Introduction

An enzyme tyrosinase (EC 1.14.18.1) shows diversity in terms of tissue distribution, cellular location and structural properties. It differs in glycosylation patterns and activation characteristics, structure and size (Zaidi et al. 2014a, b). Tyrosinase (tyrosine, L-DOPA: oxygen oxidoreductase; diphenol oxidase; catecholase; polyphenol oxidase – PPO) is a copper containing enzyme which with several phenolic substrates’ catalyses sequential oxidation steps (Faria et al. 2007; Flurkey and Inlow 2017). Tyrosinase exhibit two types of catalytic activities in the presence of oxygen (Sys et al. 2020): first is the monophenolase activity (Garcia-Molina et al. 2022), in which hydroxylation of monophenols to O-diphenols occur, second is the diphenolase activity (Li et al. 2021), involving oxidation of diphenols to their corresponding o-quinones (Cieńska et al. 2016) (Fig. 1). All tyrosinases in their active sites contain a copper centre of two copper atoms. Each copper atom is coordinated by three residues of histidine. In crystalline form, these enzymes contain six residues of histidine and three imidazoles of histidine for each copper atom, in active site. Activities of tyrosinases are widely distributed from microorganisms to mammals. They are found in mushrooms, fruits, vegetables, bacteria, fungi as well as humans (Zaidi et al. 2014a, b).

Agaricus bisporus, A. oryzae, Amanita muscaria, Neurospora crassa, Lentinula edodes, L. boryana and Pycnoporus sanguineus are different fungi that are used for isolation of tyrosinase (Zaidi et al. 2014a, b). Bacterial tyrosinases have been reported in number of species such as Bacillus thuringiensis, Marinomonas mediterranea, Pseudomonas maltophilia, P. putida, Streptomyces castaneoglobisporus, Symbiobacterium thermophilum and Verrucomicrobium spinosum (Zaidi et al. 2014a, b). Most of the current development of biotechnological applications of tyrosinases is focused on mushroom tyrosinases (Faria et al. 2007). In 1985, first biochemical investigation of tyrosinase was carried out on Russula nigricans, a mushroom, whose cut flesh turns black and red when exposed to air. Similarly, the enzyme extracted from A. bisporus, is homologous to that of mammals, and thus renders A. bisporus a well-matched model to carry out studies on melanogenesis (Chang 2009). L. edodes commonly known as shiitake mushroom is the second most commercially produced species of mushrooms (Cai et al. 2017). During post-harvest preservation, the gill of L. edodes fruit body turns brown. This gill browning of fruit bodies of shiitake is thought to be caused by biosynthesis of melanin catalysed by tyrosinase (Sato et al. 2009). Tyrosinase is responsible for generating the pigment melanin (Jimenez et al. 1988; Narasimhappa and Ramamurthy 2023) as described in Fig. 2.

The most important application of tyrosinase being investigated is the synthesis of L-DOPA from L-tyrosine using Erwinia herbicola, E. coli (Faria et al. 2007). Tyrosinase is also a potential pro-drug for melanoma treatment (Morrison et al. 1985; Jin et al. 2024). Several studies involving mushroom tyrosinase are being carried out for removal, detection and quantification of phenolic compounds present in water samples. This enzyme is being utilized for producing cross-linked protein networks (Faria et al. 2007). Herbicides are a potent hazard to human health if present in surface or ground water, thus, their presence can be detected by using biosensors based on principle of inhibition activity of tyrosinase enzyme (Wang et al. 2006). For tailoring meats’ gelation properties, cross linking enzymes play an essential role (Cirlincione et al. 2023). Recently tyrosinases have been tested for chicken proteins processing (Lantto et al. 2006; Lantto et al. 2007).

Dopamine (DA) is a major neurotransmitter belonging to monoaminergic neuroreceptor family that supports the central nervous system (Missale et al. 1998; Zhang et al. 2016). Many neurological diseases are a result of disorder in levels of dopamine (Donzella et al. 2022). These diseases include Parkinson’s disease (Nagatsu et al. 2023), Alzheimer’s, schizophrenia, sleeping and eating disorders (Zhang et al. 2016). For production of dopamine by the central nervous system, L-DOPA acts as a precursor. Due to this reason, L-DOPA is a potent drug for treating patients with Parkinson’s disease (Richmond et al. 2023). Myocardium neurogenic injury can also be regulated by using dopamine (Ali et al. 2007).

