Empirical analysis of amylolytic and proteolytic activities of microbial isolates recovered from deteriorating painted wall surfaces in Lagos Nigeria

The biodeterioration of painted walls have been associated with several biological mechanisms such as organic acid production and enzymatic activity of microorganisms amongst other factors. Therefore, this study aims to reveal the involvement of amylases and proteases from indigenous microbes on biodeteriorating painted walls. Microbial strains isolated from biodeteriorating painted walls of selected buildings in Lag os, Nigeria and previously characterized as belonging to the genera Pseudomonas, Candida, Fusarium, Aspergillus, Cerrena and Meyerozyma were used in this study . Amylolytic and proteolytic activities at varying conditions of temperature, pH, incubation time and substrate concentrations were tested. To bridge the knowledge gaps regarding statistical quantification of enzymatic mechanisms in biodeterioration, the Wilcoxon signed rank sum test was used to test the hypothesis that amylolytic/proteolytic activities are equal at all conditions tested. The conditions for optimal activity were observed to be 24h, 37 o C, pH 2 and 0.01% substrate concentration and 48h, 25 o C, pH 2, and 1% substrate concentration for amylase and protease respectively. Wilcoxon signed rank test revealed that amylolytic and proteolytic activities do not impact aesthetics on painted walls equally at all environmental conditions considered.


INTRODUCTION
Biodegradation of surf ace coatings and painted walls is occasioned most importantly because microorganisms utilize any class of material as substrate and have very simple requirements for prolif eration (Allsopp et al., 2003). Some microorganisms are also particularly very amenable to horizontal gene transf er which ehances their adaptation to dif f erent environments. This results in production of a wide variety of enzymes, such as amylases, and proteases that f acilitates their gross biodeterioration activity (Rojas et al., 2008). Microorganisms derive carbon sources as nutrients f rom the dif f erent organic constituents of paints which help to stimulate their growth in liquid and applied paints. The resulting ef f ect is a loss of aesthetics of the paint surf ace f acilitated by discoloration, reduced adhesion and detachment of the paint layer (Da Silva, 2003). Microbial enzymes theref ore, remain an important cause of discoloration (Gaylarde and Morton, 2001). Amylases are an important group of enzymes involved in the hydrolysis of polysaccharides such as starch into simple sugar components (Mitchell and Lonsane, 1990;Akpan et al., 1990a;Wahlstron and Suurnakki, 2015). Their hydrolytic properties lead to breakdown of glycosidic linkages of paint starch components into products such as maltose, glucose and maltotriose units which are of low molecular weights (Raini et al., 2016). Fungi belonging to the genus Aspergillus have been most associated with the production of α-amylase (Shivaramakrishnan et al., 2006). Microbial proteases are also of great commercial importance (Gupta et al., 2002;. For centuries, proteinaceous substances such as glues including albumin, casein and animal gelatins have been used in the paint industry as binding media in paintings to bind pigments and to protect the outer layer of f inished paintings (Peris-Vicente et al., 2005). Rojas et al., 2012. reported the ability of Aspergillus strains, Penicillium chrysogenum, P. glabrum, and Cladosporium oxysporum to degrade such protein-based components in paints.
In particular, proteases of Aspergillus species have been extensively studied due to the capacity of Aspergillus species to secrete high levels of these enzymes (Biesebeke et al., 2006). Peptidases are involved in breaking either specif ic peptide bonds (limited proteolysis), depending on the amino acid sequence of a protein, or the breaking down of a complete peptide to amino acids (unlimited proteolysis) (Hooper, 2002). Peptidase activity may be an activation of a f unction, a signal in a signaling pathway or a destructive change abolishing a protein's f unction or digesting it to its principal components (Hooper, 2002). This study aims at elucidating the involvement and impact of proteases and amylases on aesthetics of painted walls f rom a non-parametric point of view. Aspergillus aculeatus A1.9 (EU833205.1) and Fusarium proliferatum 2705 (EU272509.1) isolated f rom discolored painted buildings and submitted to GenBank in an earlier study (Obidi and Okekunjo, 2017) were used. The bacterial isolates were cultured in nutrient broth (Sigma-Aldrich) containing yeast extract, 3 g/litre; glucose, 1g/litre; peptone, 15g/litre; and NaCl, 6g/litre at 37°C while the f ungal isolates were suspended in Sabouraud Dextrose broth (Sigma-Aldrich) containing peptone 10g/litre and dextrose 20g/litre. Inoculum was standardized with 0.5 McFarland standard (1.5 x 10 8 CFU/mL) and microbial growth monitored at 600 nm (OD600) with UV Spectrophotometer (Microf ield SM23A, Microf ield Instrument England).

