The present study has undertaken the isolation of marine yeasts from mangrove sediment samples and their ability to produce alkaline protease enzymes. A total of 14 yeast isolates were recovered on yeast-malt agar (YMA) and yeast extract peptone dextrose (YEPD) agar medium. After screening for proteolytic activity on skim milk agar, marine yeast isolate, AKB-1 exhibited a hydrolysis zone of 18 mm. Optimal conditions for the enzyme production from yeast isolate AKB-1 were at 30 °C, pH 8, fructose as carbon source, potassium nitrate as nitrogen source, and 25% saline concentration. Under the optimal conditions, the protease enzyme activity of the isolate AKB-1 was observed to be 978 IU/mL. The structural and functional analysis was carried out through FTIR and HPLC analysis for the extracted protease enzyme. Furthermore, the enzyme produced was partially purified by solvent extraction using ethyl acetate and ammonium sulfate precipitation (3.4-fold) followed by dialysis (56.8-fold). The molecular weight of the purified enzyme was observed to be around 60 kDa using SDS-PAGE. The extracted protein showed good antibacterial activity against six different clinical bacterial pathogens and the highest against Bacillus cereus (16 ± 0.5 mm). The extracted protease enzyme was revealed to remove blood stains from cloth within 20 min of application similar to the commercial detergent. The marine yeast isolate was further identified as Candida orthopsilosis AKB-1 (Accession number KY348766) through 18S rRNA sequencing, and a phylogenetic tree was generated.

Marine Biotechnology Laboratory Department of Biomedical Sciences School of Biosciences and Technology Vellore Institute of Technology Vellore 632014 Tamil Nadu India

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Aamir S, Sutar S, Singh SK, Baghela A (2015) A rapid and efficient method of fungal genomic DNA extraction, suitable for PCR based molecular methods. Plant Pathol Quar 5:74–81 DOI

Aissaoui N, Abidi F, Mahat S, Marzouki MN (2014) Purification and biochemical characterization of a novel protease from Penicillium digitatum–Use in bioactive peptides production. J Basic Microbiol 54:S178–S189. https://doi.org/10.1002/jobm.201400179 PubMed DOI

Annamalai N, Kumar A, Saravanakumar A, Vijaylakshmi S, Balasubramanian T (2011) Characterization of protease from Alcaligens faecalis and its antibacterial activity on fish pathogens. J Environ Biol 32:781 PubMed

Anusha SU, Sundar SK, Williams PG (2014) Studies on the isolation and characterisation of marine yeast, β-glucan production and immunostimulatory activity on Carassius auratus. Int J Curr Microbiol Appl Sci 3:230–240

Anwar A, Saleemuddin M (1998) Alkaline proteases: a review. Bioresour Technol 64:175–183. https://doi.org/10.1016/S0960-8524(97)00182-X DOI

Banerjee S, Pandey K, Mazumdar S, Sarkar A, Rao KVB (2019) Characterization and applications of protease inhibitor compound from marine yeast Candida orthopsilosis KSSB3. Res J Biotechnol 14:43–52

Banik S, Biswas S, Karmakar S (2018) Extraction, purification, and activity of protease from the leaves of Moringa oleifera. F1000Research 7 https://doi.org/10.12688/f1000research.15642.1

Barnett JA (1975) The entry of D-ribose into some yeasts of the genus Pichia. Microbiol 90:1–12. https://doi.org/10.1099/00221287-90-1-1 DOI

Barritt MM (1936) The intensification of the Voges-Proskauer reaction by the addition of α-naphthol. J Pathol Bacteriol 42:441–454 DOI

Bhaskar N, Sudeepa ES, Rashmi HN, Selvi AT (2007) Partial purification and characterization of protease of Bacillus proteolyticus CFR3001 isolated from fish processing waste and its antibacterial activities. Biores Technol 98:2758–2764. https://doi.org/10.1016/j.biortech.2006.09.033 DOI

Buzzini P, Martini A (2002) Extracellular enzymatic activity profiles in yeast and yeast-like strains isolated from tropical environments. J Appl Microbiol 93:1020–1025. https://doi.org/10.1046/j.1365-2672.2002.01783.x PubMed DOI

