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Journal of Applied Sciences Research, 8(2): 807-814, 2012
ISSN 1819-544X
This is a refereed journal and all articles are professionally screened and reviewed

Mutation induction and protoplast fusion of Streptomyces spp. for enhanced alkaline
protease production

Khattab A.A. and 2Mohamed S. Abdel-Aziz


Genetics and Cytology Department, National Research Center, Dokki, Cairo, Egypt.
Microbial Chemistry Department, National Research Center, Dokki, Cairo, Egypt.

Five isolates of Streptomyces sp. were evaluated for alkaline protease productivity. Results showed that
Streptomyces sp. C1 is the most effective strain for alkaline protease productivity (giving 223.08 U/ml). So,
Streptomyces sp. C1 was selected and mutagenized using UV as a physical mutagen in order to induce genetic
variations. Results indicated that, the majority of mutants exhibited alkaline protease productivity higher than
the wild type strain and positive correlation between alkaline protease productivity and specific activity of the
most mutants. The highest record of the alkaline protease production was 337.97 U/ml which was obtained from
the mutant No.12. Protoplast fusion technique was carried out in order to obtain fusants with high alkaline
protease production. Results showed that, 18 out of the 22 fusants were higher alkaline protease producers than
the higher parent (P1). The highest fusants F2 and F18 produced 111.85 and 102.76 percents more alkaline
protease than its higher producer parent, respectively, and at the same time represents 58.17 and 51.38 percents
more alkaline protease than its summation of two parents.
Key words: Streptomyces, identification, alkaline protease, UV-mutagenesis, protoplast fusion.
Proteases are among the most important industrial enzymes, accounting for nearly 60% of the industrial
market in the world (Mitra et al., 1996; Layman, 1986). Alkaline proteases are of great interest because of their
high proteolytic activity and stability under alkaline conditions (Maurer, 2004; Saeki et al., 2007). These
enzymes find applications in detergents, feather processes, food processing, silk gumming, pharmaceuticals,
bioremediation, biosynthesis and biotransformation (Bhaskar et al., 2007; Jellouli et al., 2009). Furthermore,
fibrous proteins such as horn, feather, nail and hair are abundantly available in nature as waste. This waste can
be converted into useful biomass, protein concentrate or amino acids using proteases from certain microorganisms. The majority of commercial alkaline proteases are produced by bacteria, especially Bacillus sp. and
Streptomycse (Jellouli et al., 2009).
Strain improvement plays a key role in the commercial development of microbial fermentation processes.
As a rule, the wild strains usually produce limited quantities of the desired enzyme to be useful for commercial
application (Glazer and Nikaido, 1995). However, in most cases, by adopting simple selection methods, such as
spreading of the culture on specific media, it is possible to pick colonies that show a substantial increase in yield
(Aunstrup, 1974). The yield can be further improved by the use of mutagens and the adoption of special
techniques or procedures for detecting useful mutants. Shah et al., (1986) developed a cysteine auxotrophic
mutant of B. licheniformis with improved protease production. Further, increased yields of alkaline proteases
have been achieved by Bacillus mutants that were resistant to some antibiotics (Ito et al., 1991). Moreover, the
mutant of different species of Streptomyces led to produce highly enzyme activity (Catherine et al., 1992;
Michael et al., 2006 and Bushell et al., 2006). Moreover, the genetic manipulation via protoplast fusion protocol
is considered as a more recent method to promote recombinations. Furthermore, protoplast fusion led to produce
superior enzyme production fusants from Streptomyces (Teeradakorn et al., 1998 and Sivakumar et al., 2004).
In the present work we demonstrated that by induction of mutation using ultraviolet rays and protoplast fusion
we can continuously construct superior strains for alkaline protease productivity.
Materials and methods
Isolation of actinomycetes:
The sediment sample was collected from the Egyptian agriculture soil, Mansoura governate, by inserting a
sterilized polyvinyl corer (10 cm) into the sediments. The centre portion of the 2 cm sediment sample was taken
out with the help of a sterile spatula. The collected sample was transferred to a sterile polythene bag and taken
Corresponding Author: Khattab A.A., Genetics and cytology Department, National Research Center, Dokki, Cairo, Egypt.

