Oxidant and solvent stable alkaline protease from Aspergillus flavus and its characterization

The increase in agricultural practices has necessitated the judicious use of agricultural wastes into value added products. In this study, an extracellular, organic solvent and oxidant stable, serine protease was produced by Aspergillus flavus MTCC 9952 under solid state fermentation. Maximum protease yield was obtained when the strain was grown under wheat bran and corn cob mixture (1:1) incubated for 48 h at pH 9.0 and temperature 37°C with 50% of initial moisture content. The partially purified enzyme showed wide range of pH optima (8.0-12.0) and pH stability (7.0-12.0), whereas, optimum temperature was 40°C and was stable over a wide range of temperature 30-45°C. The protease was extremely stable towards several organic solvents. The enzyme retained 80% of its original activity in the presence of non ionic and ionic surfactants and 100% with 10% H2O2 after 1 h of incubation at 30°C. In addition, the enzyme showed excellent compatibility with some commercial powder detergents. The compatibility of our protease with several detergents, oxidants and organic solvents suggests its possible use in detergent industry and peptide synthesis.


INTRODUCTION
Proteases are commercially important enzymes (Ferrero et al., 1996;Kumar et al., 1999) which account for approximately 60% of global industrial enzyme sales (Rao et al., 1998).Proteases occur widely in plants and animals, but commercial proteases are produced exclusively from microorganism (Chutmanop et al., 2008).Aspergillus, Penicillium and Rhizopus are widely used for protease production since several species of these genera are regarded as safe (GRAS) strains (Pandey, 1992).Aspergillus has ideally been an organism of choice for bulk production of industrial enzymes, as the fungi can be grown on relatively inexpensive agricultural *Corresponding author.E-mail: dr_nsdarmwal@yahoo.co.in.Tel: 05278 247350.
Enzyme production can be carried out by both submerged fermentation (SmF) and solid-state fermentation (SSF), the latter being a technique of choice (Pandey et al., 2001;Sandhya et al., 2005).Alkaline serine proteases (EC 3.4.21)are active and stable at neutral to alkaline pH (7-12), and find extensive applications in protein chemistry and protein engineering as well as in industries such as detergents, leather, protein recovery, meat tenderization etc. (Lauer et al., 2000;Johnvesly and Naik, 2001).
Alkaline proteases with novel properties, such as stability with organic solvents, are in great demand for their increasing application in organic synthesis (Gupta and Khare, 2006).In view of the above, we report here the production of alkaline protease by Aspergillus flavus MTCC 9952, with an aim to find a set of culture conditions using agro industrial wastes and characterize the properties of partially purified enzyme leading to the development of detergent formulations and for peptide synthesis in non aqueous media.

Maintenance, medium and preparation of inoculums
The culture was routinely maintained on malt extract-glucose-agar slants (containing g/l: malt extract, 20; glucose, 20; KCl, 0.5; MgSO4, 0.5; KH2PO4, 1.0; pH 9.0) and stored at 4 °C.Prior to each experiment, the fungus was transferred to fresh slants and incubated at 37 °C for 4 days.The spores from fully sporulated slant were dislodged and dispersed in 10 ml of 0.1 % (v/v) Tween 80 with inoculum loop under aseptic conditions.The spore suspension was used as inoculum (1 ml), viable spores in the suspension being determined by serial dilution followed by plate count (1x10 7 spores/ml).

A Study of process variables on protease production
Effect of following variables on protease production was studied sequentially: agro-industrial wastes (wheat bran, WB; rice bran, RB; corn cob CC; pigeon pea bran, PB and black gram bran, BgB) were studied alone and in combinations (ratio of 1:1).The effect of incubation period (24 to 168 h) was studied followed by different ratios of WB and CC.The media were moistened to 44.4 to 87.5 % initial moisture and incubated at 32 to 45 °C.The effect of initial pH (8.5-10.5),different inoculum levels (1x10 4 to 1x10 9 spores/gram of solid substrate, gss) were also evaluated for the optimum enzyme yield.
Supplementation of different carbon sources were studied at the level of 100 mg/gss.Similarly several organic, inorganic nitrogen sources and metal ions were also tested at the level of 50 mg/gss, 20 mg/gss and 0.5 mg/gss, respectively.
All experiments were carried out in triplicates and the results are presented as the mean of three independent observations.

