Enhancement of protease production by Bacillus sp. and Micrococcus varians induced by UV-mutagenesis

Microbial proteases contribute nearly 40% of the total worldwide enzyme market. Hence, with the view of this significance, the main objective of the present study was to enhance protease production of two bacterial strains, Bcillus sp. and Micrococcus varians using UV mutagenesis. Induction of mutation in both strains was carried out at different exposure times: 0, 3, 6, 9, 12, 15, 18 and 21 min at a distance of 10 between UV source and treated bacteria.Two best protease producer mutants for the two bacterial strains (UV-9 for Bcillus sp.and UV-18 forMicrococcus varians) were selected based on the clearance zone diameter of mutant colonies on 1% skimmed milk agar plates. UV-9 mutant showed 1.4 fold higher protease activity than the wild type in solid and liquid medium. However UV-18 mutant was found to produce 2.5 fold increases over the wild type on agar plates and 2.1 fold enhancement in liquid-medium assay.The two mutants were very effective in feather keratin-degrading in less than two days, UV- 18 was more efficient than UV-9.


INTRODUCTION
Proteases are group of enzymes which catalyze hydrolysis of peptide bonds in proteins. They are also called as peptidases or proteinases or proteolytic enzymes (Raoet al., 1998).Among the various proteases, bacterial proteases are the most significant,compared with animal and fungal proteases and among bacteria, Bacillus sp. are the most important producers of extra-cellular proteases (Boominadhanet al., 2009). Proteases are among the most important industrial enzymes due to their biotechnological interests. They account for about 60% of the total worldwide sale of enzymes (Reddy et al., 2008), and are widely used in several industries that include detergent, food, pharmaceutical, leather, diagnostics,meat processing, waste management and silver recovery (Gupta et al., 2002;Chellappanet al., 2006). These enzymes also have potential to contribute in the development of high value added products due to their characteristic nature of aided digestion (Glazer and Nikaido, 1995). Due to their increased economic importance, research is being carried out throughout the world to isolate hyperactive strains for the production of proteases (Gupta et al., 2002). Microbial strain improvement plays a key role in the commercial development of microbial fermentation processes. As a role, the wild strains usually produce limited quantities of the desired enzyme to be useful for commercial application (Glazer and Nikaido, 1995). Mutation induction and/or selection techniques, together with cloning and protein engineering strategies have been exploited to develop enzyme production (Schallmeyet al., 2004). Ultraviolet radiation is one of the well-known and most commonly used mutagen and it is also very easy to take effective safety precautions against it. It gives a high proposition of pyrimidine dimmers and includes all types of base pair substitutions (Javedet al., 2013). The present study highlights a possible enhancement of extracellular protease production from two bacterial strains, Bcillus sp. and Micrococcus varians via UV-mutagenesis.

II. MATERIAL AND METHODS Bacterial strains used
Two bacterial strains Bacillus sp. and Micrococcus varians-producing protease were employed. Bacillus sp. was isolated from compost, whereas Micrococcus varians was isolated from soil. They were selected because of their high proteolytic activities.

Qualitative assay of proteolytic activity
The ability to produce protease enzyme was checked by transferring a single isolated colonies of both bacterial strains (wild and mutants) on 1% of skimmed milk agar plates. Plates were incubated at 37° C for 24h. The diameter (in mm) of the clear hydrolysed zone around each bacterial colony (X) was divided by the diameter of the same colony (Y). The ratio (X/Y) was taken as an indication of protease activity.
Quantitative assay of proteolytic activity Protease activity was assayed by measuring the tyrosine released in culture supernatant from the action of protease on casein substrate bya modified Anson's method (Yang and Huang, 1994).The cell free supernatant of overnight cultures was used for protease assay.The reaction mixture contains 1 ml of enzyme was added to 1 ml of casein solution (1% w/v in 50 Mm potassium phosphate buffer, pH 7.5) and the mixturewas incubated for 10 min at 37ºC. The reaction was terminatedby adding 2 ml of 10% trichloroacetic acid reagent, keptfor 30 min incubation at room temperature and then centrifugedfor 15 min at 10,000 rpm. Then 2 ml of filtrate was mixed with3 ml of 500 mM sodium carbonate solution and absorbance was measured at 280 nm. One unit of enzyme activity is definedas the amount of enzyme required to liberate 1 µmol of tyrosineper min under the defined assay conditions. Enzyme units were measured using tyrosine (0-100µmole) as standard.

Preparation of cell suspension
Cell suspension was prepared by transferring colonies from 24h Luria-Bertani Agar culture of both strains into a 100ml-Erlenmeyer flasks containing 20ml of LB broth under aseptic conditions. Flasks were placed in a shaker incubator at 37ºC, 160 rpm for 24 h. after reaching an optical density of about 1,5 at 600 nm (corresponding to approximately 10 9 -10 10 UFC/ml), it was used as source of cell suspension for irradiation.

UV mutagenesis
Ultraviolet (UV) irradiation as a physical mutagenic agent was used to select mutants which produce more protease than their parent strain. Mutagenesis was carried out according to Justin et al., (2001) using different exposure times. 5ml of bacterial suspensions prepared previously were placed into 10-cm diameter-petri dishes at a distance of 10 cm from the UV lamp (30-W germicidal lamp, 2540-2550Å) and exposed to UV radiation for 0, 3, 6, 9, 12, 15, 18 and 21 min. Portions of 0.5 ml of suitable dilutions of bacterial suspensionsstrains were spread on five LBplates and incubated at 37ºC for 24 hr.Colonies developed after incubation werecounted and transplanted onto slants forfurther studies. The survival percentage was estimated for each treatment.