The cells were grown in 50 ml of screening medium containing 0.5ml of Folin-Ciocalteu's phenol solution and kept for 30 min at room temperature. pH 7.5ml of 0. Na2CO3. Then the cells were harvested by centrifugation at 8000 rpm for 10 min and transferred to a protoplasting medium(PM) consisting of (g/liter):sucrose. Cells were subjected to hydrolysis and tested for both diaminopimelic (DAP) acid and whole cell sugar pattern (Hasegawa et al. 15-20 and distilled water.25).5M Na2CO3 and 0.. Na2CO3 and the other constituents were sterilized separately and mixed just before solidification. The medium showed a final pH of 9. all agar plates were supplemented with 20 mg/l of nystatin and cycloheximide (100 mg/l). All the colonies that were growing on the Petri plates were separately streaked in Petri plates. the treated spore suspensions were protected from light for two hours. 10-fold serial dilutions of the sediment samples were prepared.85% sterile saline solution (NaCl). After incubation for 1 hour at 50oC. Cells were then harvested by filtration. 2005). 1983). using filtered and sterilized 0. The actinomycete colonies that appeared on the Petri plates were counted from 5th day onwards. The assay was carried out routinely in a mixture containing 0. Serially diluted samples were plated in the Actinomycetes isolation agar medium in duplicate Petri plates (Starch nitrate agar plates). Skim milk. Protein was tested according to Lowry et al. 2.44 M TCA (trichloroacetic acid) solution. The developed color of the solutions were determined with respect to sample blanks at 660 nm. 3. After observation of protoplast formation   .5. 10. washed with water and ethyl alcohol and then dried in an open air at room temperature.5.0.5 ml of a suitably diluted enzyme solution and 2. 1982) was used to detect alkaline protease-producing isolates. After irradiation. yeast extract (5) and 50ml salt solution {KH2PO4 (0. MgCI2.5 ml of supernatant was mixed with 2. respectively (Kathiresan et al.5ml of 0. This medium had the following constituents (g/l): skim milk. 8(2): 807-814. UV-mutagenesis: Streptomyces spores from old slants (5 days ) were suspended in sterile distilled water and exposed to UVlight (Philips T – UV – 30 W Lamp type number 57413 p /40 ) for 0 .5 ml casein solution. 6 and 9 minutes at a distance of 20 cm.808 J. 3 .5% glycine in a 100-ml Erlenmeyer flask at 30°C for 24 h. At the end of fermentation period. Identification of the genus Streptomyces: The identification was carried out according to Lechevalier and Lechevalier (1970) by cultivating the tested microorganisms on tryptone yeast extract broth at 30oC for 5-10 days.6H20. the reaction was terminated by the addition of 2.. An aliquot of 0. Qualitative screening of alkaliphilc isolates for alkaline protease production: Skim milk agar medium described by (Ventosa et al.nitrate medium and incubated at 28 ºC for five days. 1992). The growing colonies were transplanted on slants for the forward assay of alkaline protease productivities. glucose (10).1). Alkaline protease production medium: The medium composed of (%): casein (5). MgSO4 (0. Res. Agar. (1951). the sample was air-dried aseptically for one week. 300.0. After cooling the medium was inoculated with a loopfull of Streptomyces strains and then incubated at 30oC in an orbital shaker set at 150 rpm for 4 days. 50. The pH was adjusted to be 9.5 and was sterilized in an autoclave for 15 min at 121oC. After 10min the mixture was centrifuged at 8000 rpm for 10min. Protoplast preparation. FeSO4 (0. An air-dried sediment sample was incubated at 30°C for 5 min (Balagurunathan.1)} in 1000 distilled water.. To minimize bacterial and fungal contamination.0.. yeast extract. Sci. Appl. After arrival at the laboratory.HCl(buffer.25 M TES.K2HPO4 (0. containing 10 mg /ml of lysozyme (grade 1. Then. fusion and regeneration: Different superior isolates and original strains were tested against six antibacterial agents and their responses were recorded and used as markers for fusants detection after protoplast fusion. Appropriate dilutions were spread on starch. peptone (5). 5. subcultured. Sigma Chemical Co. up to 21th day. 2012 immediately to the laboratory.0. CaCI2.). the culture was centrifuged and the supernatant was assayed for protease activity according the method of Kathiresan and Manivannan (2007).2).0. Alkaline protease assay and protein measurement: The activity of alkaline protease in the cell-free supernatant was measured by modified method of Takami et al. (1989) using casein as a substrate at a concentration of 1% w/v in 50 mM Glycine-NaOH buffer (pH 10).25). 1000ml. 0. ensured for their phenotypes and maintained in starch nitrate slants.2H2O.