Enzyme extraction
Fermented bran (5 g) was soaked in 50 ml of sterile distilled water and the resulted slurry was agitated on a rotary shaker (120 rpm, 2h, and 30 °C).Slurry was finally removed by filtering and centrifuging at 10000 xg for 10 min at 4 °C.The resulting supernatant was recovered which served as crude enzyme extract.

Enzyme assay
Protease activity was determined as described by Hagihara et al. (1958), 1ml of suitably diluted enzyme was mixed with 1ml of casein Yadav et al. 8631 (1% w/v prepared in 50 mM carbonate-bicarbonate buffer of pH 10.0).The mixture was incubated at 37 °C for 10 min.The reaction was quenched by adding 3 ml of 10 % pre-chilled trichloroacetic acid (TCA).
The reaction was allowed for 30 min to completely precipitate the proteins.The contents of the reaction tubes were filtered through Whatman No. 1 filter paper and absorbance of the filtrate was read at 275 nm which was extrapolated against tyrosine standard curve.A unit of protease activity was defined as the amount of enzyme liberating 1µg tyrosine/ml/min under the assay conditions.The protease activity was reported as per gram of solid substrate (gss) used for protease production.

Estimation of total soluble protein
The total soluble protein content was estimated following Lowry's method (Lowry et al. 1951) using BSA as standard.

Partial purification of enzyme
The supernatant was separated into three fractions by adding chilled acetone in ratio of 1: 0.8, 1:2 and 1:3 (enzyme extract: acetone).The precipitated proteins were separated by centrifugation at 10000 xg at 4 °C for 15 min, left for some time to evaporate the traces of acetone and dissolved in minimum amount of Tris-HCl buffer (50 mM, pH 9.0) and stored at 4 °C for further study.

Effect of temperature and pH on enzyme activity and stability
The optimum temperature for protease activity was determined by incubating the reaction mixture at different temperatures (20-65 °C).Thermal stability was assayed by pre-incubating the enzyme at different temperatures (30-50 °C) for 30 and 60 min and residual protease activity was determined at temperature 37 °C and pH 10.0.
The optimum pH was assayed by determining the proteolytic activity at different pH by using the following 50 mM buffer solutions (Sodium phosphate, pH 6.0 -8.0; Tris-HCl, pH 9.0; glycine/NaOH, 10.0-12.0).For pH stability the enzyme was pre-incubated (1:1) with above mentioned buffers at 30 °C for 1 h and thereafter the residual protease activity was determined under standard assay conditions.

Effect of cations, inhibitors, surfactants and oxidizing agents on enzyme activity/stability
The effect of various metal ions (5 mM) on enzyme activity was investigated using NaCl, KCl, CaCl2, MnCl2, ZnCl2, CoCl2, CuSO4, FeSO4, NiCl2, HgCl2, PbCl2 and MgSO4 in the reaction mixture.The effects of some surfactants (Triton X-100, Tween 40, Tween 80, and SDS) and oxidizing agents (H2O2 and sodium perborate) on enzyme stability were also studied by pre-incubating the enzyme for 30 min and 1 h, at 30 °C.The residual protease activity was measured under standard assay conditions.
The effects of enzyme inhibitors (2.5 mM) on protease activity were studied using phenylmethylsulfonyl fluoride (PMSF) and ethylene-diaminetetraacetic acid (EDTA).The enzyme was preincubated with inhibitors for 30 min at 30 °C and the residual enzyme activity was estimated under standard assay conditions.

Effect of organic solvents on enzyme stability
Enzyme was incubated with different hydrophilic and hydrophobic Table 1.Effect of type of solid substrate and incubation period on protease production.

Type of substrate
Mean a enzyme activity (U gss -1 )

h 72 h 96 h 120 h 144 h 168 h
a Average of three independent experiments; b The P value refers to the comparison of protease activity with wheat bran to other substrates.The comparison is among the values within the column; c All the values in the coloumn are statistically significant.WB, wheat bran; RB, rice bran; PB, pea bran; BgB, black gram bran; CC, corn cob.organic solvents (benzene, chloroform, hexane, xylene, toluene, ethanol, acetone, diethyl ether, butanol and methanol) at a concentration of 25 % at 30 °C for 1 h and residual protease activity was estimated by standard assay method.Enzyme without organic solvent was taken as a control.

