بهبود تولید پروتئاز کریزوباکتریوم ایندولوجنسیس BYK27 به منظور رنگ زدایی خون از لباس ها
محورهای موضوعی : زیست فناوری میکروبییاسمین بین آبادی 1 , ارسطو بدوئی-دلفارد 2 , عبدالحمید نمکی شوشتری 3
1 - کارشناس ارشد، گروه زیست شناسی، دانشکده علوم، دانشگاه شهید باهنر کرمان
2 - دانشیار، گروه زیست شناسی، دانشکده علوم، دانشگاه شهید باهنر کرمان
3 - دانشیار، گروه زیست شناسی، دانشکده علوم، دانشگاه شهید باهنر کرمان
کلید واژه: بهینه سازی, پروتئاز, کریزو باکتریوم ایندوجنس, حلال های آلی,
چکیده مقاله :
پروتئازها یکی از مهم ترین آنزیم های صنعتی هستند که حدود 60 درصد از فروش جهانی آنزیم ها را به خود اختصاص داده اند. پروتئاز خارج سلولی کاربردهای زیادی در صنایع مختلف مانند چرم سازی، دترجنت، آب میوه گیری، نانوایی و صنایع گوشت دارند. با این وجود، اصلی ترین چالش تولید صنعتی آنزیم ها، میزان تولید اندک شان است. این مطالعه با هدف بهینه سازی شرایط تولید آنزیم پروتئاز و ارزیابی کاربردشان در حذف لکه های خونی انجام شد. در ابتدا، نمونه های فاضلاب بر روی محیط اسکیم میلک آگار کشت داده شدند. جدایه ای که بیشترین هاله را در اطراف کلنی داشت، به منظورمطالعات بعدی انتخاب گردید. بهینه سازی عوامل موثر در تولید پروتئاز کریزو باکتریوم ایندوجنسیس BYK27 با روش تاگوچی انجام پذیرفت. همچنین قدرت پاک کنندگی پروتئاز نیز با تیمار پارچه های خونی با این آنزیم مورد بررسی قرار گرفت. پارامترهای بهینه برای تولید پروتئاز کریزو باکتریوم ایندوجنسیس BYK27، شامل گلوکز (1 درصد)، عصاره مخمر (0.06 درصد)، دمای 40 درجه سلیسیوس و 9 pH بود. میزان تولید آنزیم پروتئاز در شرایط بهینه U/ml 590 بود. این مقدار در مقایسه با محیط پایه معادل 63 درصد افزایش داشت. فعالیت پروتئازی و پایداری آنزیم توسط بتا-مرکاپتواتانل 50 درصد افزایش داشت. اما این فعالیت توسط DMF موجب مهار 88 درصدی آنزیم گردید. همچنین پروتئاز سویهBYK27 قادر بود پس از 20 دقیقه گرماگذاری، رنگ بری لکه‌های خونی را به طور کامل انجام دهد. نتایج این مطالعه نشان می دهد که پروتئاز تولید شده توسط سویه BYK27 قابلیت استفاده در بیوتکنولوژی به ویژه در صنایع شوینده و ساخت ترکیبات با ارزش را دارا می باشد.
Protease is one of the most important industrial enzymes occupying nearly 60% of global enzyme sales. Extracellular protease finds numerous applications in industrial processes like in leather tanning, detergents, dairy, brewery as well as meat tenderization industries. In spite of that, the low level of enzyme production is the main challenge of industrial production of enzyme. Therefore, optimization of industrial protease production and its application in blood de-staining were the aims of this study. The sewage samples were cultivated on the skim milk agar. BYK27 isolates with the highest clear halo around the colonies were selected for further studies. Optimization of parameters affecting protease production by Chryseobacterium indologenes BYK27 was studied by Taguchi approach. De-staining ability of protease was also investigated by de-colorization of bloody cotton cloth. The optimal factors for protease production by Ch. indologenes BYK27 were found to be the temperature of 40 ˚C, pH of 9.0, 0.06% yeast extract and 1% glucose supplements. Protease production under optimal condition was found to be 590 (U/ml) which was improved by 63%, as compared to the basal medium. The protease activity and stability were increased 50% by beta-mercaptoethanol but inhibited about 88% by DMF. In addition, BYK27 protease was able to completely de-stain blood after 20 min of incubation. The results of this study indicate that BYK27 protease has biotechnological potential, specifically in the detergent industry and provision of valuable compounds.
aeruginosa MCM B-327: enzyme production and its partial characterization. New Biotechnol.