Chi Z, Ma C, Wang P, Li HF (2007) Optimization of medium and cultivation conditions for alkaline protease production by the marine yeast Aureobasidium pullulans. Bioresour Technol 98:534–538. https://doi.org/10.1016/j.biortech.2006.02.006 PubMed DOI

Chi Z, Chi Z, Zhang T, Liu G, Li J, Wang X (2009) Production, characterization and gene cloning of the extracellular enzymes from the marine-derived yeasts and their potential applications. Biotechnol Adv 27:236–255. https://doi.org/10.1016/j.biotechadv.2009.01.002 PubMed DOI

Chi ZM, Liu G, Zhao S, Li J, Peng Y (2010) Marine yeasts as biocontrol agents and producers of bio-products. Appl Microbiol Biotechnol 86:1227–1241. https://doi.org/10.1007/s00253-010-2483-9 PubMed DOI

De Vecchi S, Coppes Z (1996) Marine fish digestive proteases—relevance to food industry and the south-west Atlantic region—a review. J Food Biochem 20:193–214. https://doi.org/10.1111/j.1745-4514.1996.tb00551.x DOI

De Viragh PA, Sanglard D, Togni G, Falchetto R, Monod M (1993) Cloning and sequencing of two Candida parapsilosis genes encoding acid proteases. Microbiol 139:335–342. https://doi.org/10.1099/00221287-139-2-335 DOI

Devi MK, Banu AR, Gnanaprabha GR, Pradeep BV, Palaniswamy M (2008) Purification, characterization of alkaline protease enzyme from native isolate Aspergillus niger and its compatibility with commercial detergents. Indian J Sci Technol 1:1–6 https://doi.org/10.17485/ijst/2008/v1i7/29599

Galanie S, Smolke CD (2015) Optimization of yeast-based production of medicinal protoberberine alkaloids. Microb Cell Fact 14:1–13. https://doi.org/10.1186/s12934-015-0332-3 DOI

Gonzalez-Lopez CI, Szabo R, Blanchin-Roland S, Gaillardin C (2002) Genetic control of extracellular protease synthesis in the yeast Yarrowia lipolytica. Genetics 160:417–427. https://doi.org/10.1093/genetics/160.2.417 PubMed DOI PMC

Harley JP (2005) Laboratory exercises in microbiology, 6th edn. McGraw Hill Publishers, New York, New York

Hassan BA, Abbas MD, Rasool SN (2022) Antibacterial activity of protease that production, purification and characterization from Proteus mirabilis against Gram Positive and Negative pathogenic bacteria. HIV Nursing 22:1741–1746

Indarmawan T, Mustopa AZ, Budiarto BR, Tarman K (2016) Antibacterial activity of extracellular protease isolated from an algicolous fungus Xylaria psidii KT30 against gram-positive bacteria. HAYATI J Biosci 23:73–78. https://doi.org/10.1016/j.hjb.2016.06.005 DOI

Karthik L, Kumar G, Keswani T, Bhattacharyya A, Chandar SS, Rao KVB (2014) Protease inhibitors from marine actinobacteria as a potential source for antimalarial compound. PLoS ONE 9:e90972. https://doi.org/10.1371/journal.pone.0090972 PubMed DOI PMC

Kim D, Shin WS, Lee KH, Kim K, Young Park J, Koh CM (2002) Rapid differentiation of Candida albicans from other Candida species using its unique germ tube formation at 39°C. Yeast 19:957–962. https://doi.org/10.1002/yea.891 PubMed DOI

Kumar SRS, Rao KVB (2016) Postprandial anti-hyperglycemic activity of marine Streptomyces coelicoflavus SRBVIT13 mediated gold nanoparticles in streptozotocin induced diabetic male albino Wister rats. IET Nanobiotechnol 10:308–314. https://doi.org/10.1049/iet-nbt.2015.0094 DOI

Kumar D, Karthik L, Kumar G, Rao KVB (2011) Biosynthesis of silver nanoparticles from marine yeast and their antimicrobial activity against multidrug resistant pathogens. Pharmacologyonline 3:1100–1111

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. https://doi.org/10.1038/227680a0 PubMed DOI

Li J, Peng Y, Wang X, Chi Z (2010) Optimum production and characterization of an acid protease from marine yeast Metschnikowia reukaufii W6b. J Ocean Uni China 9:359–364. https://doi.org/10.1007/s11802-010-1765-2 DOI