Isolation and screening
The alkaline protease production profile of forty six fungal strains was studied and the fungal strain NSD08, giving identified as Aspergillus flavus at Indian Institute of Microbial Technology, Chandigarh and given an accession No. MTCC 9952 was selected for further studies.
An effect of type of substrate and incubation time on protease production was studied.Results (Table 1) revealed a significant variation in incubation time with respect to the type of substrate used.However, a comparable level of protease activity was obtained with the substrates WB + CC (815 Ugss -1 ), BgB (814 Ugss -1 )  ), after 48 h, 72h, and 96 h, respectively, probably due to its ability to use diverse substrates.With a view to select the best combination, mixtures of WB and CC were investigated.The ratio of 1:1 (WB: CC) gave maximum production (807 U/gss) followed by 2:1 with enzyme units of 763 U/gss (Fig 1).
The effect of initial moisture content on protease production by A. flavus suggested an approximate 50 % of initial moisture content as optimum for protease production (Table 2).

Effect of inoculum size on protease production
A gradual increase in the enzyme activity was observed with increasing concentration of spores in the inoculum (data not shown).1x10 8 spores/g substrate was found to be optimum (1621 U/gss), however, further increase in the inoculum size reduced the enzyme activity.

Combined effect of pH and temperature on enzyme production
The effect of pH and temperature on alkaline protease production is shown in Fig 2 .The fungus is capable to produce significant amount of enzyme in the temperature range 32-40 °C and pH range 8.5 to 10.0.Maximum protease production was obtained at 37 °C and pH 9.0 (1640 U/gss).b the P value refers to the comparison of protease activity of control to the organic and inorganic nitrogen sources added to the medium.The comparison is among the values within the column; c All the values in the column are statistically significant.

Effect of additional carbon source on protease production
Fig 3 represents the effect of addition of carbon sources on protease production by A. flavus.Maximum yield was obtained with sorbitol (2277 U/gss), however, sufficient amount of enzyme (2054 U/gss) was produced by this strain with glucose, followed by galactose (1949 U/gss).Further increase in sorbitol concentration increased the enzyme yield (2531 U/gss) at concentration of 200 mg/gss, while higher concentration of sorbitol showed inhibitory effect on enzyme yield which might be due to catabolite repression.

Effect of additional nitrogen sources on protease production
Although seven organic nitrogen sources (50 mg/gss) were investigated to observe their effect on protease production (Table 3), only malt extract had an inductive effect on enzyme production (2923 U/gss).Therefore, different concentrations of malt extract were investigated and a 23 % increase in protease production (3136 U/gss) was obtained at the level of 100 mg/gss, over the control.Few inorganic nitrogen sources (20 mg/gss) were also incorporated in the fermentation medium and results revealed that di-ammonium hydrogen orthophosphate was the best (Table 3) with enzyme units of 3640 U/gss.

Effect of temperature on enzyme activity and stability
The temperature profile of A. flavus protease activity elaborated retention of 100 % activity at 40 °C while 92, 74 and 41 % residual activities were recorded at 45, 50 and 55 °C, respectively.However, increase in temperature beyond 55 °C resulted in complete inactivation of enzyme.
The thermal stability profile of the enzyme showed an initial retention of 96 % activity after 1 h of incubation at 40 °C, whereas, 84 and 53 % of residual activity was obtained at 45 and 55 °C, respectively (Fig 5).

Effect of pH on enzyme activity and stability
The activity profile of the A. flavus protease at different pH showed that the enzyme was active over a wide range of pH range 7-12, with maximum activity recorded between pH 8.0 -12.0 (Fig 6).
The pH stability profile also showed the considerable stability of the enzyme between pH range 7.0 to 10.0 and showed 73 and 62 % residual activity at pH 11 and 12, respectively.

