Optimisation of nutritional requirements for dopamine synthesis by calcium alginate-entrapped mutant strain of Aspergillus oryzae EMS-6

Abstract The optimisation of nutritional requirements for dopamine (DA) synthesis by calcium alginate-entrapped mutant variant of Aspergillus oryzae EMS-6 using submerged fermentation technique was investigated. A total of 13 strains were isolated from soil. Isolate I-2 was selected as a better producer of DA and improved by exposing with ethyl methylsulphonate (EMS). EMS-6 was selected as it exhibited 43 μg/mL DA activity. The mutant variable was further treated with low levels of l-cysteine HCl to make it resistant against diversion and environmental stress. The conidiospores of mutant variant were entrapped in calcium alginate beads for stable product formation. EMS-6 gave maximum DA activity (124 μg/mL) when supplemented with 0.1% peptone and 0.2% sucrose, under optimised parameters viz. pH 3, temperature of 55 °C and incubation time of 70 min. The study involves the high profile of DA activity and is needed, as DA is capable to control numerous neurogenic disorders.


Introduction
The neurotransmitter, dopamine (DA) or 2-(3, 4-dihydroxyphenyl) ethylamine is a monoamine catecholamine that is important in the regulation processes of central and peripheral nervous system. It is important for the synthesis of norepinephrine and epinephrine (Kurt et al. 2009). DA in central nervous system and periphery is important in regulation of cardiovascular homeostasis, locomotion and endocrine regulation. The defect in dopaminergic (DAergic) neurons has been linked with the aetiology of neurological disorders and psychiatric diseases (Eisenhofer et al. 2004;Goldstein 2010). DA has been used therapeutically, as a cardiovascular stimulant in treating hypotension and circulatory insufficiency (Hoffman et al. 1996). For the treatment of DA-related diseases, in vitro DA synthesis is required. But, due to the presence of tight junctions in cerebral endothelial cells (ECs), the blood brain barrier strictly limits the transfer of drug from blood to brain. l-Dopa, a precursor of DA is vigorously transported in the brain along the carriers of neutral amino acids (Pahwa & Koller 1998). However, l-dopa therapy results in side effects like dopa-induced dyskinesia (Ahmed et al. 2015). DA is produced by enzymatic and chemical methods; however, the production is restricted to chemical processes at industrial level. The enzymatic production of DA using l-tyrosine is catalysed by tyrosine hydroxylase (TH, EC 1.14.16.2), tyrosine phenol-lyase and dopa decarboxylase (DDC, EC 4.1.1.28) or tyrosine decarboxylase (Satoh et al. 2012). The pathway is shown in Figure 1.
Among the microbes' viz. fungi and bacteria, the former are easy to culture for mycelial development and thus maintain cell-free enzyme preparation (Tkacz & Lange 2004). Different isolates have been reported for the synthesis of DA i.e. Citrobacter freundii, Streptoccus faecalis and Erwinia herbicola. The analysis of catecholamines and their metabolites in samples usually aid in the examination of diseases i.e. pheochromocytoma, neuroblastoma and hypertension/hypotension (Peaston & Weinkove 2004). The present investigation describes the biotransformation of l-tyrosine to DA by A. oryzae using submerged fermentation as it is a desirable organism for metabolite production (Shaaban et al. 2014). The superior isolate was improved by chemical mutagen and made resistant using l-cysteine HCl. The conidiospores of mutant variant were entrapped in alginate beads for a stable product formation. The optimisation of nutritional requirements and cultural conditions was carried out by the potential mutant strain for better DA activity.

Isolation and screening fungi
In the present study, a total of 13 strains of A. oryzae were isolated from soil and their morphological characteristics were compared with that of the reference strain (A. oryzae FCBP-0001). The isolates were primarily screened for tyrosinase and DA activity by dye method. The secondary screening of better-isolated strains was carried out using submerged fermentation (SmF) technique. The data given in Table S1 represent secondary screening, enzyme activity and protein estimation of the selected isolates. Among the screened isolates, maximum enzyme activity (1.4 u/mg), protein content (112 mg/mL) and DA activity (21 μg/ mL) were detected by the wild-type I-2. Hence, induction of mutation was made with I-2.