Enzyme activity
Amylase activity was assayed f ollowing the method of Bernf eld (1995) using 3, 5dinitrosalicylic acid. The activity was assayed by measuring the reducing sugar f ormed by the enzymatic hydrolysis of soluble starch. The reaction mixture containing 1 ml of 1% (w/v) soluble starch in citrate phosphate buf f er (pH 6.5) and 1 ml cell-f ree enzyme extract was incubated at 40 0 C f or 30 minutes. The reaction was terminated by addition of 2 ml of dinitrosalicylic acid (DNS) reagents. The reaction mixture was then heated f or 5 min in boiling water bath and absorbance was read at 540 nm to estimate reducing sugars released. Subsequently, the solution's optical densities were read against the specif ic sample blank (glucose f or amylase and tyrosine f or protease assay) using UV Spectrophotometer (Microf ield SM23A, Microf ield Instrument England).
Protease activity was determined as described by Chopra and Mathur (1985). The reaction mixture which contained 5 ml of casein in 50 mM of Tris buf f er (pH 8.0) and an aliquot of 1.0 ml of the enzyme solution was incubated f or 30 min. The reaction was stopped by adding 5 ml of trichloroacetic acid solution (TCA) (0.11M). Af ter 30 minutes, the mixture was f iltered, 2ml of the f iltrate was then added to 5.0 ml of 0.5 M sodium carbonate and 1.0 ml of Folin-Ciocalteu's phenol reagent and kept f or 30 min at a temperature of 37°C. Absorbance was read at 660nm. One unit of enzyme activity was def ined as the amount of enzyme required to liberate 1 μg of tyrosine per ml per min under assay conditions.

Optimization for enzyme production
Incubation time: The experiment was carried out f or each organism af ter leaving the culture extract at various incubation periods; 24, 48 and 96h. The pH was lef t constant at 7, temperature at 25 o C and substrate concentration at 1%. The enzyme assay was done af ter 24, 48 and 96h of incubation.
Temperature: The ef f ect of temperature on enzyme production was studied af ter incubation of the culture extract at various temperatures of 15, 25 and 37°C. The pH was made constant at 7, incubation time was 24h and substrate concentration was 1%. The enzyme assay was carried out af ter 24h of incubation.
pH: The ef f ect of pH on amylase and protease production was determined by subjecting the culture extract to dif ferent pH levels. The experiment was carried out f or each organism at pH 2, 6, and 9 while incubation time was held constant at 24h, temperature at 25 o C and substrate concentration at 1%. The enzyme assay was carried out af ter 24h of incubation.

Substrate concentration:
The optimal production of amylase and protease by the selected experimental microorganisms was determined by the addition of starch and casein to the culture extract at dif f erent substrate concentrations which are 0.01, 0.1 and 1%. The pH was held constant at 7, temperature at 25 o C and incubation time was 24h. The enzyme activity was measured af ter 24h of incubation. Subsequently, glucose and tyrosine standard curves (Figures not shown) were plotted to calculate the reduced sugar and tyrosine concentrations f or amylase and protease respectively.

Data management and statistical analysis
Data management and statistical data analysis f or this study was carried out with the use of R sof tware (R Core Team, Version 4.1.1). Descriptive statistics such as boxplots and line charts were used to summarize the relative levels of amylase and protease production with respect to various conditions. Wilcoxon Signed Rank Test was used to investigate whether amylolytic and proteolytic activities impact aesthetics on painted walls equally. All tests were carried out at 5% level of signif icance.

RESULTS
The eight isolates used in this study which were represented as A, B, C, D, E, F, G, H in the Aspergillus sp. SL2 (KC178662.1) and M. caribbica CBS:5674 (KY104219.1) respectively. They produced amylase and protease at varying conditions. The results showed that of the microorganisms (M. guilliermondii, F. proliferatum, Aspergillus sp, Cerrena sp and M. caribbica) had optimum production of amylase at 24h while C. tropicalis, A. aculeatus and P. aeruginosa) had optimum production at 48h ( Fig  1A). M. guillermondii, M. caribbica, F. proliferatum, A. aculeatus, Aspergillus sp, Cerrena sp and C. tropicalis had optimum production of amylase at pH 2 while P. aeruginosa had the optimum production of amylase at pH 6 ( Fig 1B). P. aeruginosa, Cerrena sp., C. tropicalis and F. proliferatum had the optimum production of amylase at 37 0 C, M. caribbica and A. aculeatus had optimum amylase production at 25 0 C while M. guillermondii and Aspergillus sp had optimum amylase production at 15 0 C respectively (Fig1 C). M. guillermondii, M. caribbica and Cerrena sp. had optimum amylase production at 0.1% substrate concentration while P. aeruginosa, C. tropicalis and Aspergillus sp had the optimum production of amylase at 0.01% substrate concentration. A. aculeatus and F. proliferatum however, had optimum amylase at 1% substrate concentration (Fig 1D). For protease production, M. guillermondii, F. proliferatum, P. aeruginosa, Aspergillus sp. and A. aculeatus had the optimum production of protease in 48hours while C. tropicalis, M. caribbica and Cerrena sp had the optimum production of protease at 96 hours respectively (Fig 2A). M. guillermondii, C. tropicalis, P. aeruginosa, Cerrena sp, F. proliferatum and M. caribbica had the optimum production of protease at pH 2 respectively. A. aculeatus and Aspergillus sp had the optimum production of protease at pH 6 ( Fig 2B). Also, C. tropicalis and Cerrena sp. had the optimum production of protease in 15 0 C while M. guillermondii, P. aeruginosa , F. proliferatum , A. aculeatus, Aspergillus sp and M. caribbica had optimum production of protease in 25 0 C (Fig. 2C).