Effect of metals ions and inhibitors on enzyme activity/stability
The effect of metal ions and inhibitors on enzyme activity showed that among all the metals ions tested, only Fe ++ , stimulated the activity by 1.74 fold, whereas, Hg ++ and Cu++ showed a complete inhibition of the enzyme activity     ( 7).Despite being a serine protease addition of Ca++ ion showed only 2 % loss of enzyme activity.
In order to determine the nature of the protease, enzyme activity was measured in the presence of different enzyme inhibitors (Fig 8).The enzyme was found to be strongly inhibited by PMSF indicating that the enzyme is a serine protease.The chelating agent (EDTA) moderately inhibited (22%) the enzyme activity.However, β-mercaptoethanol and urea enhanced the protease activity by 27 and 17 %, respectively.Cystein, On the other hand, showed slight inhibition (4 %) in protease stability.

Effect of oxidizing agents and surfactants on protease stability
As shown in Table 4 the enzyme was appreciably stable in the presence of non-ionic surfactants like Tween 80 and Triton X-100.The strong anionic surfactant, sodium dodecyl sulphate (SDS, 0.1 % w/v) caused a moderate inhibition (18 %) in enzyme activity.In addition, protease retained 48 and 100 % of its activity after incubation for 1 h at 30 °C in the presence of 3 % (w/v) sodium perborate and 10 % (v/v) hydrogen peroxide, respectively.

Effect of organic solvents on enzyme stability
It is clear from Table 5 that enzyme was remarkably stable with all the solvents used.The maximum enzyme activity was found in the presence of chloroform (105.3 %) followed by acetone (97.1 %) and butanol (96.2 %).However, other solvents showed moderate inhibition (about 20 %) in enzyme activity.