Screening of higher-proteolytic mutants
Plates having between 0.1 and10 % of survival rate were selected for isolation of mutants (Hopwood et al., 1985). The isolates were selected on the basis of macroscopic differential characteristics. According to Solaimanet al., (2005), for isolation of high protease producing mutants after UV irradiation, developed colonies inoculated into skim milk agar medium and incubated at 37ºC for 24h. Depending on the zone of clearance, mutants of the two bacterial strains exhibiting maximum zone of hydrolysis as compared to the wild type were selected.

Feather-degrading capacity of wild and mutant isolates
The wild type and the best mutant of Bacillus sp. and Micrococcus varianswere tested for their ability to degrade feather by culturing both of them in modified basal medium II supplemented with 1% of chicken feather. Chicken feathers collected (medium size white hens) were chopped to small fragments, washed with distilled water and dried overnight at 60ºC (Bernhardt et al., 1978;Johnvelsy, 2002). Cultures were incubated for 3 days at 37ºCwith shaking at 160 rpm. The featherdegrading capacity was assessed according to the physical appearance of feather pieces observed by naked eyes. The bacterial strain with high keratinolytic activity is the strain that degrade feather-keratin in shorter time.

III. RESULTS AND DISCUSSION UV mutagenesis
The cost of enzymes in a bioprocess can be reduced by introducing hyper-productive strains after suitable mutagenic treatments. Results in Table 1 and 2 showed that thesurvival percentages for both isolatesdecreased by increasing the time ofexposure. The percentage of survivals has been sharply decreased from 100% to 15.07 and 35.62 % after 3min of UV treatment for Bacillus sp. and Micrococcus variansrespectively.Thenitreaches 0.001 and 0.02 % after 20 minof UV treatment for Bacillus and Micrococcus respectively. This is maybe explained by the short distance from the UV lamp (10 cm). Similar trend of decrease in survivability with increase in exposure time has also been reported by Solaimanet al., (2005) in which the distance from the UV lamp was 10 cm and the percentage of survivals was 0.12% after 10 min of UV treatment.However higher percentage of survivals has been reported by other investigations where the distance was 20 cm(Javedet al., 2013; Karn and Karn, 2014). After UV treatment plates having survival rate between 0.1 and 10 % corresponding to exposure time of 6, 9, 12 min for Bacillus and 9, 12, 15 for Micrococcus, were selected for isolation and screening of overproducing mutants.
Based onmorphology and colour differencesbetween colonies, 27 and 18 mutants for Bacillus and Micrococcus respectively were selected and transferred to skimmed milk agar plates to test proteolyticcapacities. In case of Bacillussp. and depending on their proteolytic activity (X/Y) only four UV-mutants (9, 12, 16 and 21) did exhibit higherproteolytic activity compared to the wild type

International Journal of Environment, Agriculture and Biotechnology (IJEAB)
Vol -2, Issue-5, Sep-Oct-2017  http://dx.doi.org/10.22161/ijeab/2.5.10  ISSN: 2456-1878 www.ijeab.com Page | 2350 (Table 3). Among the four mutants the most efficient strain (UV-9) was selected for further studies.Bacillussp. mutant 9(UV-9) showed 1.4 fold higher protease activity than the wild type.Similar fold increase in protease production was obtained by Nadeemet al., (2010). Concerning Micrococcusvariansresults showed that the majority of mutants were efficient in protease production. The superior protease producing mutants were 2, 8, 13, 16, 17 and 18. The X/Y values ranged from 03 to 08 for the mutant 18, so the improvement of Micrococcuswas better than that of Bacillus( Micrococcus variansmutantsshowed variable responses to UV radiation for protease production. These variations are more probably due to the differences induced in their genetic background.

Protease activity of the selected mutants in submerged culture
The best protease producing mutants UV-9 and UV-18 were further evaluated trough shack flask enzyme production studies over their wild strains. Results obtained proved that there is correlation between hydrolysis zone diameter and the ability to produce protease enzyme for Bacillus sp.and UV-9 mutant. UV-9 mutant produced almost 1.4 the yield of the wild strain.
On the other hand, in the case of Micrococcusvariansand its mutant UV-18 therewas no correlation between the clearance zone on plates and proteolytic activity in liquid assay (2.5 fold increase in solid assay whereas it was 2.1fold increase in liquid assay) ( Table 5). Similar results were found by Solaimanet al., (2005), where some potent mutants had great in protease production in plates and were not able give any proteolytic activity in shack flask.

Feather-degrading capacity of selected mutants
The two UV mutants, UV-9 and UV-18 were used for testing their ability in feather degrading. The results showed that both mutants were able to degrade feather over their wild type. The mutants grew and produced protease using chicken feather as a source of carbon, energy and nitrogen. UV-18 was more effective in keratin-degrading than UV-9 and chicken feather completely disappearedin less than two days using UV-18 mutant (Figure 1). The ability of microorganism to grow and produce appreciable levels of protease using several wastes could offer tremendous potential for development of biological methods for the hydrolysis of such products. The use of these natural residues, especially in countries where they are generated in abundance, could results in a sustainable reduction in cost of enzyme production (Wang et al., 2008).

IV. CONCLUSION
The results of the present investigation revealed that among different UV-mutants of Bacillus sp.and Micrococcusvarians UV-9 and UV-18 were selected as higher-proteolytic mutants. UV-9 and UV-18 mutants were able to increase protease production in plates and in liquid assay reaching 2.5 fold higher productions than the wild type.Hence these mutants were very effective in feather keratin degrading in two days presenting a potential use in keratin recycling and can result in a sustainable reduction in the cost of enzyme production.