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275. https://doi.org/10.1016/S0021-9258(19)52451-6 PubMed DOI

Lucia V, Daniela B, Rosalba L (2001) Use of Fourier transform infrared spectroscopy to evaluate the proteolytic activity of Yarrowia lipolytica and its contribution to cheese ripening. Inter J Food Microbiol 69:113–123. https://doi.org/10.1016/S0168-1605(01)00578-5 DOI

Ma C, Ni X, Chi Z, Ma L, Gao L (2007) Purification and characterization of an alkaline protease from the marine yeast Aureobasidium pullulans for bioactive peptide production from different sources. Mar Biotechnol 9:343–351. https://doi.org/10.1007/s10126-006-6105-6 DOI

Macfaddin JF (1980) Biochemical tests for identification of medical bacteria, 2nd edn. Williams and Wilkins, Baltimore

Macfaddin JF (2000) Biochemical tests for identification of medical bacteria, 3rd edn. Williams and Wilkins, Philadelphia (PA)

Mahakhan P, Apiso P, Srisunthorn K, Vichitphan K, Vichitphan S, Punyauppa-Path S, Sawaengkaew J (2023) Alkaline protease production from Bacillus gibsonii 6BS15-4 using dairy effluent and its characterization as a laundry detergent additive. J Microbiol Biotechnol 33:195. https://doi.org/10.4014/jmb.2210.10007 PubMed DOI

Martinez de Tejada G, Sánchez-Gómez S, Rázquin-Olazaran I, Kowalski I, Kaconis Y, Heinbockel L, Andra J, Schurholz T, Hornef M, Dupont A, Garidel P (2012) Bacterial cell wall compounds as promising targets of antimicrobial agents I. Antimicrobial peptides and lipopolyamines. Curr Drug Targ 13:1121–1130. https://doi.org/10.2174/138945012802002410 DOI

Nagarajan DR, Rajagopalan G, Krishnan C (2006) Purification and characterization of a maltooligosaccharide-forming α-amylase from a new Bacillus subtilis KCC103. Appl Microbiol Biotechnol 73:591–597. https://doi.org/10.1007/s00253-006-0513-4 PubMed DOI

Pandey A, Naik M, Dubey SK (2010) Hemolysin, protease, and EPS producing pathogenic Aeromonas hydrophila strain An4 shows antibacterial activity against marine bacterial fish pathogens. J Mar Sci 2010. https://doi.org/10.1155/2010/563205

Poole PL, Finney JL (1984) Sequential hydration of dry proteins: a direct difference IR investigation of sequence homologs lysozyme and α-lactalbumin. Biopolymers 23:1647–1666. https://doi.org/10.1002/bip.360230904 PubMed DOI

Richa K, Bose H, Singh K, Karthik L, Kumar G, Rao KVB (2013) Response surface optimization for the production of marine eubacterial protease and its application. Res J Biotechnol 8:78–85

Sarkar A, Rao KVB (2016) Marine yeast: A potential candidate for biotechnological applications-A review. Asian J Microbiol Biotechnol Environ Sci 18:627–634

Sarkar A, Rao KVB (2023) Unraveling anticancer potential of a novel serine protease inhibitor from marine yeast Candida parapsilosis ABS1 against colorectal and breast cancer cells. World J Microbiol Biotechnol 39:225. https://doi.org/10.1007/s11274-023-03670-9 PubMed DOI

Sarkar A, Philip AM, Thakker DP, Wagh MS, Rao KVB (2020) In vitro antioxidant activity of extracellular L-glutaminase enzyme isolated from marine yeast Rhodotorula sp. DAMB1. Res J Pharm Technol 13:209–215. https://doi.org/10.5958/0974-360X.2020.00042.6 DOI

Sarkar A, Ghosh D, Das S, Rao KVB (2021) Antioxidant and antibacterial activity of biogenic zirconium oxide nanoparticles from Candida orthopsilosis DSB1 isolated from backwaters of Sunderbans, West Bengal. Inter J Nanopart 13:174–194. https://doi.org/10.1504/IJNP.2021.118111 DOI

Sayali D, Sahithya K, Karthik L, Kumar G, Rao KVB (2013) Isolation, purification and application of enzymes from Bacillus subtilis. Res J Pharm Biol Chem Sci 4:1196–1204

