Investigation of extra-cellular protease in indigenous bacteria of sea cucumbers as a candidate for bio-detergent material in bio-industry

Delianis Pringgenies; Wilis Ari Setyati; Nirwani Soenardjo; Rini Pramesti; Delianis Pringgenies; Wilis Ari Setyati; Nirwani Soenardjo; Rini Pramesti

doi:10.3934/environsci.2020022

AIMS Environmental Science

2020, Volume 7, Issue 4: 335-349. doi: 10.3934/environsci.2020022

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Research article

Investigation of extra-cellular protease in indigenous bacteria of sea cucumbers as a candidate for bio-detergent material in bio-industry

Department of Marine Science, Faculty of Fisheries and Marine Science, Diponegoro University, Jl. Prof. H. Soedarto, S.H. No.1. Tembalang, Kota Semarang, Jawa Tengah, Indonesia

Received: 07 July 2020 Accepted: 06 September 2020 Published: 21 September 2020

This research aims to determine the characteristics of extra-cellular protease enzymes derived from the intestines of sea cucumbers as a candidate for bio-detergent, to optimize the growth of extra-cellular protease-producing bacteria and to identify the enzymes producing bacteria by DNA sequencing. The study consisted of 5 stages, namely isolation, selection and identification of extra-cellular protease-producing bacterial enzymes, bacterial growth optimization test in the production of extra-cellular protease enzymes, enzyme isolation and characterization, enzyme ability testing of various detergent components and determining the highest conditions of enzyme activity, and purification and identification from the extra-cellular protease enzyme. The results showed that bacteria isolated from the sea cucumbers Stichopus hermanni, Holothuria atra and H.leucopilota. Bacterial isolation derived from the contents of all these sea cucumbers produced 60 isolates and 22 isolates were active against the enzyme protease. Optimization test of carbon compounds (C), namely glucose, fructose, and molasses produces the enzyme molasses. Nitrate (N) compounds optimization test, namely: Ammonium chloride, Ammonium nitrate, Urea found Ammonium nitrate as the best. Optimization test results of the concentration of substances C, N and optimization on the parameters of salinity (25, 30, 35 ppt), pH (6, 7, 8) and temperature (25, 30, 35℃), produced the best salinity at 30 ppt, the best ph at 8 and the best temperature at 30 ℃. The results of the fermentation test showed that protein hydrolysis in TH.IP.4 isolate media was better than that in THDM.IP.3. There was a positive correlation between cell density and the presence of protease enzymes, where the higher the number of cells, the higher the production of protease enzymes. Protease activity in TH.IP.4 isolates was better than THDM.IP.3 isolates. Molecular identification results showed that isolate 0TH.IP.4 had the closest match (99%) with Bacillus cereus and THDM.IP.3 was a complete match (100%) with Bacillus thuringiensis.
- bacteria,
- bio-detergent,
- enzyme,
- protease,
- sea cucumber
Citation: Delianis Pringgenies, Wilis Ari Setyati, Nirwani Soenardjo, Rini Pramesti. Investigation of extra-cellular protease in indigenous bacteria of sea cucumbers as a candidate for bio-detergent material in bio-industry[J]. AIMS Environmental Science, 2020, 7(4): 335-349. doi: 10.3934/environsci.2020022

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Abstract

This research aims to determine the characteristics of extra-cellular protease enzymes derived from the intestines of sea cucumbers as a candidate for bio-detergent, to optimize the growth of extra-cellular protease-producing bacteria and to identify the enzymes producing bacteria by DNA sequencing. The study consisted of 5 stages, namely isolation, selection and identification of extra-cellular protease-producing bacterial enzymes, bacterial growth optimization test in the production of extra-cellular protease enzymes, enzyme isolation and characterization, enzyme ability testing of various detergent components and determining the highest conditions of enzyme activity, and purification and identification from the extra-cellular protease enzyme. The results showed that bacteria isolated from the sea cucumbers Stichopus hermanni, Holothuria atra and H.leucopilota. Bacterial isolation derived from the contents of all these sea cucumbers produced 60 isolates and 22 isolates were active against the enzyme protease. Optimization test of carbon compounds (C), namely glucose, fructose, and molasses produces the enzyme molasses. Nitrate (N) compounds optimization test, namely: Ammonium chloride, Ammonium nitrate, Urea found Ammonium nitrate as the best. Optimization test results of the concentration of substances C, N and optimization on the parameters of salinity (25, 30, 35 ppt), pH (6, 7, 8) and temperature (25, 30, 35℃), produced the best salinity at 30 ppt, the best ph at 8 and the best temperature at 30 ℃. The results of the fermentation test showed that protein hydrolysis in TH.IP.4 isolate media was better than that in THDM.IP.3. There was a positive correlation between cell density and the presence of protease enzymes, where the higher the number of cells, the higher the production of protease enzymes. Protease activity in TH.IP.4 isolates was better than THDM.IP.3 isolates. Molecular identification results showed that isolate 0TH.IP.4 had the closest match (99%) with Bacillus cereus and THDM.IP.3 was a complete match (100%) with Bacillus thuringiensis.