EMS-induced mutagenesis of selected A. oryzae isolate
Incubation of mycelia with 2.5 mM of EMS for 5 min resulted in seven different types of EMStreated variants (Table S1), as point mutations were induced by EMS in A. oryzae (ravinder et al. 2003). In a study, Abdullah and Ikram-ul-Haq (2015) reported high proportion of induced chromosomal aberrations and base-pair substitution by EMS. A maximal of 154 mg/mL of protein content was estimated by EMS-6. The selected variant (EMS-6) was made resistant by treating it with low levels of l-cysteine HCl. The treatment with l-cysteine HCl helped the fungal cultures to maintain their growth in strict environmental conditions (Karmakar et al. 2012).

Calcium alginate entrapment of A. oryzae conidiospores for stable DA activity
Various immobilisation parameters including concentration of sodium alginate, inoculum size and bead size of calcium alginate were investigated for product stability. The optimised conditions for entrapment of conidiospores were found to be 1.5% alginate, 2 mL inoculum size and 2 mm bead size.

Evaluation of additional nitrogen sources for DA activity
Nitrogen source is one of the important nutritional factors for the active metabolism of micro-organisms (Lee et al. 1999). The effect of different organic and inorganic nitrogen sources (peptone, urea, ammonium nitrate, ammonium chloride, sodium nitrate and sodium nitrite) was examined for improved activity of DA using entrapped mutant variant of A. oryzae EMS-6. The data are given in Table S2. It was observed that maximum activity (76 μg/ mL) was obtained from the culture supplemented with peptone and from the study, it was demonstrated that peptone increased the metabolic activity of resistant A. oryzae EMS-6 greater than any other nitrogen source. Similarly, El-Hadi et al. (2014) reported peptone as a better nitrogen source using A. hortai as the organism of choice. The concentration of peptone (0.05-0.3%) was also examined for enhanced activity of DA and the data are represented in Figure S2. The DA activity increased (74 μg/mL) up to the concentration of 0.1%. Further increase in peptone concentration, however resulted in the decreased activity.

Evaluation of additional carbon sources for DA activity
The effect of different additional carbon sources like glucose, fructose, sucrose, maltose and cellulose on the activity of DA was also evaluated. The data are shown in Table S2. It was observed that among other carbon sources, the culture grown on 0.1% sucrose gave a maximum of 84 μg/mL DA activity. The activity under different concentration of sucrose (0.1-0.6%) was also assessed. The increase in product activity was observed as the concentration was increased from 0.1 to 0.2% which gave a maximum of 90 μg/mL DA activity ( Figure S3). The protein content and enzyme activity by EMS-6 supplemented with nitrogen and carbon sources were also estimated (Table S2). Further increase in concentration decreased the activity. In a similar study, Maresma et al. (2010) maintained the growth of mutated strain of A. oryzae for the mass production of particular metabolite on media supplemented with sucrose as carbon source.

Effect of buffer pH on DA activity
The pH of buffer in the fermentation reaction was changed from 2.5 to 5 and its effect on DA activity was observed. The activity of the product was not effective at pH 2.5; however, it was maximum at pH 3 ( Figure S4). Further increase in pH resulted in the decreased DA activity. results of the study showed that low pH-favored l-tyrosine dissolution in the production medium. The increase in pH decreased the activity, because the product formed readily decomposed into melanin. Although the immobilisation of cells allowed them to show their activity at broader range of pH (Munjal & Sawhney 2002), it was evaluated that at high values of pH, the metabolic activity of immobilised spores was greatly affected. Contrary to the observations, Surwase et al. (2012) reported maximum production of their metabolite at pH 8 using l-tyrosine as a substrate.

Effect of temperature of the reaction procedure on DA activity
In the study, the effect of different temperature (45-70 °C) of the fermentation reaction on the activity of DA was studied ( Figure S5). Initially, the activity increased. Further increase in temperature decreased the activity from 108 to only 50 μg/mL. The temperature optimised for maximum DA activity was 55 °C. In contrast to the experimental result, Krishnaveni et al.
(2009) optimised a temperature of 25 °C for better activity of tyrosinase and maximum product formation. Koyanagi et al. (2012) reported the best enzyme stability by Pseudomonas putida at temperature less than 45 °C for the transformation of DA from l-dopa.