Materials and Methods

The chemicals including ethylenediaminetetraacetic acid (EDTA), L-catechol, L-ascorbic acid, L-tyrosine, sodium nitrite (NaNO₂), sodium molybdate (Na₂MoO₄), sodium acetate (CH₃COONa.3H₂O), sodium hydroxide (NaOH), sodium dodecyl sulphate (SDS) and chitosan were of analytical grade and were purchased from Sigma-Aldrich (Germany).

Collection of mushrooms

Black mushroom commonly known as shiitake mushroom (L. edodes) was purchased from a store in dried form. The mushrooms were of two types differing in their developmental and morphological stages thus named as Shii-I and Shii-II.

Pretreatment of mushroom

The mushroom biomass was thoroughly dried and grinded by using mortar and pestle. The grinded biomass was further treated with 1-butanol. Organic solvent was added in a conical flask till mushroom was completely dipped in it and was incubated for 20 min in a shaking incubator (VS-8480, Vision Scientific, Daejeon-Si, Korea) at 160 rpm at 30°C. After incubation mushroom biomass was filtered with muslin cloth and double washed with ice cold water (4°C) to remove any residual organic solvent. The biomass was oven dried (DHG-9202, SANFA, Yangzhou, China) at 105°C for 2 h.

Enzyme harvesting and filtration

Pretreated mushroom biomass was incubated in saline water as an extractant at 37°C in a shaking incubator for 24 h. After incubation, the extractant solution was filtered to remove mushroom biomass and centrifuged (1020 D.E, Centurion Scientific, West Sussex, U.K) at 3500 rpm for 15 min to remove any residual contents of biomass (Zaidi et al. 2014a, b).

Reaction procedure

Reaction mixture containing L-tyrosine, as a substrate was used for the biosynthesis of DA. For biosynthesis of DA the reaction was carried out using 2.5 mg/mL L-tyrosine as a substrate. Reaction mixture consisted of 50 mM of acetate buffer (pH 3.5) in which 1 mL of extract, 2.5 mg/mL of L-tyrosine and 5 mg/mL of ascorbic acid were added. The reaction was carried out at 50°C in a water bath (MaxTurdy 18, Dhaihan Scientific, Wonju-si, South Korea) at 120 rpm for 60 min (Koyanagi et al. 2012).

Enzyme extraction

The reaction mixture was centrifuged at 3000 rpm for 20 min. The supernatant was separated in a clean falcon and pellet was discarded. Supernatant was stored at -4°C in a refrigerator (Koyanagi et al. 2012).

Analytical techniques

Tyrosinase assay

The method reported by Kandaswami and Vaidyanathan (1973) was used to determine the tyrosinase activity. Enzyme extract (0.5 mL), 1 mL of 50 mM phosphate buffer and 0.1 mL of 1% L-catechol was added in a test tube. This mixture was incubated for 10 min at 30°C. After completion of incubation, 0.1 mL of 1% L-ascorbic acid and 0.1 mL EDTA of 100 ppm concentration was added in the same test tubes. The contents were mixed thoroughly. Distilled water (1.2 mL) was added to raise volume to 3 mL. Absorbance was noted at a wavelength of 390 nm with spectrophotometer (UV-1700, Shiimadzu Corp., Kyoto, Japan). The activity of tyrosinase was calculated by using the following formula:

Tyrosinase activity (U/mg) =

One unit of enzyme activity was equal to a ∆A_390nmof 0.001 per min at pH 7 (25°C) in 3 mL of reaction mixture containing reagents, L-catechol and L-ascorbic acid.

DA assay

Enzyme extract of about 1 mL was taken in a test tube. In the same test tube 1 mL of 0.5 N HCl and 1 mL of nitrite molybdate reagent was added. The contents were mixed thoroughly. After mixing, 1 mL of 1 N NaOH was added to the same tube. The volume of this mixture was raised to 5 mL by using distilled water. The absorbance was analyzed at 460 nm (A₄₆₀) by using spectrophotometer and the amount of DA produced was determined (Arnow 1937; Fan et al. 2021).

Optimization of reaction conditions

Different organic solvents (acetone, 1-propanol, 1-butanol, ethanol, n-hexane and methanol) were used for pretreatment of mushroom biomass. The effect of level of different extracting agents (distilled water, saline water, 0.1 N H₂SO₄, phosphate buffer pH 7.2, acetate buffer pH 4.8 and 0.1 N tris-HCl) on tyrosinase activity and production of DA was studied. By varying the level of mushroom biomass (0.5 g, 1, 1.5, 2, 2.5 and 3 g) and changing the time of incubation (12 h, 24, 36, 48, 60 and 72 h), the production of DA was investigated. The effect of various extractants (SDS, MOT, Chitosan, Tween-80, Triton X-100 and PEG) along with their different concentration was determined. The tyrosinase assay was later performed at different temperature (20–30°C) and DA assay was performed at one of the optimum temperatures by varying the time of incubation by an interval of 10 min (10–60 min) (El-Hadi et al. 2014).