Singh K, Richa K, Bose H, Karthik L, Kumar G, Rao KVB (2014) Statistical media optimization and cellulase production from marine Bacillus VITRKHB. 3 Biotech 4:591–598. https://doi.org/10.1007/s13205-013-0173-x PubMed DOI PMC

Singh S, Sharma S, Agarwal SK (2020) A simple purification procedure of buffalo lung cathepsin H, its properties and influence of buffer constituents on the enzyme activity. Biochem Biophys Rep 22:100739. https://doi.org/10.1016/j.bbrep.2020.100739 PubMed DOI PMC

Sneha S, Palsokar M, Jahnavi VS, Sarkar A, Rao KVB (2021) Isolation, characterization and application of protease enzyme from marine bacteria. Res J Pharm Technol 14:4236–4240. https://doi.org/10.52711/0974-360X.2021.00735 DOI

Spalding M (2010) World atlas of mangroves. A collaborative project of ITTO, ISME, FAO, UNEP-WCMC, UNESCO-MAB, UNU-INWEH and TNC. Routledge, London (UK): Earthscan, London, p 319. https://doi.org/10.4324/9781849776608

Sun B, Zou K (2023) The fermentation optimization for alkaline protease production by Bacillus subtilis BS-QR-052. Front Microbiol 14:1301065. https://doi.org/10.3389/fmicb.2023.1301065 PubMed DOI PMC

Takami H, Nakamura S, Aono R, Horikoshi K (1992) Degradation of human hair by a thermostable alkaline protease from alkaliphilic Bacillus sp. no. AH-101. Biosci Biotechnol Biochem 56:1667–1669. https://doi.org/10.1271/bbb.56.1667 DOI

Taniguchi Y (1992) Pressure-induced secondary structure of proteins studied by FT-IR spectroscopy. High Pres Biotech 224:115–122

Tarek H, Nam KB, Kim YK, Suchi SA, Yoo JC (2023) Biochemical characterization and application of a detergent stable, antimicrobial and antibiofilm potential protease from Bacillus siamensis. Inter J Mol Sci 24(6):5774. https://doi.org/10.3390/ijms24065774 DOI

Ullah N, Rehman MU, Sarwar A, Nadeem M, Nelofer R, Shakir HA, Irfan M, Idrees M, Naz S, Nabi G, Shah S (2022) Purification, characterization, and application of alkaline protease enzyme from a locally isolated Bacillus cereus strain. Fermentation 8:628. https://doi.org/10.3390/fermentation8110628 DOI

Velho S, D’Souza J (1982) Studies on pectinolytic fungi from the mangrove sediments. Mahasagar 15:167–173

Vishalakshi N, Lingappa K, Amena S, Prabhakar M, Dayanand A (2009) Production of alkaline protease from Streptomyces gulbargensis and its application in removal of blood stains. Indian J Biotechnol 8:280–285

Vishwanatha KS, Rao AA, Singh SA (2009) Characterisation of acid protease expressed from Aspergillus oryzae MTCC 5341. Food Chem 114:402–407. https://doi.org/10.1016/j.foodchem.2008.09.070 DOI

Zhang X, Shuai Y, Tao H, Li C, He L (2021) Novel method for the quantitative analysis of protease activity: the casein plate method and its applications. ACS Omega 6:3675–3680. https://doi.org/10.1021/acsomega.0c05192 PubMed DOI PMC

Zhenming C, Zhiqiang L, Lingmei G, Fang G, Chunling MA, Xianghong W, Haifeng LI (2006) Marine yeasts and their applications in mariculture. J Ocean Univ China 5:251–256. https://doi.org/10.1007/s11802-006-0010-5 DOI

Zhou C, Qin H, Chen X, Zhang Y, Xue Y, Ma Y (2018) A novel alkaline protease from alkaliphilic Idiomarina sp. C9–1 with potential application for eco-friendly enzymatic dehairing in the leather industry. Sci Rep 8:16467. https://doi.org/10.1038/s41598-018-34416-5 PubMed DOI PMC

Zhou J, Cai Y, Liu Y, An H, Deng K, Ashraf MA, Zou L, Wang J (2022) Breaking down the cell wall: still an attractive antibacterial strategy. Front Microbiol 13:952633. https://doi.org/10.3389/fmicb.2022.952633 PubMed DOI PMC