Production and characterization of β-galactosidase from the fungus Thielaviopsis ethacetica Went.

: β-galactosidase (EC 3.2.1.23) is an industrially important enzyme. It can catalyse two different types of reactions, namely lactose hydrolysis and transgalactosylation. β-galactosidase enzyme production by Thielaviopsis ethacetica , was investigated and the enzyme was partially purified and characterized in the present study. Qualitative assay for the pre β-galactosidase activity indicated the formation of water insoluble blue-green colour, dichloro-dibromo-indigo zone towards the edges of the fungal colony after a 72-hour incubation period. Quantitative assay recorded a maximum enzyme activity of 0.599 U/mL at 72 hours. The enzyme was partially purified from the crude enzyme using ammonium sulphate precipitation and dialysis. Fraction 40-60 % (fraction 2) had the highest specific enzymes activity of 1.65 U/mg with 8.68 purification fold and 28.3 % recovery. SDS-PAGE analysis of fraction 2 resulted three separate protein bands above 40 kDa. The protein band corresponding to 50 kDa molecular weight was confirmed as β-galactosidase. The optimum temperature and pH for the enzyme was at 60 ̊ C and 7.0, respectively. The present study reveals the fungal strain Thielaviopsis ethacetica with β-galactosidase activity. To our knowledge this is the first report about β-galactosidase activity of the strain T. ethacetica. This investigation suggests the potential use of this species in industrial applications.

β-galactosidases are widely distributed in different biological systems, e.g. microorganisms, plants such as almonds, peaches, apricots, and animal tissues such as brain and placenta (Kayukawa et al., 2020). β-galactosidases are members of the family of hydrolases that belongs to the group of saccharide-converting enzymes. Properties, activity and the structure of the enzyme such as position of the active site, molecular weight of the enzyme, length of the amino acid chain, thermal and pH stability vary with the sources from which the enzyme is obtained (Zhou & Chen, 2001). Therefore, source of the enzyme can be selected according to one's choice, depending on reaction conditions. Among all the natural sources, production of the enzyme is focused on microorganisms as they have the ability to grow on low-cost substrates. Moreover, compared to plants and  Journal of the National Science Foundation of Sri Lanka 49 (4) animal sources, higher yields can be obtained while minimizing the cost of production (Santos et al., 1998). Microorganisms including yeast, fungi, bacteria and actinomycetes have been exploited for β-galactosidase production (Zadow, 1992;Fernandez, 1999;Bhanwar & Ganguli, 2014;Panesar et al., 2016;Xu et al., 2019). Among these microorganisms special attention is still paid to fungal sources as their enzymes are thermostable.
Since the enzyme is extracellular, additional cost for the extraction is very low. Furthermore, fungi give higher yields, and their enzymes show broad stability profiles. Therefore, the commercially available β-galactosidases have been mainly obtained from fungi: yeast and moulds. The most important sources are Aspergillus and Kluyveromyces species including A. oryzae, K. lactis, K. marxianus and K. fragilis (Zhou &d Chen, 2001;Roy & Gupta, 2003;Johnson & Echavarri-Erasun, 2011;Maccaferri et al., 2012;Martarello et al., 2019). As the demand is high, finding new fungal source for the production of the enzyme is of current interest.
The objective of the present study was the optimisation of production, partial purification and characterisation of β-galactosidase secreted by Thielaviopsis ethacetica.

The microorganism and culture conditions
Fungal strain Thielaviopsis ethacetica has been previously isolated from the topsoil and identified to exhibit alpha-amylase activity (Dissanayaka et al., 2019). The internal transcribed spacer (ITS) sequences have been deposited in the GenBank/NCBI (Accession no; MG062870.1). The fungus was stored in potato dextrose agar (PDA) slants for long term storage and subcultures were prepared once a month throughout the study period. After reviving on PDA supplemented with tetracycline, spores were picked up using a sterile moistened loop and streaked on to 2 % sterile agar plates and incubated at 26 °C for 18 h. Germinated spores were transferred aseptically onto fresh PDA plates to get a single spore culture.