2011; 28 (2), 173-181.
2. Haddar A, Sellami-Kamoun A, Fakhfakh-Zouari N, Hmidet N, Nasri M. Characterization of
detergent stable and feather degrading serine protease from Bacillus mojavensis A21. Biochem
Eng J. 2010; 51(1-2): 53-63.
3. Rao M, Tanksale A, Ghatge M, Deshpande, V. Molecular and biotechnological aspects of
microbial protease. Microbiol Mol Biol Rev. 1988; 62(3): 597-635.
4. Anbu P. Characterization of solvent stable extracellular protease from Bacillus koreensis
(BK-P21A). Int J Biological Macromolecules. 2013; 56: 162-168.
5. Ghafoor A, Hasnain Sh. Purification and characterization of an extracellular protease from
Bacillus subtilis EAG-2 strain isolated from ornamental plant nursery. Pol J Microbiol. 2010;
59(2): 107-112.
6. Gupta A, Khare SK. A protease stable in organic solvents from solvent tolerant strain of
Pseudomonas aeruginosa. Bioresour Technol. 2006; 97(15): 1788-1793.
7. Hajji M, Kanoun S, Nasri M, Gharsallah N. Purification and characterization of an alkaline
serine-protease produced by a new isolated Aspergillus clavatus ES1. Process Biochem. 2007;
42(5): 791-797.
8. Chaudhari P, Chaudhari B, Chincholkar S. Iron containing keratinolytic metallo-protease
produced by Chryseobacterium gleum. Process Biochem. 2013; 48(1): 144-151.
9. Lv LX, Sim MH, Li YD, Min J, Feng WH, Guan WJ, Li YQ. Production, characterization and
application of a keratinase from Chryseobacterium l99 sp. nov. Process Biochem. 2010; 45 (8):
1236-1244.
10. Wang S, Yang Ch, Liang T, Yen Y. Optimization of conditions for protease production by
Chryseobacterium taeanense TKU001. Bioresour Technol. 2008, 99(99): 3700-3707.
11. Riffel A, Brandelli A, Bellato MC, Souza, HMFG, Eberlin NM, Tavares CAF. Purification and
characterization of a keratinolytic metalloprotease from Chryseabacterium sp.Kr6. J Biotechnol.
2007; 128(3): 693-703.
12. Wang S, Hsu W, Liang T, Yen Y, Wang Ch. Purification and characterization of three novel
keratinolytic metalloprpteases produced by Chryseobacterium indologenes TKU014 in a shrimp
shell powder medium. Bioresour Technol. 2008; 99(13): 5679-5686.
13. Prakasham RS, Subba-Rao Ch, Sreenivas R, Rajesham S, Sarma PN. Optimization of alkaline
protease production by Bacillus sp. using Taguchi methodology. Appl Biochem Biotechnol.
2004; 120(2): 133-144.
14. Beinabadi Y, Namaki-Soushtari AH, Badoei-Dalfard A. Isolation and characterization of
protease producing Chryseobacterium indologenes strain BYK27 from Kerman, s dairy industry
sewage. J Cell Mol Res. 2016; 29: 33-47.
15. Badoei-Dalfard A, Karami Z. Screening and isolation of an organic solvent tolerant-protease
from Bacillus sp. JER02: Activity optimization by response surface methodology. J Mol
Catalysis B: Enzymatic. 2013; 89: 15-23.
16. Badoei-Dalfard A, Karami Z, Ravan H. Purification and characterization of a thermo- and
organic solvent-tolerant alkaline protease from Bacillus sp. JER02. Preparative Biochem
Biotechnol. 2015; 45: 128-143.
17. Meena P, Dutt Tripathi A, Srivastava SK, Jha, A. Utilization of agro-industrial waste (wheat
bran) for alkaline protease production by Pseudomonas aeruginosa in SSF using Taguchi
(DOE) methodology. Biocatal Agric Biotechnol. 2013; 2(3): 210-216.
18. Badoei-Dalfard A, Amiri-Bahrami M, Riahi-Madvar A, Karami Z, Ebrahimi M A. Isolation,
identification and characterization of organic solvent tolerant protease from Bacillus sp.
DAF-01. Biological J Microorganism. 2012; 1(2): 37-48.