Results

Pre-treatment of mushroom biomass with different organic solvents

The two strains of L. edodes or shiitake mushroom (Shii-I and Shii-II) were pretreated with organic solvents (acetone, 1-propanol, 1-butanol, ethanol, n-hexane and methanol) to study their effects on the DA activity as shown in Fig. 3. Shii-I and Shii-II pretreated with 1-butanol exhibited maximum DA activity of 17 and 15 µg/mL respectively. Both the mushrooms had tyrosinase activity of 18 and 16 U/mg respectively with 1-butanol. However, there was an exception for the highest tyrosinase activity of 23 U/mg with Shii-I using n-hexane. The lowest DA activity for Shii-I (10 µg/mL) and Shii-II (3 µg/mL) was shown by acetone and methanol.

Evaluation of different extracting agents

The biomass pretreated with 1-butanol was further assessed with various extracting agents (distilled water, saline water, 0.1N H₂SO₄, acetate buffer (pH 4), phosphate buffer (pH 7.2) and 0.1 N Tris-HCl). The DA activity of 25 µg/mL by Shii-I and of 20 µg/mL by Shii-II, using saline water was exhibited to be the highest as shown in Fig. 4a. The tyrosinase activity corresponding to the highest DA activity for both the mushrooms was 23 and 18 U/mg, respectively. The least DA activity by Shii-I was depicted by 0.1 N H₂SO₄(3 µg/mL) and in case of Shii-II (5 µg/mL) it was depicted with acetate buffer, pH 4.8. Saline water was observed to be the optimal extracting agent. Nitrogen source is important for active metabolism of mushrooms, peptone being a nitrogen source is present in saline water making it efficient medium for DA activity.

Once saline water was optimized as an effective extractant, its levels were varied i.e., 12.5, 25, 37.5, 50, 62.5 and 75 mL. The Fig. 4b demonstrates the DA activity for Shii-I and Shii-II, as comparison. The maximum DA activity by Shii-I (28 µg/mL) and Shii-II (22 µg/mL) was found at 25 mL. The enzyme activity at 25 mL of saline water by Shii-I and Shii-II was 26 and 17 U/mg, respectively. The increase in level of saline water from 37.5 to 75 mL showed a gradual decline in DA activity having minimum activity of 6 and 2 U/mg, respectively at 75 mL for both mushrooms.

Effect of different levels of mushroom biomass

The amount of mushroom biomass was varied with a difference of 0.5 g that is 0.5, 1, 1.5, 2, 2.5 and 3 g. The DA activity was observed for both the mushroom as given in Fig. 5. The maximum DA activity of 35 µg/mL (Shii-I) and 28 µg/mL (Shii-II) was observed at 2 g along with increased tyrosinase activity. The DA activity was increased gradually from 12 to 35 µg/mL with Shii-I and from 13 to 28 µg/mL with Shii-II as biomass was increased from 0.5 to 2 g. The lowest DA activity was observed at 3 g. Thus, 2 g of mushroom biomass was optimal for DA activity which was highly encouraging (P ≤ 0.05).

Effect of time of incubation

The DA activity was observed with a difference of 12 h in incubation time as given in Fig. 6. Time was increased from 12, 24, 36, 48, 60 until 72 h. The maximum DA activity of 43 µg/mL and 40 U/mg of tyrosinase activity was shown by Shii-I after incubation time of 36 h. In case of Shii-II, again maximum DA activity of 29 µg/mL was observed at 36 h with 32 U/mg of tyrosinase activity. The DA activity increased gradually from 12 to 36 h, however, further increase in time of incubation lowered the DA activity. At 72 h, DA activity of Shii-I and Shii-II was 11 and 7 µg/mL, respectively. The 36 h time of incubation was revealed to be

Fig. 1: Structure of oxy-form of tyrosinase

Fig. 2: Melanin biosynthetic pathway

optimal. Copeland (2023) described that if substrate concentration was kept constant throughout the reaction with increase in incubation period, after sometime substrate will become inadequate, thus, causing a decrease in nutrients for microbes to survive. Microorganisms reached the decline phase of their growth causing a reduction in enzyme formation.