Determining the time for optimal enzyme production
Pre-inoculum was prepared in 50 mL of fermentation medium comprising 2% soluble starch, 0.5% yeast extract, 0.03% MgSO 4 , 0.03% CaCl 2 and 0.7% KH 2 PO 4 at pH 4. Sterile medium was inoculated with 3 agar plugs of profusely growing fungi (6 mm diameter) and incubated at room temperature with shaking at 120 rpm. After 96 hours of incubation, 5 mL of homogenized pre-inoculum (5 ×10 4 -6 ×10 4 spore/mL) was carefully withdrawn and added to 50 mL of the fermentation medium.
Fermentation medium was incubated at 32 o C for 72 h in a rotary shaker at 120 rpm (Dissanayaka et al., 2019). β-galactosidase activity and total protein concentration was measured at 12-hour intervals by removing 150 µL from the fermentation medium. All assays were carried out in triplicate.
Appropriately diluted 0.15 mL crude enzyme was mixed with 0.6 mL of substrate solution. 1 mM o-NPG was prepared in 0.1 M sodium phosphate buffer at pH 7.0. The reaction mixture was incubated at 40 o C for 30 min. The reaction was terminated by adding 1 mL of 1 m of Na 2 CO 3. The concentration of o-nitrophenol (oNP) released from o-NPG was determined by measuring the absorbance at 420 nm by UV spectrophotometer using the standard calibration curve (Soydan, 2006;Konsoula & Liakopoulou-Kyriakides, 2007). For enzyme blank, 0.15 mL of sample was mixed with 0.6 mL of buffer and 1 mL of 1 M Na 2 CO 3 . For substrate blank, 0.15 mL of buffer was mixed with 0.6 mL of substrate solution and 1 mL of 1 M Na 2 CO 3 . Mean absorbance value of the triplicates was taken to calculate the unit enzyme activity. One unit of β-galactosidase activity was defined as the amount of enzyme required to release 1 µmol o-nitrophenol per minute under assay conditions (Soydan, 2006, Konsoula & Liakopoulou-Kyriakides, 2007

Total protein content assay
Total protein concentration was assayed in triplicate using Qubit 3 Invitrogen™ Qubit™ 3.0 Fluorometer according to the given instructions in Qubit assay kit (Invitrogen™).

Partial purification of the β-galactosidase enzyme
Five hundred millilitres from the submerged fermentation medium containing 2% soluble starch were inoculated with the fungal strain Thielaviopsis ethacetica and incubated at 32 o C for 72 h. After the incubation, culture medium was centrifuged at 6000 rpm for 15 min at 40 o C and the supernatant was subjected to gradient ammonium sulphate fractionation. The supernatant was brought into 40% (Fraction 1) saturation followed by 60% (Fraction 2), 80% (Fraction 3), and 100% (Fraction 4). Ammonium sulphate was gradually added into the medium while stirring continuously using a magnetic stirrer (Wingfield, 2001). Saturated protein samples were then centrifuged at 12000 rpm for 15 min, at 40 o C and the protein pellets were recovered. Before the dialysis, each pellet was dissolved in a minimum volume of 0.1 M phosphate buffer at pH 7 separately. Dialysis was accompanied by continuous stirring over 24 hours at 40 o C against the same buffer and the buffer was changed intermittently. Finally, all the fractions were assayed for enzyme activity and total protein concentration.

Investigation the effect of temperature on β-galactosidase activity
To find the effect of temperature and pH on β-galactosidase activity, Fraction 2 (40-60%) with the highest enzyme activity was collected and used. The effect of temperature was evaluated at 20, 37, 40, 60 and 80 o C by incubating the reaction mixture for 30 min. β-galactosidase enzyme activity was determined as previously described.

Investigation of the effect of pH on β-galactosidase activity
The effect of pH on enzyme activity was evaluated by adjusting the pH of the substrate at 4, 5, 6, 7, and 8. The reaction mixture was incubated at 60 o C for 30 min under specific pH value and enzyme activity was determined as previously described.

Determination of the molecular weight of Thielaviopsis ethacetica β-galactosidase
Partially purified protein fraction 2 with the highest enzyme activity was freeze-dried (CHRIST Alpha 1-2 LDplus, Germany) and 1 mg of lyophilized protein sample was dissolved in an appropriate volume of sample buffer (Laemmli, 1970). To separate proteins in the sample, 10 % sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) stained with Coomassie Brilliant Blue R-250 was used. To determine the molecular weight of the protein, a broad range molecular weight protein marker ranging from 10-100 kDa was used.

Protein identification using mass spectrometry peptide sequencing (MALDI/MS) method
Molecular weight of many fungal β-galactosidases lies around 40-75 kDa. Therefore, three separated protein bands representing more than 40 kDa were used for MALDI/MS identification. Immediate gel surface around these three bands were carefully separated and stored in an appropriate amount of sterilized distilled water. Three protein samples were sent to ALPHALYSE, Denmark for protein identification using Bruker Autoflex Speed MALDI TOF/TOF instrument. The resulted spectra were searched against available protein databases from UniProtKB using the Mascot software.

