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The objective of the present study was to investigate the potential application of alkaline protease as a laundry additive in detergent formulations. It is widely preferred over the conventional synthetic detergents for their better cleaning properties in addition to various other applications. A microorganism PP16 which produces protease was isolated from the gut of Portunus pelagicus and it was identified as Bacillus subtilis based on 16S rRNA sequencingEnzyme yield was maximized by optimizing the composition of a culture medium. The optimum fermentation conditions for maximum protease production was 254.98 U/ml at 42°C. Fructose was a good carbon source while casein was utilized as a good nitrogen source for enzyme production. The partially purified enzyme was shown to have a relative molecular weight of 75 kDa. Crude protease enzyme extract activity against hydrolysis of stains like chocolate and grass were examined by enzyme compatibility analysis and wash performance test. Protease showed its stability at 40–50°C with most of the detergents, which enabled it as a choice for application in detergent industries.

Bacillus subtilis, protease, SDS-PAGE, enzyme compatibility, detergent, laundry additive.

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Zhao XQ. Genome-based studies of marine microorganisms to maximize the diversity of natural products discovery for medical treatments. Evid. Based Complement. Alternat. Med. 2011;1–11.

Zhang C, Kim SK. Research and application of marine microbial enzymes: Status and prospects. Marine Drugs. 2010;8(6):1920–1934.

Ilona K, Zdzislaw ES. Neutral and alkaline muscle proteases of marine fish and invertebrates a review. J. Food Biochem. 2007; 20:349–364.

Godfrey T, West S. Industrial Enzymology, 2nd ed., New York: Macmillan Publishers Inc.; 1996.

Geethanjali S, Subash A. Optimization of protease production by Bacillus subtilis isolated from mid gut of fresh water fish Labeo rohita. World J. Fish and Marine Sci. 2011;3: 88-95.

Venugopal M, Saramma AV. Characterization of alkaline protease from Vibrio fluvialis strain VM10 isolated from a mangrove sediment sample and its application as a laundry detergent additive. Process Biochemistry. 2006;41(6):1239-1243.

Ao X, Yu, X, Wu, D, Li, C, Zhang T, Liu S, Zou L. Purification and characterization of neutral protease from Aspergillus oryzae Y1 isolated from naturally fermented broad beans. AMB Expr. 2018;8(1):96.

Parekh S, Vinei VA, Stroobel RJ. Alkaline Protease Production by Batch Culture of Bacillus sp. Appl Microbial Biotechnol. 2002; 54:287:301.

Suganthi C, Mageswari A, Karthikeyan S, Anbalagan M, Sivakumar A, Gothandam KM. Screening and optimization of protease production from a halotolerant Bacillus licheniformis isolated from saltern sediments Journal of Genetic Engineering and Biotechnology. 2013;11(1):47-52.

Bairagi A, Ghosh KS, Sen SK, Ray AK. Enzyme producing bacterial flora isolated from fish digestive tracts. Aquacul. Int. 2002;10: 109-121.

Ringo E, Strom E. Microflora of Arctic charr, Salvelinus alpines the gastrointestinal microflora of free-living fish and the effect of diet and salinity on intestinal microflora. Aquacul. Fish. Manage. 1994;25:623-629.

Dalal R. Screening and isolation of protease producing bacteria from soil collected from Different Areas of Burhanpur Region (MP) India, IJGMAS. 2015;597-606.

Abidi F, Limam F, Nejib MM. Production of alkaline protease by Botrytis cinera using economic raw materials: Assay as biodetergent. Proc. Biochem. 2008;43:1202-1208.

Sivasubramanian KS, Ravichandran S, Karthick Rajan D. Isolation of gut associated bacteria from mangrove crabs collected from different mangrove regions of Tamil Nadu, South east coast of India, African Journal of Microbiology Research. 2017;11(14):586-595.

Talpur AD, Memon AJ, Khan MI, Ikhwanuddin M, Daniel MD, Abol-Munafi AB. A novel of Gut Pathogenic bacteria of blue swimming crab, Portunus pelagicus (Linneaus, 1758) and pathogenicity of Vibrio harveyi a transmission agent in larval culture under hatchery conditions. Res. J. Appl. Sci. 2011;6: 116-127.

Olsson JC, Westerdahl A, Conway PL, Kjelleberg S. Intestinal colonization potential of turbot (Scophthalmus maximus) - and dab (Limannda limanda)-associated bacteria with inhibitory effects against Vibrio anguillarum. Appl. Environ. Microbiol. 1992;58(2):551-556.

Harrigan WF, McCance, ME. Laboratory methods of Microbiology. London. Academic Press. 1972;362.

Koneman EW, William MJ, Stephen DA, Schreeken B, Washington CW. Laboratory and clinical diagnosis of infectious diseases. In Introduction to diagnostic Microbiology. J.B. Lippincott Company,1 Philadelphia. 1994; 1-19.

Anson ML. Estimation of Pepsin, Papain and Cathepsin with haemoglobin. Journal of General Physiology. 1938;22:79-89.

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent, J. Biol. Chem. 1951;193:265-275.

Mohammed YHI. Isolation of protease producing microorganisms from food waste. J. Bio. Innov. 2015;4(6):322-346.

Gomori G. Preparation of buffers for use in enzyme studies. In Methods in Enzymology, Colwick SP.; and Kaplan NO. (Eds.). New York: Academic Press. 1955;1:138-146.

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685.

Lantz MS, Ciborowski P. Zymographic techniques for detection and characterization of microbial proteases. Methods Enzymol. 1994; 235:563-594.