Discussion
Forty six fungal strains were screened for their alkaline protease production profile.Fungal strain Aspergillus flavus (NSD 08), was identified and selected for further studies.
The composition of substrate in the SSF and incubation time has a marked effect on the protease production.Our results revealed that a ratio of 1:1 (WB: CC) gave maximum production (815 U/gss) followed by the ratio 2:1 with enzyme units of 763 U/gss recorded after 48 h of incubation.Malathi and Chakraborty (1991) and Agrawal et al (2004) obtained maximum protease yield using wheat bran as substrate after 48 and 72h of incubation, with A. flavus and Penicilium sp, respectively.However, Sumantha et al (2005) reported production of a metalloprotease on mixed substrate after 48 h of incubation.
The moisture content of the fermentation medium is well known to have a profound effect on both, the fungal growth as well as the enzyme production under SSF.Since presence of water in the medium makes the nutrients more readily accessible.The effect of initial moisture content on protease production by A. flavus suggested an approximate 50 % of initial moisture content as optimum for protease production.Maximum protease production with 55 and 63 % initial moisture content have also been reported earlier by Penicillium LPB-9 (Germano et al, 2003) and A. flavus IMI 327634 (Malathi and Chakraborty, 1991), respectively.Protease yield was found to reduce sharply below or above optimum level, as at lower moisture content the substrate would dry out and inhibit the growth of the fungus, while higher level of moisture content might waterlog the substrate which in turn affects the O 2 diffusion resulting in lower level of protease production (Bogar et al, 2003).
We observed a gradual increase in the enzyme activity with increasing concentration of spores in the inoculum.An inoculum size of 1x10 8 spores/g substrate was found to be optimum (1621 U/gss), however, further increase in the inoculum size reduced the enzyme activity.
Conflicting reports are available on protease activity of fungi in relation to inoculum size.For R. oryzae NRRL 21498, 2x10 5 spores/g wheat bran has been reported as optimum spore concentration (Tunga et al, 1998), whereas, Agrawal et al (2005) reported 1.0x10 10 spores/g substrate as optimum.
It is well established that extracellular pH and temperature is important for cell growth and enzyme production (Kumar and Tagaki 1999).Our fungal isolate was capable of producing significant amount of enzyme in the temperature range 32-40 °C and pH range 8.5 to 10.0.Maximum protease production (p < 0.02) was obtained at 37 °C and pH 9.0 (1640 U/gss).Anandan et al (2007) also reported maximum protease production by A. tamarri at pH 9.0 but at temperature 30 °C, however, Negi and Banerjee (2006) found optimum protease production at temperature 37 °C and pH 7.0 for A. awamori.
Glucose has been universally suggested as the best carbon source (Mehrotra et al., 1999, Srinubabu et al, 2007) as it can be easily metabolized by the microorganisms (Ashour et al, 1996).Adequate amount of enzyme (2054 U/gss) was produced by the isolated strain with glucose, however, sorbitol was found to be best source and maximum yield of enzyme (2531 U/gss) was obtained at a concentration of 200 mg/gss, while the inhibitory effect at higher concentrations might be due to catabolite repression.
Out of the seven organic nitrogen sources investigated, only malt extract had an inductive effect and production increased by 23 % (2923 U/gss).Our results are in accordance with earlier reports where complex nitrogen sources induced protease production by different microbial species (Pandey et al, 2000, Prakasham, et al, 2006).
Few inorganic nitrogen sources were also incorporated in the fermentation medium and results revealed that diammonium hydrogen orthophosphate was the best with enzyme units 3640 U/gss, while urea was the poorest one and other sources did not have any significant effect.Our report is in accordance with Srinubabu et al. (2007) who also reported di-ammonium hydrogen orthophosphate for protease production by A. oryzae, whereas, Johnvesly and Naik (2001) observed nitrates as best nitrogen sources.
It is well known fact that the metal ions in the fermentation medium greatly influence the protease production by microbes (Varela et al. 1996).Results revealed that MnCl 2 stimulated the enzyme production (4091 U/gss), followed by FeSO 4 (3855 U/gss), whereas, CuSO 4, ZnSO 4 and CoCl 2 inhibited the production.CaCl 2, however , showed insignificant effect.In earlier studies different metals have been reported for protease production with different microbes (Abidi et al. 2008;Vijayanand et al. 2010).
The partially purified enzyme elaborated a 3 fold increase increase in specific activity.A 100 % retention of protease activity was recorded at 40 °C while 92, 74 and 41 % residual activities were recorded at 45, 50 and 55 °C, respectively.However, an increase in temperature beyond 55 °C resulted in the inactivation of enzyme.Wang et al (2005) and Tremacoldi et al (2007) also reported alkaline protease from Aspergillus sp.having optimum activity at 40 °C.
The thermal stability profile of the enzyme showed an initial retention of 96% activity after 1 h of incubation at 40 °C, whereas, 84 and 53 % of residual activities were scored at 45 and 55 °C, respectively.It also showed that protease from A. flavus was relatively thermostable when compared to other reports for the same organism (Tremacoldi et al 2007, Tunga et al, 2003).
The activity profile of the A. flavus protease at different pH showed that the enzyme was active over a wide range of pH 7-12, with maximum activity at pH 12.With regard to pH stability the enzyme was stable between pH range 7.0 to 10.0 and showed 73 and 62% residual activity at pH 11 and 12, respectively.These findings are in accordance with earlier studies (Miyaji et al, 2006), where the optimum pH for alkaline protease was 11-12, whereas, Devi et al (2008) and Charles et al (2008) reported pH optima 10.0 and 8.0, for A. niger and A. niduans HA01, respectively.
The effect of metal ions and inhibitors on enzyme activity showed that among all the metals ions tested, only Fe ++ , stimulated the activity by 1.74 fold, whereas, Hg ++ and Cu ++ showed a complete inhibition of the activity.Despite being a serine protease addition of Ca ++ ion showed 2% loss of enzyme activity, which reflected the independency of the enzyme on Ca ++ ion, as most of the serine proteases have Ca 2+ binding site(s) (Vielli and Zeikus, 2001).
The inhibition of the enzyme activity by serine protease inhibitor (PMSF) indicated that the enzyme was a serine protease.The enzyme was moderately inhibited by the chelating agent EDTA, with 78 % residual activity.However, β-mercaptoethanol and urea enhanced the protease activity by 27 and 17%, respectively, and cystein showed slight inhibition of activity.The high activity of enzyme in the presence of EDTA is very useful for application as detergent additive because chelating agents are components of most of the commonly used household detergents (Hajji et al, 2007).
A good detergent protease must be compatible and stable with all commonly used detergent compounds such as surfactants, bleaches, oxidizing agents and other additives, which could be present in the formulation (Gupta et al, 2002).The enzyme produced by the isolated strain was appreciably stable in the presence of non-ionic surfactants like Tween 80 and Triton X-100.The strong anionic surfactant (SDS) at 0.1 % (w/v) caused a moderate inhibition (18 %) in enzyme activity on the other hand Tremacoldi et al. (2007) reported that the protease from A. clavatus CCT2759 was strongly inhibited by SDS, Tween 80 and carbonate ion.The stability of the enzyme against SDS was lower than A. parasiticus protease which retained about 97% of its Yadav et al. 8639 initial activity after 1 h incubation with 2 % SDS at room temperature (Tunga et al. 2003).In addition, protease retained 100 and 48% of its activity after incubation for 1 h at 30 °C in the presence of 10 % (v/v) hydrogen peroxide and 3 % (w/v) sodium perborate, respectively.Mei and Jiang (2005) reported valuable stability with H 2 O 2 at concentration 1%, whereas, Joo et al (2003) reported 110% activity with 10% H 2 O 2 after 72 h.Most of the organic solvent stable proteases are reported from bacteria, particularly by Pseudomonads and Bacillus sp.(Gupta and Khare 2006;Fang et al. 2009).Though the alkaline protease produced by our strain is stable in presence of different organic solvents but not the strain, hence, it could be used as a biocatalyst for peptide synthesis in organic media.

