Abstract
The aim of present study was to analyze the prevalence of protease diversity among psychrotrophic bacteria in Lahaul and Spiti of the Western Himalayas. A total of 459 bacteria were screened and protease activity was observed in 150 isolates at 5 °C. Furthermore, 55 isolates showed protease activity up to pH 10 at 5 °C. Based on the hydrolytic zone, 22 isolates were selected for protease quantification. The protease activity varied from 58–377 U mL−1 at 10 °C, 49–396 U mL−1 at 28 °C and 31–407 U mL−1 at 37 °C. Similarly, protease activity ranged from 36–353 U mL−1 at pH 7, 40–306 U mL−1 at pH 9 and 33–304 U mL−1 at pH 10. The isolates were identified based on 16S rRNA gene sequencing and showed phylogenetic relationship to Arthrobacter belonging to the class Actinobacteria, Bacillus, Exiguobacterium, Paenibacillus, and Planomicrobium to Bacilli, and Pseudomonas, Serratia, and Stenotrophomonas to Gammaproteobacteria. Zymogram analysis revealed variations in protease isoforms ranging from 20 to 250 kDa which were strongly inhibited in the presence of phenylmethylsulfonyl fluoride, thus indicated serine-type nature. The extensive number of serine proteases among these bacteria was confirmed by annotating genomes of the reported genera for prevalence of protease isoforms. The properties of proteases including low-temperature activity with alkaline stability and detergent compatibility suggested their suitability as bio-additives in laundry.
Similar content being viewed by others
References
Kuddus M, Ramteke PW (2011) Production optimization of an extracellular cold-active alkaline protease from Stenotrophomonas maltophilia MTCC 7528 and its application in detergent industry. Afr J Microbiol Res 5(7):809–816
Adrio JL, Demain AL (2014) Microbial enzymes: Tools for biotechnological processes. Biomolecules 4:117–139
Pant G, Prakash A, Pavani JVP, Bera S, Devirama GVNS, Kumar A, Panchpuri M, Prasuna RG (2015) Production, optimization and partial purification of protease from Bacillus subtilis. J Taibah Univ for Sci 9:50–55
Maurer KH (2004) Detergent proteases. Curr Opin Biotechnol 15:330–334
Ghorbel S, Kammoun M, Soltana H, Nasri M, Hmidet N (2014) Streptomyces flavogriseus HS1: isolation and characterization of extracellular proteases and their compatibility with laundry detergents. BioMed Res Int. https://doi.org/10.1155/2014/345980
Barberis S, Quiroga E, Barcia C, Liggieri C (2013) Effect of laundry detergent formulation on the performance of alkaline phytoproteases. Electron J Biotechnol 16(3):3458
Feller G, Gerdey C (2003) Psychrophilic enzymes: hot topics in cold adaptation. Nature 1:200–208
Sigurðardottir AG, Arnorsdottir J, Thorbjarnardottir SH, Eggertsson G, Suhre K, Kristjansson MM (2009) Characteristics of mutants designed to incorporate a new ion pair into the structure of a cold adapted subtilisin-like serine proteinase. Biochim Biophys Acta 1794:512–518
Wang Q, Hou YH, Xu Z, Miao JL, Li GY (2008) Purification and properties of an extracellular cold-active protease from the psychrophilic bacterium Pseudoalteromonas sp. NJ276. J Biochem Eng 38:362–368
Xin L, Meng Z, Zhang L, Cui Y, Han X, Yi H (2014) The diversity and proteolytic properties of psychrotrophic bacteria in raw cows' milk from North China. Int Dairy J 66:34–41
Zhang S, Li H, Uluko H, Liu L, Pang X, Lv J (2015) Investigation of peptidase production by Pseudomonas fluorescens BJ-10 and degradation on milk proteins. J Food Process Pres 39:2466–2472