Alkaline protease was the most preferable type of proteases as it is used as detergent additive. classified belong to the genus Streptomyces.97 U/ml which was obtained from the mutant No. four mutants. Results and discussion Protease was considered as industrially important enzyme with a wide range of applications in pharmaceutical. 15% dimethyl sulfoxide (DMSO). 43 and 48 proved to have a little bit more efficiency alkaline protease productivity than the original strain. 2012 with a phase contrast microscope. and 10 mM CaCl2. Isolates that showed positive results were screened quantitatively using broth medium containing casein. On the other hand. The protoplast suspension was diluted and plated and the plates were incubated for 5 to 8 days at 30°C. 2006.e. Spores suspension of Streptomyces sp C1was exposed to UV light as mentioned previously in the adopted methods. 2007. (1965) and Lechevalier and Lechevalier (1970). Results presented in Table (2) clearly showed that. The following superior mutant for alkaline protease   C1 87A B2 83A 37 Fig. Identification of selected isolates: The whole cell hydrolysates of the tested isolates were examined to determine the isomers of diaminopimelic acid (DAP) and sugar pattern of isolates following the methods of Becker et al. After gentle shaking for 30 min at 0°C. Rao and Narasu. 2007). The chromatographic analysis of the cell wall hydrolysates revealed that the all tested isolates contain LL-DAP and no characteristic sugars and were. therefore. Isolate C1 overproduce alkaline protease than the others at pH 9 as shown in Table(1). The highest record of the alkaline protease production was 337.The growing recombinant colonies (fusants) were transplanted on slants for the forward assay of alkaline protease productivities. Alkaline protease productivity after UV-treatments: The physical mutagen ultraviolet light (UV) was used for the induction of genetic variabilities. 8(2): 807-814..12. In our search for enhancement of alkaline protease. Several investigators used different screening plate agar media in their search for alkaline proteases (Aftab et al. Meanwhile. peptone and yeast extract as nitrogen source and the enzyme was assayed at different pHs.809 J. i. 9.   . food.. only 7 mutants produced alkaline protease lower than their wild type strain. out of selected 24 mutants which was exposed to UV-light for different time periods. protoplasts were fused by suspension in 10 ml of PM containing 30% PEG 4000. Sci. five Streptomyces strains isolated from soil were selected as they were able to produce alkaline protease on skim milk agar plate screening medium as shown in Figure(1). laundry. Res. 1: Photographs of wild types strains of Streptomyces produced alkaline protease as a clear area around the growth area on the skim milk agar medium. 13 mutants of the tested 24 ones produced alkaline protease higher than their original parental strain. Appl.. Kasana and Yadav. 27. Protoplasts were harvested by centrifugation at 2500 rpm for 5 min at 4°C and then resuspended in 10 ml of regeneration medium. the suspension was diluted 10-fold with PM buffer. leather. waste processing as well as in textile industry.