Optimization for enzyme production
Incubation time: Result of the ef f ect of incubation time revealed that optimum amylase production was obtained at 24h f or 62.5% (5) of the microorganisms while 37.5% (3) showed optimum production at 48 hours. Proteolytic activity was observed to be optimum at 48h for 62.5% (5) of the microorganisms while others (37.5%) (3) showed optimum production at 96 h ( Fig. 1A and 2A). pH: Amylolytic activity was optimum at pH 2 (87.5%) and 6 (12.5%) f or all the microorganisms. The ef f ect of pH showed optimum protease activity at 2 f or 6 of the microorganisms (75%) and two of the organisms (25%) had optimum pH of 6 ( Fig. 1B and 2B).
Temperature: 37.5% of the microorganisms showed optimum amylase activity at 37 o C. Also, 37.5% (3) showed optimum amylase activity at 15 o C while 25% (2) of the microorganisms had optimum amylase activity at 25 0 C. Protease activity was optimal at 25 o C f or 75% (6) of isolates while others (25%) showed optimum protease activity at 15 0 C ( Fig. 1C and 2C).

Results of Wilcoxon rank sum test
In this section, we present the results obtained f rom the various tests of hypotheses to test whether enzyme activities are equal at the various environmental conditions considered at 5% signif icant levels.

Hypothesis one.
Ho: enzyme (amylase and protease) activities are equal at the time levels (24h, 48h and 96h). H1 : enzyme (amylase and protease) activities are not equal at the time levels (24h, 48h and 96h). Figure 3A shows the boxplot of the distribution of amylase and protease at the various times. It reveals that the location parameter (median) of the two enzymes is not the same.
To verif y this dif f erence, Wilcoxon signed rank te st result (W = 559, p-value = 2.434e-08) showed that amylase and protease activities are not equ al at the time levels (24hours, 48hours and 96 ho urs).

Hypothesis two.
Ho: enzyme (amylase and protease) activities are equal at the temperature levels (15 o C, 25 o C and 37 o C). H1 : enzyme (amylase and protease) activities are not equal at the temperature levels (15 o C, 25 o C and 37 o C).
To verif y this variation statistically, Wilcoxon Sign ed Rank Test result (W =576, p-value = 2.891e-0 9) reveals that enzyme (amylase and protease) a ctivities are not equal at the temperature levels ( 15 o C, 25 o C and 37 o C) since the p-value is greate r than 0.05.
To verif y this dif ference statistically, Wilcoxon Sig ned Rank Test result (W = 576, p-value = 3.04e-09) showed that the test is signif icant. We theref ore reject Ho and conclude that enzyme (amylas e and protease) activities are not equal at the pH levels (2, 6 and 9) since the p-value is greater th an .05. The above tests show the comparisons b etween the ef f ects of amylase and protease activ ities on the measured environmental parameters . The results f rom the box plots showed amylase activity to be more prominent f or all the environm ental conditions considered, suggesting clearly t hat amylolytic and proteolytic activities do not im pact aesthetics on painted walls equally. All the t ests were signif icant.