References

[1]	Olsen HS, Falholt P (1998) The role of enzymes in modern detergency. J Surfactants Deterg 1: 555-567.
[2]	Singh R, Kumar M, Mittal A, et al. (2016) Microbial enzymes: industrial progress in 21st century. 3 Biotech 6: 1-15.
[3]	Zhang X, Nakahara T, Miyazaki M, et al. (2012) Diversity and function of aerobic culturable bacteria in the intestine of the sea cucumber Holothuria leucospilota. J Gen Appl Microbiol 456: 447-456.
[4]	Girsang PH, Pringginies D, Yudianti E, et al. (2020) Exploration of sea cucumber intestinal symbiont microbe as probiotic microbe candidate in healthcare products. JFMR-Journal Fish Mar Res 4: 27-34.
[5]	Naiola E, Widyastuti N (2002) Isolation, selection and optimalization of protease production of some bacterial isolates. Ber Biol 6: 467-473.
[6]	Irfan M, Safdar A, Syed Q, et al. (2012) Isolation and screening of cellulolytic bacteria from soil and optimization of cellulase production and activity. Türk Biyokim Derg [Turkish J Biochem J Biochem 37: 287-293.
[7]	Bairagi A, Ghosh KS, Kumar S, et al. (2002) Enzyme producing bacterial flora isolated from fish. Aquac Int 10: 109-121.
[8]	Polak-berecka M, Ko AWA, Kordowska-wiater M (2010) Optimization of medium composition for enhancing growth of lactobacillus rhamnosus PEN using response surface methodology. Polish J Microbiol 59: 113-118.
[9]	Doresti L, Setyati WA, Widowati I (2018) Optimasi sumber karbon dan nitrogen sebagai co-substrat untuk pertumbuhan bakteri probiotik pseudomonas sp. J Mar Res 7: 178-184.
[10]	Mahdhi A, Á ngeles M, Hmila Z, et al. (2012) Research in veterinary science survival and retention of the probiotic properties of Bacillus sp. strains under marine stress starvation conditions and their potential use as a probiotic in Artemia culture. Res Vet Sci 93: 1151-1159.
[11]	Marchesi JR, Sato T, Weightman AJ, et al. (1998) Design and Evaluation of Useful Bacterium-Specific PCR Primers That Amplify Genes Coding for Bacterial 16S rRNA. Appl Environ Microbiol 64: 795-799.
[12]	Isnansetyo A, Kamei Y (2009) Bioactive substances produced by marine isolates of Pseudomonas. J Ind Microbiol Biotechnol 36: 1239-1248.
[13]	Keshwani A, Malhotra B, Kharkwal DH (2015) Natural polymer based detergents for stain removal. World J Pharm Pharm Sci 4: 490-508.
[14]	Li S, Yang X, Yang S, et al. (2012) Technology prospecting on enzymes: Application, marketing and engineering. Comput Struct Biotechnol J 2: 1-11.
[15]	Guinebretière MH, Auger S, Galleron N, et al. (2013) Bacillus cytotoxicus sp. nov. is a novel thermotolerant species of the Bacillus cereus group occasionally associated with food poisoning. Int J Syst Evol Microbiol 63: 31-40.
[16]	Tewari A, Abdullah S (2015) Bacillus cereus food poisoning: international and Indian perspective. J Food Sci Technol 52: 2500-2511.
[17]	Bottone EJ (2010) Bacillus cereus, a volatile human pathogen. Clin Microbiol Rev 23: 382-398.
[18]	Ehling-Schulz M, Lereclus D, Koehler TM (2019) The Bacillus cereus Group: Bacillus Species with Pathogenic Potential. Gram-Positive Pathogens. 2019: 875-902.
[19]	Alseth I, Rognes T, Lindbä ck T, et al (2006) A new protein superfamily includes two novel 3-methyladenine DNA glycosylases from Bacillus cereus, AlkC and AlkD 59: 1602-1609.
[20]	Brar SK, Verma M, Tyagi RD, et al (2007) Bacillus thuringiensis proteases: Production and role in growth, sporulation and synergism. Process Biochem 42: 773-790.
[21]	Furhan J, Sharma S (2014) Microbial alkaline proteases : Findings and applications. Int J Invent Pharm Sci 2: 823-834.

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