Qualitative assay for the pre β-galactosidase activity
Although X-Gal is a commonly associated colourimetric method with lac operon in E. coli, the applicability of X-Gal for the detection of fungal β-galactosidase is still under assessment. In the present study, it has been attempted to determine the presence of an active fungal β-galactosidase in culture plates containing X-Gal. IPTG was not added to the culture plates as an inducer. After a 72-hour incubation period, a blue-green colour zone formation was observed towards the edges of the fungal colony. A similar finding has been reported by Fischer et al. (1995a), Soydan (2006), and Kishore and Kayastha (2012) with fungal β-galactosidase enzyme. This method will be a great advantage during the early screening procedures when the number of testing samples are high.

β-galactosidase enzyme assay
Thielaviopsis ethacetica containing fermentation medium gave a prominent peak for β-galactosidase enzyme activity during the 3 rd day of the incubation.
During the first 24 hours protein concentration gradually increased and then peaked at 48 hours ( gradually increased reaching its maximum at 72 hours giving 0.599 U/mL maxima with a simultaneous drop in protein content to 67 μg/mL (Figure 1). After 72 hours, enzyme activity remained static where the protein concentration started to increase. Static enzyme activity may probably be due to the denaturation of protein resulted from unfavourable culture conditions during the latter part of the incubation period. Moreover, due to the depletion of initial substrate concentration, newly synthesised mycelia may be secreting other proteins extracellularly to degrade substrates other than starch, resulting in an increased protein concentration after 72 hours.
During the hydrolysing process it can form galactose and o-nitrophenol (oNP) with a yellow colour. oNP is capable of absorbing light at 420 nm, therefore the colour intensity can be used to detect β-galactosidase activity of complex biological systems (Miller, 1972).
The present study used starch as the substrate, instead of well-known β-galactosidase substrates containing b-Dgalactopyranoside moiety or lactose. The results suggest that one of the intermediate or an end product such as maltose or malto dextrin produced during the starch hydrolysis may act as the substrate for β-galactosidase enzyme production. Konsoula and Liakopoulou-Kyriakides (2007) reported that there can be about 1.5 fold increament of β-galactosidase enzyme activity when using soluble starch as the fermentation medium instead of lactose under the co-production culture conditions of alpha-amylase enzyme. Further, it has been reported that the enzyme production is induced in the presence of yeast extract (Konsoula & Liakopoulou-Kyriakides, 2007). Nitrogen dependancy and carbon independancy of β-galactosidase have opened many arguments among the scientific community and still there is no strong conclusion regading its substrate specificity (Lind et al., 1989;Fischer et al, 1995;Konsoula & Liakopouloukyriakides, 2007). Optimum β-galactosidase enzyme activity was observed at 72 hours incubation period. Therefore, the extraction of β-galactosidase from T. ethacetica was fixed to 72 hours of incubation period. Late secretion of β-galactosidase enzyme was observed in many other organisms; for example, Bacillus sp., Rhizomucor sp. and Aspergillus sp. (Shaikh et al., 1997;Akcan, 2011;Martarello et al., 2019). Quantitative determination of the β-galactosidase activity is commonly accomplished by using colourimetric method.
β-galactosidase is capable of catalysing substrates with b-D-galactopyranoside residues like onitrophenol-galactopyranoside (o-NPG). During the hydrolysing process it can form galactose and onitrophenol (oNP) with a yellow colour. oNP is capable of absorbing light at 420 nm, therefore the colour intensity can be used to detect β-galactosidase activity of complex biological systems (Miller, 1972).
The present study used starch as the substrate, instead of well-known β-galactosidase substrates containing b-D-galactopyranoside moiety or lactose. The results suggest that one of the intermediate or an end product such as maltose or malto dextrin produced during the starch hydrolysis may act as the substrate for β-galactosidase enzyme production. Konsoula and Liakopoulou-Kyriakides (2007) reported that there can be about 1.5 fold increament of β-galactosidase enzyme activity when using soluble starch as the fermentation medium instead of lactose under the co-production culture conditions of alphaamylase enzyme. Further, it has been reported that the enzyme production is induced in the presence of yeast extract (Konsoula & Liakopoulou-Kyriakides, 2007). Nitrogen dependancy and carbon independancy of β-galactosidase have opened many arguments among the scientific community and still there is no strong conclusion regading its substrate specificity (Lind et al., 1989;Fischer et al, 1995;Konsoula & Liakopou-loukyriakides, 2007).

