi.org/10.1016/j.jece.2020.104607 https://doi.org/10.1080/07388550601168223 https://doi.org/10.1080/07388550601168223 https://doi.org/10.1016/0003-9861(91)90572-z https://doi.org/10.1016/0003-9861(91)90572-z https://doi.org/10.1186/2191-0855-3-8 https://doi.org/10.1016/j.wasman.2016.10.006 https://doi.org/10.1016/j.wasman.2016.10.006 https://doi.org/10.1186/s12866-018-1320-7 https://doi.org/10.1186/s12866-018-1320-7 https://doi.org/10.1007/s00253-011-3527-5 https://doi.org/10.1007/s00253-011-3527-5 https://doi.org/10.1007/s13205-015-0287-4 https://doi.org/10.1007/s13205-015-0287-4 https://doi.org/10.1525/bio.2012.62.9.7 https://doi.org/10.1042/bj2800309 https://doi.org/10.1042/bj2800309 https://doi.org/10.1002/pi.6131 https://doi.org/10.1016/j.polymdegradstab.2018.06.002 https://doi.org/10.1016/j.polymdegradstab.2018.06.002 https://doi.org/10.1038/nprot.2012.085 https://doi.org/10.1007/s00253-002-1205-3 https://doi.org/10.1007/s00253-002-1205-3 https://doi.org/10.1093/molbev/msy096 https://doi.org/10.1038/227680a0 https://doi.org/10.1038/227680a0 https://doi.org/10.1016/j.mib.2013.08.005 https://doi.org/10.1016/j.mib.2013.08.005 https://doi.org/10.1186/s12864-015-1570-9 Page 11 of 11Urbanek et al. AMB Express (2022) 12:12 Liu Z, Gosser Y, Baker PJ, Ravee Y, Lu Z, Alemu G, Li H, Butterfoss GL, Kong XP, Gross R, Montclare JK (2009) Structural and functional studies of Aspergillus oryzae cutinase: enhanced thermostability and hydrolytic activity of synthetic ester and polyester degradation. J Am Chem Soc 131(43):15711–15716. https:// doi. org/ 10. 1021/ ja904 6697 Liu M, Zhang T, Long L, Zhang R, Ding S (2019) Efficient enzymatic degrada- tion of poly (3-caprolactone) by an engineered bifunctional lipase-cuti- nase. Polym Degrad Stab 160:120–125 Madeira F, Park YM, Lee J, Buso N, Gur T, Madhusoodanan N, Basutkar P, Tivey ARN, Potter SC, Finn RD, Lopez R (2019) The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res 47(W1):W636– W641. https:// doi. org/ 10. 1093/ nar/ gkz268 Maeda H, Yamagata Y, Abe K, Hasegawa F, Machida M, Ishioka R, Gomi K, Naka- jima T (2005) Purification and characterization of a biodegradable plastic- degrading enzyme from Aspergillus oryzae. Appl Microbiol Biotechnol 67:778–788. https:// doi. org/ 10. 1007/ s00253- 004- 1853-6 Malecki PH, Bejger M, Rypniewski W, Vorgias CE (2020) The crystal structure of a Streptomyces thermoviolaceus thermophilic chitinase known for its refolding efficiency. Int J Mol Sci 21(8):2892. https:// doi. org/ 10. 3390/ ijms2 10828 92 Mao H, Liu H, Gao Z, Su T, Wang Z (2005) Biodegradation of poly(butylene succinate) by Fusarium sp. FS1301 and purification and characterization of poly(butylene succinate) depolymerase. Polym Degrad Stab 114:1–7. https:// doi. org/ 10. 1016/j. polym degra dstab. 2015. 01. 025 Mao Y, Yin Y, Zhang L, Alias SA, Gao B, Wei D (2015) Development of a novel Aspergillus uracil deficient expression system and its application in expressing a cold-adapted α-amylase gene from Antarctic fungi Geomy- ces pannorum. Process Biochem 50:1581–1590. https:// doi. org/ 10. 1016/j. procb io. 2015. 06. 