Effect of incubation time on DA and enzyme activity
In the experiment, fermentation medium was incubated for different time intervals and DA activity was determined ( Figure S6). The incubation time was varied from 50 to 75 min. After an incubation of 50 min, a minimal activity was observed. Further increase in the time of incubation, resulted in two-fold increase in the activity of DA (124 μg/mL). When the incubation time was increased from 70 to 75 min, the product activity declined significantly (p ≤ 0.05) to 98 μg/mL. The effect of incubation time on the activity of tyrosinase was also determined. Initially, the activity of enzyme was nominal (15 u/mg) which was also observed by minimum product activity. With the increase in incubation time, the activity improved gradually (54 u/mg) up to an incubation of 70 min. Further increase in incubation period, decreased the activity. In a previous study, Surwase et al. (2012) reported maximum product formation by incubating the strain of Brevundimonas sp. for 60 min.

Experimental
The chemicals including ethyl methylsulphonate (EMS), sodium thiosulphate, l-cysteine HCl, sodium alginate, l-tyrosine and l-ascorbic acid were of analytical grade and procured from E-Merck (Germany).

Isolation of A. oryzae strains
The fungal cultures capable of producing tyrosinase were isolated from soil using serial dilution method. Malt extract (ME) agar medium containing 20 g/L ME, 20 g/L agar, pH 4.8 was autoclaved at 15 lbs/in 2 (121 °C) for 15 min. one millilitre of the diluted soil suspension (10 −3-10−5 ) was transferred to solidified ME agar plates. The plates were incubated for 3 to 5 days at 30 °C.

Preliminary screening and maintenance of the selected A. oryzae isolates
The preliminary screening of isolates was accomplished by inoculating ME-agar plates supplemented with 0.1% l-tyrosine. After incubation, the initial colonies bearing larger zones of tyrosine hydrolysis were selected, maintained and later stored at 4 °C.

Preparation of conidial suspension
The conidial suspension was prepared by transferring 10 mL of sterilised 0.05% (w/v) MoT to a 3-5 days old slant of fungi having profuse conidial growth. The spore clumps were disrupted with the help of a sterile inoculating needle. The tube was swirled to form homogenous spore suspension. The spore count of inoculum was made using a haemocytometer slide bridge (Neubaur improved HBG, Marinefield, Germany) and found to be 2.5 × 10 6 CFu/ mL.

Harvesting the fungal biomass
The mycelial biomass was obtained by inoculating 50 mL of sterilised cultivation medium comprising 20 g/L glucose, 10 g/L peptone, 3 g/L KH 2 Po 4, 3 g/L NH 4 Cl, 10 g/L yeast extract and 0.2 g/L MgSo 4 .7H 2 o at pH 5 in an Erlenmeyer flask of 250 mL capacity with 1 mL of spore suspension, aseptically. The flasks were placed at 30 °C for 72 h at 160 rpm in a rotary shaking incubator (MIr-153, Sanyo, Japan). The mycelial morphology was noted. The biomass was filtered, washed with ice cold water (4 °C) and dried at 105 °C for 30 min in an oven (Memmort-455i, Munich, Germany).

Induction of mutation
The selected strain of A. oryzae was improved by EMS-induced mutagenesis. The dried mycelia were exposed to EMS (0.5-3.0 mM), for different time intervals (5-30 min). The reaction was terminated with 0.5 mL of sodium thiosulphate prepared in 0.05 M of phosphate buffer (pH 7.2). The tubes were centrifuged (centrifuge refrigerated, D-78532, Hettich zentrifugen EBA 20 , Tuttligen, Germany) at 4500 × g for 15 min. The supernatant was discarded and pellet was washed twice with phosphate buffer (pH 7.2).