Fig. 3: Pre-treatment of mushroom biomass with different organic solvents for higher tyrosinase production and subsequent dopamine activity. Time of incubation 24 h, Temperature 37°C, Reaction temperature 50°C, Reaction time 1 h. Y-error bars indicate the values of standard deviation (± SD) from the sum mean at a level of 5%. The values in each set differ significantly at P ≤ 0.05

Effect of different surfactants and their concentrations

Different surfactants (tween-80, SDS, triton X-100, PEG, chitosan and MOT) were added in saline water (0.1 % w/v) to investigate the changes in DA activity. The Fig. 7a reveals that both mushrooms showed optimum DA activity with different surfactants. It was observed that with Shii-I, maximum DA activity of 58 µg/mL and 60 U/mg of tyrosinase activity was found when saline water was supplemented with SDS. The maximum DA activity of 50 µg/mL and 47 U/mg of tyrosinase activity was exhibited by Shii-II with chitosan. The minimal DA activity was exhibited with saline water containing tween-80, 15 µg/mL by Shii-I and even lower by Shii-II (11 µg/mL). Thus, SDS was optimized for Shii-I and chitosan for Shii-II.

The effect of different concentrations of SDS (2.5, 3, 3.5, 4, 4.5 and 5 mM) on DA activity was investigated for Shii-I. The data is represented in Fig. 7b. The maximum DA activity (64 µg/mL) was exhibited by SDS at concentration of 4.5 mM. The DA activity increased from 2.5 to 4.5 mM,

Fig. 4a: Evaluation of different extracting agents for higher tyrosinase production and subsequent dopamine activity. *Pretreatment 1-butanol, Time of incubation 24 h, Temperature 37°C, Reaction temperature 50°C, Reaction time 1h.Y-error bars indicate the values of standard deviation (± SD) from the sum mean at a level of 5%. The values in each set differ significantly at P ≤ 0.05

31 to 64 µg/mL, respectively. However, DA activity was minimum at 5 mM SDS, showing a sharp decline to 39 µg/mL. The effect of different concentrations of chitosan (0.2, 0.4, 0.6, 0.8, 1 and 1.2 mM) on DA activity is shown in Fig. 8. Saline water supplemented with 1 mM of chitosan in case of Shii-II had maximum DA activity of 59 µg/mL and 57 U/mg of tyrosinase activity. The lowest DA activity, 38 µg/mL was at 0.2 mM. An increasing trend was perceived from 0.2 mM till 1 mM, whereas, further increase in chitosan concentration showed a noticeable decline in DA activity.

Fig. 4b: Effect of different level of saline water on higher tyrosinase production and subsequent dopamine activity. *Pretreatment 1-butanol, Saline water pH 7, Time of incubation 24 h, Temperature 37°C, Reaction temperature 50°C, Reaction time 1 h. Y-error bars indicate the values of standard deviation (± SD) from the sum mean at a level of 5%. The values in each set differ significantly at P ≤ 0.05

Effect of different temperatures on tyrosinase activity

The effect of different incubation temperatures (20, 40 and 60ºC) on tyrosinase activity was studied as depicted in Fig. 8. From 20 to 60ºC, the tyrosinase activity increased from 30 to 66 U/mg, respectively for Shii-I. However, Shii-II exhibited maximum tyrosinase activity (60 U/mg) at 40ºC followed by a sharp decline at 60ºC. This decline is because of denaturation of tyrosinase at high temperature and it is unable to convert L-tyrosine to L-DOPA. The temperature optimized for tyrosinase activity of Shii-I and Shii-II mushrooms was 60 and 40ºC, respectively.

Effect of different time of incubation on DA activity

The effect of time of incubation of reaction mixture on the DA activity was determined. The incubation time was varied from 10 to 60 min with a difference of 10 min at each

Fig. 5: Varying levels of mushroom biomass for higher tyrosinase production and subsequent dopamine activity. *Pretreatment 1-butanol, Saline water (pH 7) 25 mL, Time of incubation 24 h, Temperature 37°C, Reaction temperature 50°C, Reaction time 1 h. Y-error bars indicate the values of standard deviation (± SD) from the sum mean at a level of 5%. The values in each set differ significantly at P ≤ 0.05

interval as presented by data in Fig. 9. Maximum DA activity of 68 µg/mL was exhibited by Shi-I at 20 min whereas at 40 min Shi-II displayed maximum DA activity of 62 µg/mL. The DA activity for both mushrooms increased from 10 min onward, but increase was more gradual for Shii-II. From 30 to 60 min, the DA activity of Shii-I decreased from 56 to 25 µg/mL, respectively. The incubation time optimized for both strains was 20 and 40 min, respectively.