016 Mouyna I, Aimanianda V, Hartl L, Prevost MC, Sismeiro O, Dillies MA, Jagla B, Legendre R, Coppee JY, Latgé JP (2016) GH16 and GH81 family β-(1,3)- glucanases in Aspergillus fumigatus are essential for conidial cell wall morphogenesis. Cell Microbiol 18:1285–1293. https:// doi. org/ 10. 1111/ cmi. 12630 Nandanwar SK, Borkar SB, Lee JH, Kim HJ (2020) Taking advantage of prom- iscuity of cold-active enzymes. Appl Sci 10(22):8128. https:// doi. org/ 10. 3390/ app10 228128 Nawaz A, Hasan F, Shah AA (2015) Degradation of poly(ε-caprolactone) (PCL) by a newly isolated Brevundimonas sp. strain MRL-AN1 from soil. FEMS Microbiol Lett 362:1–7. https:// doi. org/ 10. 1093/ femsle/ fnu004 Oda Y, Oida N, Urakami T, Tonomura K (1997) Polycaprolactone depolymerase produced by the bacterium Alcaligenes faecalis. FEMS Microbiol Lett 152:339–343. https:// doi. org/ 10. 1111/j. 1574- 6968. 1997. tb104 49.x Pedraza-Reyes M, Lopez-Romero E (1991) Detection of nine chitinase species in germinating cells of Mucor rouxii. Curr Microbiol 22:43–46 Poveda G, Gil-Durán C, Vaca I, Levicán G, Chávez R (2018) Cold-active pecti- nolytic activity produced by filamentous fungi associated with Antarctic marine sponges. Biol Res. https:// doi. org/ 10. 1186/ s40659- 018- 0177-4 Sameshima-Yamashita Y, Koitabashi M, Tsuchiya W, Suzuki K, Watanabe T, Shi- nozaki Y, Yamamoto-Tamura K, Yamazaki T, Kitamoto H (2016) Enhance- ment of biodegradable plastic-degrading enzyme production from Paraphoma-like fungus, Strain B47–9. J Oleo Sci 65:257–262. https:// doi. org/ 10. 5650/ jos. ess15 207 Sándor E, Pusztahelyi T, Karaffa L, Karányi Z, Pócsi I, Biró S, Szentirmai A (1998) Allosamidin inhibits the fragmentation of Acremonium chrysogenum but does not influence the cephalosporin-C production of the fungus. FEMS Microbiol Lett 164:231–236. https:// doi. org/ 10. 1111/j. 1574- 6968. 1998. tb130 91.x Santiago M, Ramírez-Sarmiento CA, Zamora RA, Parra LP (2016) Discovery, molecular mechanisms, and industrial applications of cold-active enzymes. Front Microbiol 7:1408. https:// doi. org/ 10. 3389/ fmicb. 2016. 01408 Schrödinger L (2010) The PyMOL molecular graphics system, version 2.1. DeLano Scientific LLC, San Carlos Takaya N, Yamazaki D, Horiuchi H, Ohta A, Takagi M (1998) Cloning and characterization of a chitinase-encoding gene (chiA) from Aspergillus nidulans, disruption of which decreases germination frequency and hyphal growth. Biosci Biotechnol Biochem 62:60–65. https:// doi. org/ 10. 1271/ bbb. 62. 60 Urbanek AK, Rymowicz W, Strzelecki MC, Kociuba W, Franczak Ł, Mirończuk AM (2017) Isolation and characterization of Arctic microorganisms decomposing bioplastics. AMB Express 7:148. https:// doi. org/ 10. 1186/ s13568- 017- 0448-4 Urbanek AK, Mirończuk AM, García-Martín A, Saborido A, de la Mata I, Arroyo M (2020) Biochemical properties and biotechnological applications of microbial enzymes involved in the degradation of polyester-type plastics. BBA Proteins Proteom 2:140315. https:// doi. org/ 10. 1016/j. bbapap. 2019. 140315 Urbanek AK, Strzelecki MC, Mirończuk AM (2021) The potential of cold- adapted microorganisms for biodegradation of bioplastics. Waste Manag 119:72–81. https:// doi. org/ 10. 