Sellami-Kamoun A, Haddar A, Ali NEH, Ghorbel-Frikha B, Kanoun S,

Nasri M. Stability of thermostable alkaline protease from Bacillus licheniformis RP1 in commercial solid laundry detergent formulations. Microbiological Research. 2008; 163(3):299–306.

Sila A, Nasri R, Bougatef A, Nasri, M. Digestive Alkaline Proteases from the Goby (Zosterisessor ophiocephalus): Characterization and potential application as detergent additive and in the deproteinization of shrimp wastes. Journal of Aquatic Food Product Technology. 2012;21(2):118-133.

Kuddus M, Ramtake PW. Optimization of an extracellular cold-active alkaline protease from Stenotrophomonas maltophilia MTCC 7528 and its application in detergent industry. African Journal of Microbiology Research. 2011;5(7):809-816.

Rajkumar R, Jayappriyan KR, Ramesh Kannan P, Rengasamy R. Optimization of culture conditions for the production of protease from Bacillus megaterium. Journal of Ecobiotechnology. 2010;2(4):40-46.

Hakim A, Bhuiyan FR, Iqbal A, Emon TH, Ahmed J, Azad AK. Production and partial characterization of dehairing alkaline protease from Bacillus subtilis AKAL7 and Exiguobacterium indicum AKAL11 by using organic municipal solid wastes. Heliyon. 2018; 4(6):1-27.

Sarker P, Talukdar S, Deb P, Sayem S, Mohsina K. Optimization and partial characterization of culture conditions for the production of alkaline protease from Bacillus licheniformis P003. SpringerPlus. 2013;2(1): 506: 1-11.

Pant G, Prakash A, Pavani J, Bera S, Deviram G, Kumar A, Prasuna, RG.

Production, optimization and partial purification of protease from Bacillus subtilis. Journal of Taibah University for Science. 2015; 9:50-55.

Agrawal R, Singh R, Verma A, Panwar P, Verma AK. Partial purification and characterization of alkaline protease from Bacillus sp. isolated from soil. World J. Agricult. Sci. 2012;8(1):129-133.

Shine K, Kanimozhi K, Panneerselvam A, Muthukumar C, Thajuddin N. Production and optimization of alkaline protease by Bacillus cereus RS3 isolated from desert soil, Int. J. Adv. Res. Biol. Sci. 2016;3(7): 193-202.

Nisha NS, Divakaran J. Optimization Parameters for Alkaline protease production using bacterial isolates from different coastal regions of Tamil Nadu, India. Int. J. Curr. Microbiol. App. Sci. 2014;3(8):500-505.

Vasantha S, Subramanian A. Optimization of cultural conditions for the production of an extracellular protease by Pseudomonas species. International Current Pharmaceutical Journal. 2012;2(1):1-6.

Suganthi C, Mageswari A, Karthikeyan S, Anbalagan M, Sivakumar A, Gothandam, KM. Screening and optimization of protease production from a halotolerant Bacillus licheniformis isolated from saltern sediments. Journal of Genetic Engineering and Biotechnology. 2013;11:47–52.

Dissanayaka DMS, Rathnayake, IVN. Effect of temperature, pH, carbon and nitrogen sources on extracellular protease production by four Geobacillus species isolated from Maha Oya geothermal springs in Sri Lanka. Appli Microbiol Open Access. 2019; 5:160.

Ibrahim AS, Al-Salamah SS, Elbadawi AA, El-Tayeb YB, Ibrahim SSS. Production of extracellular alkaline protease by new halotolerant alkaliphilic Bacillus sp. NPST-AK15 isolated from hyper saline soda lakes. Electronic Journal of Biotechnology. 2015;18 (3):236–243.

Asha B, Palaniswamy M. Optimization of alkaline protease production by Bacillus cereus FT 1 isolated from soil. J App Pharm Sci. 2018;8(02):119-127.

Putatunda C, Kundu, BS. Optimization of cultural characteristics for alkaline protease production by Bacillus. Asian Journal of Microbiology, Biotechnology and Environmental Science. 2015;17(3):695-702.

Anandkumar, Ramanadevi V, Ajithkumar, TT, Lipton AP. Thangaraj M. Production and Partial Characterization of Protease from Staphylococcus sp. Isolated from the Mud Crab, Scylla serrata. Int. J. Pure App. Biosci. 2014; 2 (2): 213-220.

Meera Venugopal AV. Saramma. Characterization of alkaline protease from Vibrio fluvialis strain VM10 isolated from a mangrove sediment sample and its application as a laundry detergent additive. Process Biochemistry. 2006;41:1239–1243.

Banerjee UC, Sani RK, Azmi W, Soni R. Thermostable alkaline protease from Bacillus brevis and its characterization as a laundry detergent additive. Process Biochem. 1999;35: 213–219.

Anwar A, Saleemuddin M. Alkaline pH acting digestive enzymes of the polyphagous insect pest Spilosoma obliqua: Stability and potential as detergent additives. Biotechnol. Appl. Biochem. 1997;25:43–46.

Najafi MF, Deobagkar D, Deobagkar D. Potential application of protease isolated from Pseudomonas aeruginosa PD100. Elect J Biotechnol. 2005;8:197–203.

Adinarayana K, Ellaiah P, Prasad SD. Purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11. AAPS Pharma Sci Technol. 2003;4:1–9.

Naidu KSB, Devi KL. Optimization of thermostable alkaline protease production from species of Bacillus using rice bran. African J. Biotechnol. 2005;4:724-726.