19. Ward OP. Proteolytic enzymes. Comprehensive Biotechnol. 1985; 8: 789-818.
20. Sarkar PK, Cook PE, Owens JD. Bacillus fermentation of soybeans. World J
Microbiol Biotechnol. 1993; 9(3): 295-299.
21. Sinagh S, Sinagh S, Tripathi V, Grag S. Purification, characterization and secondary structure
elucidation of a detergent stable, halotolerant, thermoalkaline protease from Bacillus cereus
SIU1. Process Biochem. 2012; 47(10): 1479-1487.
22. Venil CK, Lakshmanaperumalsamy P. Taguchi experimental design for medium optimization
for enhanced protease production by Bacillus subtilis HB04. J Sci Technol. 2009; 2: 54-59.
23. Ghaemi Oskouie SF, Tabandeh F, Yakhchali B, Eftekhar F. Enhancement of alkaline protease
production by Bacillus clausii using Taguchi experimental design. Afr J Biotechnol. 2007; 6
(22): 2559-2564.
24. Raut GR, Chakraborty S, Chopade BA, Kokare CR. Isolation and characterization of organic
solvent stable protease from alkaliphilic marine Saccharopolyspora species. Ind J Geo-Mar Sci.
2013; 42 (1): 131-138.
25. Annamalai N, Veeramuthu Rajeswari M, Kumar Sahu S, Balasubramanian Th. Purification
and characterization of solvent stable, alkaline protease from Bacillus firmus CAS 7 by
microbial conversion of marine wastes and molecular mechanism underlying solvent stability.
Process Biochem. 2014; 49(6): 1012-1019.
26. Paul T, Das A, Mandal AK, Halder S, Jana A, Maity Ch, Kumar DasMohapatra, P, Pati RB,
Mondal CK. An efficient cloth cleaning properties of a crude keratinase combined with
detergent: towards industrial viewpoint. J Clean Prod. 2014; 66(1): 672-684.
27. Ubba-Rao Ch, Sathish T, Ravichandra P, Prakasham RS. Characterization of thermos- and
detergent stable serine protease from isolated Bacillus circulans and evaluation of eco-friendly
applications. Process Biochem. 2009; 44(3): 262-268.
_||_
aeruginosa MCM B-327: enzyme production and its partial characterization. New Biotechnol.
2011; 28 (2), 173-181.
2. Haddar A, Sellami-Kamoun A, Fakhfakh-Zouari N, Hmidet N, Nasri M. Characterization of
detergent stable and feather degrading serine protease from Bacillus mojavensis A21. Biochem
Eng J. 2010; 51(1-2): 53-63.
3. Rao M, Tanksale A, Ghatge M, Deshpande, V. Molecular and biotechnological aspects of
microbial protease. Microbiol Mol Biol Rev. 1988; 62(3): 597-635.
4. Anbu P. Characterization of solvent stable extracellular protease from Bacillus koreensis
(BK-P21A). Int J Biological Macromolecules. 2013; 56: 162-168.
5. Ghafoor A, Hasnain Sh. Purification and characterization of an extracellular protease from
Bacillus subtilis EAG-2 strain isolated from ornamental plant nursery. Pol J Microbiol. 2010;
59(2): 107-112.
6. Gupta A, Khare SK. A protease stable in organic solvents from solvent tolerant strain of
Pseudomonas aeruginosa. Bioresour Technol. 2006; 97(15): 1788-1793.
7. Hajji M, Kanoun S, Nasri M, Gharsallah N. Purification and characterization of an alkaline
serine-protease produced by a new isolated Aspergillus clavatus ES1. Process Biochem. 2007;
42(5): 791-797.
8. Chaudhari P, Chaudhari B, Chincholkar S. Iron containing keratinolytic metallo-protease
produced by Chryseobacterium gleum. Process Biochem. 2013; 48(1): 144-151.
9. Lv LX, Sim MH, Li YD, Min J, Feng WH, Guan WJ, Li YQ. Production, characterization and
application of a keratinase from Chryseobacterium l99 sp. nov. Process Biochem. 2010; 45 (8):
1236-1244.
10. Wang S, Yang Ch, Liang T, Yen Y. Optimization of conditions for protease production by
Chryseobacterium taeanense TKU001. Bioresour Technol. 2008, 99(99): 3700-3707.