mutant No.98 192.794 303.69 3.637 283.11 4.46 101. data in Table (2) showed that positive correlation between alkaline protease productivity and specific activity of the most mutants.13 119.04 3.treated mutants were selected to determine their antibiotic resistance or sensitivity as an additional selective marker for fusant(s) detection and isolation.50 Specific activity U/mg protein 33.64 25. the other wild type strains showed different responses.39 120.99 77.171 248.77 111.25 6 44 77.42 3.92 38.144 266.04 83. the strain 87A was resistant to Nb but it was sensitive to the others. 100.07 83A 4.32 114. it was sensitive to the others.745 193.22 4. Nb and Lm while. exposure time.26 4.159 337.94 4.60 4.77 151.19 130.36 4. three original strains and three UV.93 3.893 236.989 235.70 86.63 19.810 J.81 3. exposure time.57 12 10 51.T.. Finally.57 9 28 65.94 percent higher than the original strain.074 164. To study the effect of protoplast fusion on alkaline protease productivity.69 3. C1.074 267. cross between mutant No.00 93.18 6 23 57. Res.67 192. UV-exposure Productivity Protein Specific activity time(min.99 9 36 64.e.94 127. we can fuse different strains in the protoplast fusion experiment.90 4. In order to investigate the effect of protoplast fusion on alkaline protease production.016 290.57 130.16 3. Moreover.242 291.58 9 46 72.31 38.773 248.18 4.08 53.22 pH10 Activity (U/ml) 132.26 9 43 79.21   .) (U/ml) (mg/ml) (U/mg protein) 55. From these antibiotic responses differences.37 107. it could be clearly concluded that the highest promising mutants for alkaline protease production counted as the follows: i) One mutant as a result of 12 min.31 9 40 59.08 55. Protoplast fusion and alkaline protease productivity: Protoplast fusion was the main method for exchanging genetic material between two cell types and subsequently obtaining the new gene recombinants towards the isolation of higher alkaline protease producing fusant(s). Results showed that.04 3.931 208. ii) One mutant as a result of 6 min.031 208.92 35.73 3.110 238.938 227.097 272.61 B2 4.92 3.53 4. Table 2: Alkaline protease production using UV.019 185.31 9 29 51. 87A which varied in both their productivity and antibiotic responses was done.05 192.90 12 49 66.92 192.45 47.83 9 47 58.57 12 11 81.97 12 12 40.07 118.31 9 42 60.38 223..17 194.50 percent higher than the original strain.68 105.31 93.049 165. i. The strain B2 was resistant to Ap.08 0 Wild type 53.44 which produced 35.42 4.42 47.71 74.43 9 18 40.125 259. 8(2): 807-814. Tc and Em.12 which produced 51.31 105.31 113.46 139.63 216.85 6 48 65.60 6 30 62.59 9 20 69.42 26.04 191.51 116.04 109.92 47.mutants obtained from Streptomyces strain No.91 135.75 4.40 6 45 46.73 4.935 273.50 73.e. Out of the previous obtained results of Streptomyces sp C1exposed to different UV-exposure times.78 4.12 and wild strain No.64 6 31 65.14 production was No. they were sensitive to Nb.44 which produced 303. Sci. Km.83 34.91 86. (mg/ml) U/mg U/mg protein protein 37 3. Appl. 2012 Table 1: Alkaline protease activity from positive Streptomyces isolates at different incubation pHs.34 6 27 50.29 122.023 263. pH 8 pH 9 Total Activity Specific Activity Specific Streptomyces isolate protein (U/ml) activity (U/ml) activity no. the strain 83A was resistant to Ap but it was sensitive to the others.04 223.45 48.25 U/ml. i.26 4.98 12 1 59.71 26.98 12 50 Productivity (%) to W.51 12 9 68.38 111. mutant No. the original strain and all UV-mutants were resistant to Ap and Lm.868 193. Also. Meanwhile. On the other hand. Table (4) presents the alkaline protease productivity of the parents and the protoplast fusants which were obtained following the protoplast fusion cross and detection on the regeneration medium containing 50µg/ml of Lincomycin and 20 µg/ml of Novobiocin.35 C1 4..97 173.30 122. Table (3) presents the selected strains and mutants response to six different types of antibiotics.58 4. Mutant No.82 87A 4.