Discussion

Shiitake mushrooms are second most cultivated species. In current study cultivation and processing parameters were optimized for enhanced dopamine activity. Pre-treatment of mushroom biomass with different organic solvents was evaluated. 1-butanol was revealed to be the optimum organic solvent for shiitake mushrooms pretreatment. The study revealing that cultures exposed to organic solvents show a significant increase in dopamine synthesis was investigated, hypothesizing, that the rate of dopamine synthesis may increase on exposure to organic solvents without affecting dopamine receptors as reported by Edling et al. (1997).

Fig. 6: Effect of time incubation on tyrosinase production and subsequent dopamine activity. *Pretreatment 1-butanol, Biomass 2 g, Saline water (pH 7) 25 mL, Time of incubation 24 h, Temperature 37°C, Reaction temperature 50°C, Reaction time 1 h. Y-error bars indicate the values of standard deviation (± SD) from the sum mean at a level of 5%. The values in each set differ significantly at P ≤ 0.05

Evaluation of different extracting agents was carried out in current study. El-Hadi et al. (2014) reported that peptone was a good nitrogen source for tyrosinase production from A. horta comparable to current results. The decrease in DA activity was due to increase in dilution of saline water as reported by El-Hadi et al. (2014). Thus, 25 mL of saline water was found to be the optimal extracting agent. As far as effect of different levels of mushroom biomass is concerned the increase in biomass other than the optimal limit caused an inhibition of enzyme and subsequent reduction of DA activity as described by Copeland (2023). Effect of time of incubation was an evaluating parameter in this study. This study is strengthened by Copeland (2023). Who described that if substrate concentration was kept constant throughout the reaction with increase in incubation period, after some time substrate will become inadequate, thus, causing a decrease in nutrients for microbes to survive. Microorganisms reached the decline phase of their growth causing a reduction in enzyme formation. Effect of different surfactants and their concentrations was observed and comparable with Yang et al. (2007). He reported that different surfactants i.e., aerosol OT, Brij 52 and CTAB, act differently on the activity of tyrosinase. When AOT was

Fig. 7a: Effect of different surfactants on tyrosinase production and subsequent dopamine activity. *Pretreatment 1-butanol, Biomass 2 g, Saline water (pH 7) 25 mL, Time of incubation 36 h, Temperature 37°C, Reaction temperature 50°C, Reaction time 1 h. Y-error bars indicate the values of standard deviation (± SD) from the sum mean at a level of 5%. The values in each set differ significantly at P ≤ 0.05

added, the mushroom tyrosinase activity increased with increase in concentration of AOT. Brij-52 and AOT both showed a positive response by displaying the ability to activate the enzyme while CTAB sharply inhibited the tyrosinase activity. Some other studies also reported the effect of different temperatures on tyrosinase activity. Koyanagi et al. (2012) reported that the stability of tyrosinase obtained from Pseudomonas putida for DA production from L-DOPA was at temperatures less than 45ºC. The maximum tyrosinase activity of Lentinula boryana was at 40°C as reported by Zaidi et al. (2014a, b). Halaouli et al. (2006) exhibited 30°C to be the optimum incubation temperature for tyrosinase production by P. sanguineus CBS 614.73. As far as effect of different time of incubation on DA activity, Ali and Nawaz (2016) reported that DA activity was lowest at the start of reaction. As incubation period was increased from 50 to 70 min there was a two-fold increase in DA activity. However, when the time of incubation was increased above 70 min, the activity of DA declined effectively. Pre-treatment of mushroom biomass with different organic solvents revealed that 1-butanol was the optimum organic solvent for shiitake mushrooms pretreatment. The study revealing that cultures exposed to organic solvents show a significant increase in dopamine synthesis was investigated, hypothesizing, that the rate of dopamine synthesis may increase on exposure to organic solvents without affecting dopamine receptors as reported by Edling et al. (1997). Evaluation of different extracting agents was evaluated. El-Hadi et al. (2014) reported that peptone was a good nitrogen source for tyrosinase production from A. horta comparable to current results.