Jackson ML (1973) Soil chemical analysis. Prentice Hall, New Delhi, p 187
Salwan R, Gulati A, Kasana RC (2010) Phylogenetic diversity of alkaline protease-producing psychrotrophic bacteria from glacier and cold environments of Lahaul and Spiti, India. J Basic Microbiol 50:150–159
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30(12):2725–2729
Salwan R and Kasana RC (2013) Purification and characterization of cold active and alkaline stable peptidase from Acinetobacter sp. MN12 (MTCC 10786). Indian J Microbiol 53(1):63–69
Saba I, Qazi PH, Rather SA, Dar RA, Qadri QA, Ahmad N, Johri S, Taneja SC, Shawl S (2012) Purification and characterization of a cold active alkaline protease from Stenotrophomonas sp., isolated from Kashmir, India. World J Microbiol Biotechnol 28:1071–1079
Razzaq A, Shamsi S, Ali A, Ali Q, Sajjad M, Malik A, Ashraf M (2019) Microbial proteases applications. Front Bioeng Biotechnol 7:1–20. https://doi.org/10.3389/fbioe.2019.00110
Vishnivetskaya T, Kathariou S, McGrath J, Gilichinsky D, Tiedje JM (2000) Low-temperature recovery strategies for the isolation of bacteria from ancient permafrost sediments. Extremophiles 4(3):165–173
Zhang G, Ma X, Niu F, Dong M, Feng H, An L, Cheng G (2007) Diversity and distribution of alkaliphilic psychrotolerant bacteria in the Qinghai-Tibet Plateau permafrost region. Extremophiles 11:415–424
Kuddus M, Ramteke PW (2008) A cold–active extracellular metalloprotease from Curtobacterium luteum (MTCC 7529), enzyme production and characterization. J Gen Appl Microbiol 54(6):385–392
Guoying XU, Xuezheng LIN, Nengfei W, Qizhong ZHU (2011) Optimization of cold-active protease production by polar psychrotrophic bacterium Pseudoalteromonas sp. QI-1. Chin J Bioprocess Eng 2:5–11
Zeng R, Zhang R, Zhao J, Lin N (2003) Cold-active serine alkaline protease from the psychrophilic bacterium Pseudomonas strain DY-A, enzyme purification and characterization. Extremophiles 7:335–433
Huston AL, Methe B, Deming JW (2004) Purification, characterization, sequencing of an extracellular cold–active aminopeptidase produced by marine psychrophile Colwellia psychrerythraea strain 34H. Appl Environ Microbiol 70:2321–2328
Bekker A, Steyn L, Charimba G, Jooste P, Hugo C (2015) Comparison of the growth kinetics and proteolytic activities of Chryseobacterium species and Pseudomonas fluorescens. Can J Microbiol 61:977–982
Machado SG, Silva FL, Bazzolli DMS, Heyndrickx M, Costa PMA, Vanetti MCD (2015) Pseudomonas spp. and Serratia liquefaciens as predominant spoilers in Brazilian cold raw milk. J Food Sci 80:M1842–M1849
Joshi GK, Kumar S, Sharma V (2007) Production of moderately halotolerant, SDS stable alkaline protease from Bacillus cereus MTCC 6840 isolated from lake Nainital, Uttaranchal state, India. Braz J Microbiol 38:773–779
Rodarte MP, Dias DR, Vilela DM, Schwan RF (2011) Proteolytic activities of bacteria, yeasts and filamentous fungi isolated from coffee fruit (Coffea arabica L.). Acta Sci Agron 33(3):457–464
Martins ML, Pinto UM, Riedel K, Vanetti MCD (2015) Milk-deteriorating exoenzymes from Pseudomonas fluorescens 041 isolated from refrigerated raw milk. Braz J Microbiol 46:207–217
Vazquez S, Ruberto L, Cormack WM (2005) Properties of extracellular proteases from three psychrotolerant Stenotrophomonas maltophilia isolated from Antarctic soil. Polar Biol 28:319–325
Baweja M, Tiwari R, Singh PK, Nain L, Shukla P (2016) An alkaline Protease from Bacillus pumilus MP 27: Functional analysis of its binding model toward its applications as detergent additive. Front Microbiol 7:1195