deletion etc.59 86.62 145. 13 fusants of the tested 22 ones produced alkaline protease higher than its summation of two parents.15 64.54 173..76 Productivity (%) to (P1+ P2) 74. Km(Kanamycin): 50µg/ml.12 4. For many reasons.40 F3.23 178.00 P2(87A) 4. 18 out of the 22 fusants were higher alkaline protease producers than the higher parent (P1).04 F3.38 95. The efficiency of induced mutation depends on the type of damage (base pair substitution.76 percents more alkaline protease than its higher producer parent.06 120.08 160.69 F8 4.17 and 51.23 107. UV-light is safety usage and causing no pollution.86 F3 4.05 103..4.17 200. For instance. Lm(Lincomycin): 50µg/ml.83 150.53 603.66 370.16 F3.84 147.9 5. Parents &Fusant No.89 486. Sci.24 81.55 F16 4.94 F1 4.38 percents more alkaline protease than its summation of two parents.66 25.27 130.02 62.49 138.92 F3. UV-light also induced tolerance to different environmental stresses and changes in protein synthesis (Hartke et al. F3 and F17 showed less alkaline protease production than the higher parent (P1) but these fusants exhibited higher alkaline protease production than the lower parent (P2).1 4. Tc(tetracycline): 50µg/ml. Michael et al. Furthermore.97 F2.69 F17 3.47 F2. only four fusants. Using UVlight as a mutation-inducer has been recommended as the method of choice for increasing the enzyme productivity from Streptomyces (Catherine et al..67 F2 4.77 108. The results obtained after UV-mutagenesis were in agreement with those obtained by Bushell et al.23 124.85 and 102.5 5. In addition.65 82.61 377.13 393. Res. Mutation is the ultimate source of all genetic variation. Nb(Novobiocin): 20 µg/ml.32 89.17 81.47 679.60 F2.52 58.7.49 109.47 81.01 293.52 133.2 5.74 542.84 66.29 599. Em(Erythromycin):100 µg/ml.10 5. 2012 Table 3: Response of the superior mutants for alkaline protease production against five antibacterial agents. Isolates that showed positive results were screened at different pHs to detect the best isolate for enzyme production.7 5. Appl.04 202.47 64.13 337.93 F2.42 97.51 145.13 544. 1992.15 55.15 3.8 4.50 109.49 546.75 F2.46 121.44 F2.56 674.53 115. F2. 2006).05 211.90 120. Protein Productivity Specific activity Productivity (%) to (mg/ml) (U/ml) (U/mg protein) parent(P1) P1(12) 4. respectively.) a given mutagen causes on DNA and the cellular mechanism(s) involved in the repair of that damage.71 90. 9 fusants of the tested 22 ones produced alkaline protease lower than its summation of two parents.03 586. Meanwhile.e.11 4.39 40.24 151. on skim milk agar plate screening medium.85 468.85 F2.27 685. In our search for alkaline protease five Streptomyces strains isolated from soil were selected as they were able to produce alkaline protease.4 5.74 715.811 J. 8(2): 807-814.13 4.6 5.97 81.38 72.34 149.26 106.68 368.28 327.62 119. Results in this table showed that. i.88 111. (2006) who obtained a zwf mutant   . it appeared that UV-induced mutants were more stable through long term of generation and subculturing (Thoma. Moreover.25 116.87 83..00 61.30 182.45 143.72 129.30 496.09 161.68 33.47 F4 4.43 402.20 53. while.69 133.36 177.56 86.71 161.3 4.99 151.54 F3.71 28.82 88.76 299.93 96. F2. Antibacterial agents Wild types* &Mutant strains Ap Nb Km Tc Lm Em Original strain* + + 12 + + 44 + + 45 + + B2* + + + 83A* + 87A* + Ap(Ampicillin): 125µg/ ml.57 F3..90 F18 5.47 146.. the highest fusants F2 and F18 produced 111.38 Discussion: The importance of the isolation of new strains of Streptomyces from the nature become a global programs to produce new type of alkaline protease with high activity.99 F3.00 96.91 119.83 100. 1971). and at the same time represents 58.68 113. 1995).00 114.33 132.99 199. Before the advent of protoplast fusion technology.08 158. random mutagenesis with UV-rays and screening was the main technique applied for strain improvement. insertion. Table 4: Alkaline protease production using fusants obtained after protoplast fusion crosses.

Appl.. the highest fusants F2 and F18 produced 111.N. BTS-24 for enhanced lipase productivity. Industrial Aspects of Biochemistry. In addition. Razoo.A. The results obtained confirmed that UV-mutagenesis technique is an important tool in Streptomyces improvement for increasing production of alkaline protease enzyme. Technol. N. the fusants appeared similar to one of the parental strains. Screening. Lechevalier and H. (1990) isolated fusants which exhibited recombination between a range of chromosomal marker after intraspecific protoplast fusion between isogenic Lactobacillus plantarum strains. editor.. Ellaiah and T. And also. (1998) who isolated new fusants. Moreover. S. protoplast fusion is used to combine genes from different isolates for creating strains with high activity and productivity of alkaline protease. North HollandAmerican Amsterdam: Elsevier. 1965.P. India. Res. Biol.. 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