Development of resistance in selected mutant variants
The selected mutant variant was made resistant by treating with various concentrations of l-cysteine-HCl (0.1-1.2 μM). For this, mycelia were added in l-cysteine-HCl solution and incubated at 30 °C for 5 min. The tube was centrifuged at 3000 × g for 15 min. The supernatant was discarded and the mycelia were washed with buffer. The treated mycelia were plated on ME agar and incubated at 30 °C for 3-5 days.

Immobilisation of conidiospores by entrapment in calcium alginate
Sodium alginate solution (3%) was prepared in the treated spore suspension. Calcium alginate beads were prepared by dripping the homogenous alginate suspension in sterilised 0.2 M CaCl 2 at 4 °C from the height of 10 cm. The beads were allowed to cure in CaCl 2 solution, filtered and washed twice with ice cold water.

Optimisation of nutritional requirements and cultural conditions
The resistant mutant variant was cultured with NH 4 Cl, NaNo 3 , NaNo 2 , (NH 4 ) 2 Co, NH 4 No 3 and peptone was added solely. The concentration of nitrogen source was ranged from 0.05 to 3%. The level of individual carbon sources viz. fructose, glucose, maltose, sucrose, cellulose and starch was varied from 0.1 to 0.6% at optimal fermentation conditions. The reaction mixture was incubated at different pH level (2.5-5) and temperature (40-65 °C) for different time intervals (50-75 min) to obtain maximum product formation.

Protein estimation
The protein content in the reaction mixture was determined using Bradford (1976) method. The enzyme supernatant (0.1 mL) was taken in a test tube along with 5 mL of Bradford reagent. Blank was run in parallel by replacing enzyme with equal quantity of water. The contents in the test tube were mixed and incubated at room temperature for 15 min. A 595 nm was measured by a uV/VIS spectrophotometer (Irmeco GmbH, D-21,496, Heidelberg, Germany).

Reaction procedure
The reaction for DA synthesis was carried out in a medium fed with 5 mg/mL l-ascorbic acid and 2.5 mg/mL l-tyrosine in 50 mM acetate buffer (pH 3.5). The medium was inoculated with treated entrapped conidiospores and incubated at 50 °C for 60 min in a water bath at 160 rpm. The mixture was filtered using a Whatman filter paper 44 and centrifuged at 5000 × g for 20 min. The clear supernatant was taken for DA analysis.

Analytical techniques
3.11.1. DA assay one milliliter of filtrate was transferred to a test tube followed by the addition of 1 mL 0.5 N HCl and 1 mL nitrite molybdate reagent. After mixing, 1 mL of 1 N NaoH was added. The final volume of assay mixture was raised to 5 mL with distilled water. The reaction was analysed (A 460 nm ) by a spectrophotometer and the DA activity was determined (Arnow 1937).

Tyrosinase assay
The tyrosinase activity was determined by a method described by Kandaswami and Vaidyanathan (1973). l-ascorbic acid (0.1 mL), EDTA (0.1 mL) and l-catechol (0.1 mL) was added in a test tube containing 2.6 mL of phosphate buffer (50 mM). The contents were mixed and A 265 nm was observed until it became constant. The decrease in absorption was monitored for 5 min after equal time interval (1 min).

Enzyme unit
one unit of enzyme activity is equal to a ΔA 265 nm of 0.001 per min at pH 6.5 (25 °C) in a 1 mL reaction mixture containing reagents (l-catechol and l-ascorbic acid).

Statistical analysis
Treatment effects were compared by SPSS (version 24) after Snedecor and Cochran (1980). The significant differences among replicates have been represented as probability (p) value.

Conclusion
In the present study, a strain of A. oryzae I-2 was isolated and improved for the bioconversion of l-tyrosine to DA. At initial stages, 44 μg/mL of DA was produced by the EMS-6. The spores of the improved strain were entrapped in calcium alginate beads for the product stability. The optimisation of nutritional requirements (additional carbon and nitrogen sources) and cultural conditions (pH, temperature and time of incubation) by the immobilised mutant strain of A. oryzae EMS-6 improved three-fold DA activity (124 μg/mL) in the production medium, which is highly significant (HS, p ≤ 0.05).

Supplementary material
Supplementary material relating to this article is available online, along with Figures (S2-S5) and Tables (S1-S2).