DISCUSSION
Prof ound enzymatic activities have been linked to biodeterioration and biodegradation (Teyjgeler, 2001;Cappitelli and Sorlini, 2005;Rojas et al., 2008). This paper describes a novel research into the authentication of the deterioration mechanism as well as quantif ying its contribution to obvious discolorations and stains using a non-parametric approach. Such studies have not been previously reported in Nigeria, and this represents the f irst study that reports such investigations. Three of the microorganisms (37.5%) used in this study produced amylase optimally at 37 0 C. Amylase activity has been reported to be optimum at 35 o C (Singh and Kumari, 2016) in an assay involving temperature ranges of 35 o C, 40 o C and 45 o Crespectively. Similarly, protease was produced optimally by 75% (6) of the organisms at 25 0 C. This suggests the contributions of these enzymes to the heavy discoloration on the painted walls where they were isolated . At the isolation sites, the prevailing temperature ranged f rom 25-30 0 Cwhich supports active metabolism and optimum enzymatic production by the producing organisms leading to obvious discolorations (Obidi and Okekunjo, 2017). The f act that amylases are produced optimally at such temperature range shows that amylase activity would decrease as the temperature increases above 37 o C. In this study, majority of the microorganisms (62.5%) (5) screened had their optimum incubation time to be 24 hours for amylase production. This is in line with the study of Koel et al. (2014) who reported an optimum amylase production at 24 h f or bacteria. We observed an optimum pH of 6 f or P. aeruginosa. Similarly, Raju and Divakar (2003) in a previous study, observed a maximum amylase production at pH of 7.0 f or Pseudomonas aeruginosa. The substrate concentration f or optimum amylase production, however, varies with 37.5% (3) having 0.01; 37.5% (3) having 0.1 and 25% (2) having 1% respectively. Previous reports on ef f ects of other physico-chemical parameters such as substrate concentration on amylase production varied when compared with our study. This observation agrees with an earlier report that optimum amylase production dif f er in other physicochemical properties depending on their origin (Arti et al., 2010.).
Pseudomonas aeruginosa CH01 (KY511067.1), the only bacterium tested in this study produced protease optimally at 25 o C while the f ungi had temperatures ranging f rom 15-25 0 C. This agrees with the study of Sharma et al. (2017) who reported an optimum temperature range of 28 o C-30 o C f or f ungi and lower temperature of 25 o C for some bacteria during protease production. Also, our study showed an optimum protease production at incubation time of 48 hours for Aspergillus sp. while no microorganism showed optimum protease production at an incubation time lesser than 48 hours. This agrees with previous studies that reported that the time generally, f or optimum protease production by bacteria or f ungus was 48 h to 9 days (Sharma et al., 2017). The study reported optimum incubation time of 48 hours and 72 h for Aspergillus oryzae and Aspergillus niger respectively. Furthermore, the study reported an optimum pH range between 5.0 and 10 as optimum f or protease production. In the present study, all the microorganisms except A. aculaetus and Aspergillus sp. had an optimum pH of 2 for protease production. Most studies did not report lower (acidic) pH probably because the pH range was not extended to a lower value during laboratory experiments as it was done in the present study. Protease activity increased as substrate concentration increased f or Cerrena sp. N10CC2a (FJ010208.1), M. carribica and Candida tropicalis. This corroborates the report by Priyanka et al. (2015). This study also revealed that the microorganisms utilized minimal substrate concentrations f or their amylolytic and proteolytic activities and produced optimally within the range of 0.01% to 1% substrate concentration respectively. This is particularly important since wall paints contain several organic and inorganic compounds which are utilizable as substrates [Rojas et al., 2008;Obidi et al., 2009a] by the isolated microorganisms. Previous workers have shown that microorganisms on painted surf aces utilize nutrients, substrates and carbon sources which enhances their prolif eration and enzymatic activities resulting in aesthetic discoloration on painted walls (Ravikumar et al., 2012).
M. guilliermondii had the highest amylolytic and proteolytic activity at 15 o C and 25 o C respectively. C. tropicalis had the highest proteolytic activity at 15 o C. This corroborates their greater abundance on deteriorated wall paints as reported by Obidi and Okekunjo (2017). The study detected them (M. guilliermondii and C. tropicalis) at 0.4-0.6 x 10 2 CFU/g compared to others which had their population densities ranging f rom 0.02-0.2 x 10 2 CFU/g. This agrees with the f act that prevailing environmental conditions in such tropical environments (temperature of ~ 26 0 C, average rainf all of ~186.8mm and humidity of ~ 86% (Obidi and Okekunjo, 2017) will f avour the growth of M. guilliermondii and C. tropicalis. The present study shows that amylolytic and proteolytic activities were achieved within an optimum period of 24-48h suggesting that most painted surf aces in tropical countries with such environmental conditions are at risk of microbial colonization as well as susceptible to serious discoloration over time.
Lagos is a typical tropical city which experiences a tropical savanna climate according to the Köppen climate classif ication because there are f our months under 60 millimetres or 2.4 inches of rain (Awe and Gil-Alana, 2021). Environmental conditions such as 37 0 C, pH 2-6 and substrate concentration of 0.01%during a period of 24 hour provides optimal amylase production. Similarly, a temperature range of 15-25 0 C, pH 2 and substrate concentration of 1% during a 48-hour period provides optimal protease production in organisms isolated f rom discolored painted buildings with visible stains. Wilcoxon Signed Rank test however, revealed that amylolytic and proteolytic activities in these organisms do not impact aesthetics on painted walls equally at all environmental conditions considered.

CONCLUSION
The study concludes that variations in environmental conditions af f ect the amylolytic and proteolytic activities of various microorganisms dif f erently.

Conflicts of interests:
The authors have no conf lict of interest to declare