Poza M, Miguel T, Siero C, Villa TG (2001) Characterization of a broad pH range protease of Candida caseinolytica. J Appl Microbiol 91(5):916–921
Champasri C, Champasri T (2017) Biochemical characterization, activity comparison and isoenzyme analysis of amylase and alkaline proteases in seven cyprinid fishes. J Fish Aquat Sci 12:264–272
Zacaria J, Delamare APL, Costa SOP, Echeverrigaray S (2010) Diversity of extracellular proteases among Aeromonas determined by zymogram analysis. J Appl Microbiol 109:212–219
Tripathi LP, Sowdhamini R (2008) Geome-wide survey of prokaryotic serine proteases: Analysis of distribution and domain architectures of five serine protease families in prokaryotes. BMC Genom 9:549
Secades P, Alvarez B, Guijarro JA (2001) Purification and characterization of a psychrophilic calcium induced, growth-phase dependent metalloprotease from the fish pathogen Flavobacterium psychrophilum. Appl Environ Microbiol 67(6):2436–2444
Singh J, Batra N, Sobti RC (2001) Serine alkaline protease from a newly isolated Bacillus sp. SSR1. Process Biochem 36:781–785
Venugopal M, Saramma AV (2006) Characterization of alkaline protease from Vibrio fluvialis strain VM10 isolated from a mangrove sediment sample and its application as a laundry detergent additive. Process Biochem 41:1239–1243
Liu D, Wu CL, Yang XH, Wu RB, Zhang J, Huang JH, Liao BQ, Bian F, He HL (2016) Intrinsic properties analysis of multiproteases system from marine bacteria by inhibitor-subsatrate immersing zymography. Am J BioSci 4(3):20–27
Khajuria V, Sharma K, Slathia P, Razdan K, Singh S, Bajaj BK (2015) Production of a detergent-compatible alkaline protease from Bacillus cereus K-3. J Mater Environ Sci 6:2089–2096
Sellami-Kamoun A, Haddar A, Ali NE, Ghorbel-Frikha B, Kanoun S, Nasri M (2008) Stability of thermostable alkaline protease from Bacillus licheniformis RP1 in commercial solid laundry detergent formulations. Microbiol Res 163:299–306
Ramesh S, Rajesh M, Mathivanan N (2009) Characterization of a thermostable alkaline protease produced by marine Streptomyces fungicidicus MML1614. Bioprocess Biosyst Eng 32:791–800
Zambare VP, Nilegaonkar SS, Kshirsagar PR, Kanekar PP (2014) Scale production of protease using Pseudomonas aeruginosa MCM B-327 and its detergent compatibility. J Biochem Technol 5(2):698–707
Acknowledgements
The authors acknowledge the Director, CSIR-Institute of Himalayan Bioresource Technology, Palampur, for support and encouragement during the course of this investigation. The financial support received from the CSIR and award of Senior Research Fellowship to Richa Salwan is gratefully acknowledged. We gratefully acknowledge the late Dr. R.D. Singh for his wonderful suggestions in the applications of statistical analysis.
Author information
Authors and Affiliations
Contributions
RS executed experiments and MS writing. VS assisting in planning experiments and MS writing. AG and RK were involved in MS editing.
Corresponding author
Ethics declarations
Conflicts of interest
The authors declare that they have no conflicts of interest.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Salwan, R., Sharma, V., Kasana, R.C. et al. Bioprospecting Psychrotrophic Bacteria for Serine-Type Proteases from the Cold Areas of Western Himalayas. Curr Microbiol 77, 795–806 (2020). https://doi.org/10.1007/s00284-020-01876-w
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00284-020-01876-w