11. Riffel A, Brandelli A, Bellato MC, Souza, HMFG, Eberlin NM, Tavares CAF. Purification and
characterization of a keratinolytic metalloprotease from Chryseabacterium sp.Kr6. J Biotechnol.
2007; 128(3): 693-703.
12. Wang S, Hsu W, Liang T, Yen Y, Wang Ch. Purification and characterization of three novel
keratinolytic metalloprpteases produced by Chryseobacterium indologenes TKU014 in a shrimp
shell powder medium. Bioresour Technol. 2008; 99(13): 5679-5686.
13. Prakasham RS, Subba-Rao Ch, Sreenivas R, Rajesham S, Sarma PN. Optimization of alkaline
protease production by Bacillus sp. using Taguchi methodology. Appl Biochem Biotechnol.
2004; 120(2): 133-144.
14. Beinabadi Y, Namaki-Soushtari AH, Badoei-Dalfard A. Isolation and characterization of
protease producing Chryseobacterium indologenes strain BYK27 from Kerman, s dairy industry
sewage. J Cell Mol Res. 2016; 29: 33-47.
15. Badoei-Dalfard A, Karami Z. Screening and isolation of an organic solvent tolerant-protease
from Bacillus sp. JER02: Activity optimization by response surface methodology. J Mol
Catalysis B: Enzymatic. 2013; 89: 15-23.
16. Badoei-Dalfard A, Karami Z, Ravan H. Purification and characterization of a thermo- and
organic solvent-tolerant alkaline protease from Bacillus sp. JER02. Preparative Biochem
Biotechnol. 2015; 45: 128-143.
17. Meena P, Dutt Tripathi A, Srivastava SK, Jha, A. Utilization of agro-industrial waste (wheat
bran) for alkaline protease production by Pseudomonas aeruginosa in SSF using Taguchi
(DOE) methodology. Biocatal Agric Biotechnol. 2013; 2(3): 210-216.
18. Badoei-Dalfard A, Amiri-Bahrami M, Riahi-Madvar A, Karami Z, Ebrahimi M A. Isolation,
identification and characterization of organic solvent tolerant protease from Bacillus sp.
DAF-01. Biological J Microorganism. 2012; 1(2): 37-48.
19. Ward OP. Proteolytic enzymes. Comprehensive Biotechnol. 1985; 8: 789-818.
20. Sarkar PK, Cook PE, Owens JD. Bacillus fermentation of soybeans. World J
Microbiol Biotechnol. 1993; 9(3): 295-299.
21. Sinagh S, Sinagh S, Tripathi V, Grag S. Purification, characterization and secondary structure
elucidation of a detergent stable, halotolerant, thermoalkaline protease from Bacillus cereus
SIU1. Process Biochem. 2012; 47(10): 1479-1487.
22. Venil CK, Lakshmanaperumalsamy P. Taguchi experimental design for medium optimization
for enhanced protease production by Bacillus subtilis HB04. J Sci Technol. 2009; 2: 54-59.
23. Ghaemi Oskouie SF, Tabandeh F, Yakhchali B, Eftekhar F. Enhancement of alkaline protease
production by Bacillus clausii using Taguchi experimental design. Afr J Biotechnol. 2007; 6
(22): 2559-2564.
24. Raut GR, Chakraborty S, Chopade BA, Kokare CR. Isolation and characterization of organic
solvent stable protease from alkaliphilic marine Saccharopolyspora species. Ind J Geo-Mar Sci.
2013; 42 (1): 131-138.
25. Annamalai N, Veeramuthu Rajeswari M, Kumar Sahu S, Balasubramanian Th. Purification
and characterization of solvent stable, alkaline protease from Bacillus firmus CAS 7 by
microbial conversion of marine wastes and molecular mechanism underlying solvent stability.
Process Biochem. 2014; 49(6): 1012-1019.
26. Paul T, Das A, Mandal AK, Halder S, Jana A, Maity Ch, Kumar DasMohapatra, P, Pati RB,
Mondal CK. An efficient cloth cleaning properties of a crude keratinase combined with
detergent: towards industrial viewpoint. J Clean Prod. 2014; 66(1): 672-684.
27. Ubba-Rao Ch, Sathish T, Ravichandra P, Prakasham RS. Characterization of thermos- and
detergent stable serine protease from isolated Bacillus circulans and evaluation of eco-friendly
applications. Process Biochem. 2009; 44(3): 262-268.