Fig. 7b: Effect of different concentrations of surfactants on tyrosinase production and dopamine activity. *Pretreatment 1-butanol, Biomass 2 g, Saline water (pH 7) 25 mL, Time of incubation 36 h, Temperature 37°C, Reaction temperature 50°C, Reaction time 1 h, Surfactant SDS/chitosan. Y-error bars indicate the values of standard deviation (± SD) from the sum mean at a level of 5%. The values in each set differ significantly at P ≤ 0.05

The decrease in DA activity was due to increase in dilution of saline water as reported by El-Hadi et al. (2014). Thus, 25 mL of saline water was found to be the optimal extracting agent. Effect of different levels of mushroom biomass revealed that the increase in biomass other than the optimal limit caused an inhibition of enzyme and subsequent reduction of DA activity as described by Copeland (2023). Copeland (2023) described that if substrate concentration was kept constant throughout the reaction with increase in incubation period, after sometime substrate will become inadequate, thus, causing a decrease in nutrients for microbes to survive. Microorganisms reached the decline phase of their growth causing a reduction in enzyme formation. Yang et al. (2007) reported that different surfactants i.e., aerosol OT, Brij 52 and CTAB, act differently on the activity of tyrosinase. When

Fig. 8: Effect of different temperatures on tyrosinase activity. ^*Pretreatment 1-butanol, Biomass 2 g, Saline water (pH 7) 25 mL, Time of incubation 36 h, Temperature 37°C, Reaction temperature 50°C, Reaction time 1 h, Surfactant SDS (Shi-I) Chitosan (Shi-II). Y-error bars indicate the values of standard deviation (± SD) from the sum mean at a level of 5%. The values in each set differ significantly at P ≤ 0.05

Fig. 9: Effect of different time of incubation on DA activity. *Pretreatment 1-butanol, Biomass 2 g, Saline water (pH 7) 25 mL, Time of incubation 36 h, Temperature 37°C, Reaction temperature 50°C, Reaction time 1 h, Surfactant SDS (Shi-I) Chitosan (Shi-II), Tyrosinase assay 60°C (Shi-I) 40°C (Shi-II).Y-error bars indicate the values of standard deviation (± SD) from the sum mean at a level of 5%. The values in each set differ significantly at P ≤ 0.05

AOT was added, the mushroom tyrosinase activity increased with increase in concentration of AOT. Brij-52 and AOT both showed a positive response by displaying the ability to activate the enzyme, while CTAB sharply inhibited the tyrosinase activity. Effect of different temperatures on tyrosinase activity was a part od current study and reported by others too. Koyanagi et al. (2012) reported that the stability of tyrosinase obtained from Pseudomonas putida for DA production from L-DOPA was at temperatures less than 45ºC. The maximum tyrosinase activity of Lentinula boryana was at 40°C as reported by Zaidi et al. (2014a, b). Halaouli et al. (2006) exhibited 30°C to be the optimum incubation temperature for tyrosinase production by P. sanguineus CBS 614.73. Ali and Nawaz (2016) reported that DA activity was lowest at the start of reaction. As incubation period was increased from 50 to 70 min there was a two-fold increase in DA activity. However, when the time of incubation was increased above 70 min, the activity of DA declined effectively.

Conclusion

In the present study, two strains of L. edodes, commonly known as shiitake mushroom were used as a potential source of enzyme tyrosinase for enhanced activity of dopamine (DA). Prior to the optimization of some significant parameters, the mushroom types i.e., Shii-I and Shii-II exhibited 17 and 15 µg/mL of DA, respectively in the reaction mixture. The optimization of harvesting and reaction conditions particularly pretreatment of mushroom biomass by 1-butanol, SDS & chitosan as surfactants and 36 h of incubation improved the DA activity by 4-fold. The maximal DA activity was found to be 68 µg/mL during the course of study. Overall, the results both in terms of enzyme and DA activity of shii-I and shii-II mushrooms are highly encouraging (P ≤ 0.05), thus indicating the viability of the process used.

Acknowledgements

The authors highly acknowledge Higher Education Commission of Pakistan and GC University Lahore to provide necessary facilities for conduct of this research work.

Author Contributions

SA and MUA planned the experiments, HZ and HU conducted the experiments. SA, MUA and SS supervised the experimental work. MS and AI facilitated in data analysis. HZ and SA managed the article write up. SS and MUA helped in article formatting.

Conflicts of Interest

The authors declare that there is no conflict of interest.

Data Availability

Data presented in this study will be available on a fair request to the corresponding author.

Ethics Approval

Not applicable to this paper.

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