Effect of pH on β-galactosidase activity
Thielaviopsis ethacetica exhibited two prominent enzyme activity peaks for β-galactosidases at distinct pH values. The highest activity peak with 0.083 U/mL was observed at pH 7 and slightly lower second peak of 0.054 U/mL at pH 5 ( Figure 3).
These results show similarities to the observations made by Fisher et al. (1995) for Thermomyes sp. In the literature it is reported that most fungal β-galactosidase showed high activity in two pH ranges from 3-5 and 6-7. Borglum and Sternberg (1972), Cardoso et al. (2017), Gekas and Lopez-Leiva (1985), Niu et al. (2017), andO'Connell andWalsh (2008) reported that most of the Aspergillus and Mucor species exhibited enhanced enzyme activity under low pH values. On the other hand, Đfrij and Ögel (2002), Fischer et al. (1995), Martarello et al. (2019) and Matioli et al. (2001) reported that the highest enzyme activity was near neutral pH with Aspergillus, Thermomyces and Kluyveromyces species. However, due to having two enzyme activity peaks in the acidic and neutral pH ranges, the present strain can give an additional advantage for use in industry. Considering the acid stability nature of the enzyme, it can be useful in the pharmaceutical industry, while its activity near neutral pH values can be useful in the dairy industry.

Determination of the molecular weight of T. ethacetica β-galactosidase
Partially purified lyophilised fraction 2 was run with a marker protein in SDS-PAGE. The resulted gel image is given in Figure 4. Three proteins bands designated by arrows were used for MALDI-MS/MS identification. MALDI-MS/MS analysis involved 15 partially sequenced peptides and identity searching against UniProtKB using Mascot software. The protein band corresponded to 50 kDa molecular weight was matched to E. coli β-galactosidase with 729 Mascot score value (> 95% confidence level). Identified peptides corresponding to E. coli β-galactosidase are shown in Figure 5 with 25% sequence coverage. MALDI/MS peptide mapping and sequencing permit the identification of peptide sequences in a mixture especially when it is difficult to obtain one single pure protein. During the SDS-PAGE, Coomassie Brilliant Blue R-250 was used for staining to minimize the interference during mass spectrometry. According to available data on the molecular weight summary report on β-galactosidase, its molecular weight can range from 12 kDa to 270 kDa depending on the species from which it has been isolated. β-galactosidase isolated from Thermomyces and Thermotoganaphthophila species had molecular weights between 75-80 kDa (Fischer et al., 1995) and 70 kDa (Kong et al., 2014), respectively, whereas the enzyme 1975;Park et al., 1979;Gonzalez & Monsan, 1991;Saad, 2004;Todorova-Balvay et al., 2006).
Considering the results of the protein sequencing analysis of the present study, it is indicated that majority of the starch hydrolase at 72 hours represent β-galactosidase enzyme.

CONCLUSION
The present study identified a fungal strain Thielaviopsis ethacetica with β-galactosidase activity and to the best of our knowledge this is the first report on β-galactosidase activity by T. ethacetica. Enzyme activity at high temperature, pH preference and substrate independency make the β-galactosidase produced by this strain suitable for industrial applications. Further, we emphasize the co-production of Aspergillus species reported to have a molecular weight range of 27-126 kDa (Tanaka et al., 1975;Park et al., 1979;Gonzalez & Monsan, 1991;Saad, 2004;Todorova-Balvay et al., 2006). Considering the results of the protein sequencing analysis of the present study, it is indicated that majority of the starch hydrolase at 72 hours represent β-galactosidase enzyme.

CONCLUSION
The present study identified a fungal strain Thielaviopsis ethacetica with β-galactosidase activity and to the best of our knowledge this is the first report on β-galactosidase activity by T. ethacetica. Enzyme activity at high temperature, pH preference and substrate independency make the β-galactosidase produced by this strain suitable for industrial applications. Further, we emphasize the coproduction ability of alpha amylase and β-galactosidase under starch fermentation medium, which can be readily substituted with low-cost substrates such as rice bran, banana peel and cassava starch (Dissanayaka et al., 2019). During the initial stages of growth, T. ethacetica secretes extracellular alpha-amylase to the medium and involves starch hydrolysing process. Subsequently, it begins and continues to secrete β-galactosidase up to its death phase. Fungal strains having such beneficial enzyme production characteristics would have a significant impact on industrial-scale applications. Therefore, new opportunities exist for further research on enzyme production abilities of T. ethacetica and optimization of such capabilities to fulfil the industrial demands on starch processing.

Conflict of interest statement
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.