Conclusion
The A. flavus serine protease in the present study retained its activity even in the presence of EDTA and was an oxidant stable as was evident by 100% retention of its activity in the presence of hydrogen peroxide, which makes its use ideal for detergent formulations.Agro industrial waste combinations such as wheat bran and corn cob could also be exploited as a cost effective substrate for pilot and large scale industrial applications.The application profile of this protease is extended by its stability in the presence of organic solvents, due to which it can potentially be used in organic synthesis.

Figure 1 .
Figure 1.Effect of ratio of wheat bran and corn cob on protease production.An average of three observations, bars indicate the standard error, p < 0.02.The p value refers to the comparison of protease activity at 1:1 ratio of WB to CC with other combinations.All the comparisons are statistically significant.

Figure 2 .
Figure 2. Effect of pH and temperature on alkaline protease production.An average of three observations, bars indicate the standard error, p < 0.02.The p value refers to comparison of protease activity at different pH and temperatures.All comparisons are statistically significant.

Figure 3 .
Figure 3.Effect of additional carbon sources on protease production.An average of three observations, bars indicate the standard error, p< 0.02.The p value refers to the comparison of protease activity at control (without any carbon source) with other carbon sources amended in the medium.All the comparisons are statistically significant.

Figure 4 .
Figure 4. Effect of metal salts on protease production.An average of three observations, bars indicate the standard error, p< 0.02.The p value refers to the comparison of protease activity at control with other metals added in the medium.All the comparisons are statistically significant.

Figure 5 .
Figure 5.Effect of temperature on enzyme activity and stability.An average of three observations, standard deviation for all values is < ±5 %.

Figure 6 .
Figure 6.Effect of pH on enzyme activity and stability.An average of three observations, standard deviation for all values is < ±5 %.

Figure 8 .
Figure 8.Effect of inhibitors on enzyme activity.An average of three observations, standard deviation for all values is < ±5 %.

Table 2 .
Effect of initial moisture content on protease production

Table 3 .
Effect of nitrogen sources on enzyme production by A. flavus

Organic nitrogen Mean a enzyme activity (Ugss 1 ) Inorganic nitrogen Mean a enzyme activity (U gss -1 )
c; a Average of the three independent experiments;

Table 4 .
Effect of detergents and oxidizing agents on enzyme activity.The P value refers to the comparison of protease activity of control residual activity after incubation with the reagents.The comparison is among the values within the column;C all the values in the column are statistically significant.
a Average of the three independent experiments; b

Table 5 .
Effect of organic solvents on enzyme stability the P value refers to the comparison of protease activity of control residual activity after incubation with the reagents.The comparison is among the values among the column; a Average of the three independent experiments; b c all the values in the column are statistically significant.