1016/j. wasman. 2020. 09. 031 van Munster JM, Nitsche BM, Akeroyd M, Dijkhuizen L, van der Maarel MJ, Ram AF (2015) Systems approaches to predict the functions of glycoside hydrolases during the life cycle of Aspergillus niger using developmental mutants ∆brlA and ∆flbA. PLoS ONE 10(1):e0116269. https:// doi. org/ 10. 1371/ journ al. pone. 01162 69 Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJ (2009) Jalview version 2–a multiple sequence alignment editor and analysis workbench. Bioinformatics 25:1189–1191. https:// doi. org/ 10. 1093/ bioin forma tics/ btp033 Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L, Lepore R, Schwede T (2018) SWISS- MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 46:W296–W303. https:// doi. org/ 10. 1093/ nar/ gky427 Wojtusik M, Yepes CM, Villar JC, Cordes A, Arroyo M, Garcia-Ochoa F, Ladero M (2018) Kinetic modeling of cellobiose by a beta-glucosidase from Aspergillus fumigatus. Chem Eng Resh Des 136:502–512. https:// doi. org/ 10. 1016/j. cherd. 2018. 06. 020 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. https://doi.org/10.1021/ja9046697 https://doi.org/10.1093/nar/gkz268 https://doi.org/10.1007/s00253-004-1853-6 https://doi.org/10.3390/ijms21082892 https://doi.org/10.3390/ijms21082892 https://doi.org/10.1016/j.polymdegradstab.2015.01.025 https://doi.org/10.1016/j.procbio.2015.06.016 https://doi.org/10.1016/j.procbio.2015.06.016 https://doi.org/10.1111/cmi.12630 https://doi.org/10.1111/cmi.12630 https://doi.org/10.3390/app10228128 https://doi.org/10.3390/app10228128 https://doi.org/10.1093/femsle/fnu004 https://doi.org/10.1111/j.1574-6968.1997.tb10449.x https://doi.org/10.1186/s40659-018-0177-4 https://doi.org/10.5650/jos.ess15207 https://doi.org/10.5650/jos.ess15207 https://doi.org/10.1111/j.1574-6968.1998.tb13091.x https://doi.org/10.1111/j.1574-6968.1998.tb13091.x https://doi.org/10.3389/fmicb.2016.01408 https://doi.org/10.3389/fmicb.2016.01408 https://doi.org/10.1271/bbb.62.60 https://doi.org/10.1271/bbb.62.60 https://doi.org/10.1186/s13568-017-0448-4 https://doi.org/10.1186/s13568-017-0448-4 https://doi.org/10.1016/j.bbapap.2019.140315 https://doi.org/10.1016/j.bbapap.2019.140315 https://doi.org/10.1016/j.wasman.2020.09.031 https://doi.org/10.1371/journal.pone.0116269 https://doi.org/10.1371/journal.pone.0116269 https://doi.org/10.1093/bioinformatics/btp033 https://doi.org/10.1093/bioinformatics/btp033 https://doi.org/10.1093/nar/gky427 https://doi.org/10.1016/j.cherd.2018.06.020 https://doi.org/10.1016/j.cherd.2018.06.020 Identification of novel extracellular putative chitinase and hydrolase from Geomyces sp. B10I with the biodegradation activity towards polyesters Abstract Introduction Materials and methods Fungal strain and culture media Materials Partial protein purification steps Biodegradation activity Matrix-assisted laser desorption-time-of-flight (MALDI-TOF) analysis Phylogenetic analyses Amino acid sequences Results Inoculum and enzyme production Partial protein purification steps Matrix-assisted laser desorption-time-of-flight (MALDI-TOF) analysis Discussion Acknowledgements References