The psychrotrophic yeast Sporobolomyces roseus LOCK 1119 as a source of a highly active aspartic protease for the in vitro production of antioxidant peptides

Abstract

A psychrotrophic yeast strain producing a cold-adapted protease at low temperature was classified as Sporobolomyces roseus. In standard YPG medium, S. roseus LOCK 1119 synthesized an extracellular protease with an activity of approximately 560 U/L. Optimization of medium composition and process temperature considerably enhanced enzyme biosynthesis; an approximate 70% increase in activity (2060 U/L). The native enzyme was purified to homogeneity by cation exchange chromatography followed by a size exclusion step, resulting in a 103-fold increase in specific activity (660 U/mg) with 25% recovery. The enzyme displayed 10%-30% of its maximum activity at 0-25 °C, with the optimum temperature being 50°C. Protease G8 was strongly inactivated by pepstatin A, an aspartic protease inhibitor. The enzyme was used to hydrolyze four natural substrates, and their antioxidant activities were evaluated against 1,1-diphenyl-2-picrylhydrazyl. The highest antioxidant activity (69%) was recorded for beef casein.

Adaptation, production, and biotechnological potential of cold-adapted proteases from psychrophiles and psychrotrophs: recent overview

“…Cold-adapted protease producers have been isolated from soil samples of various Indian Himalayan regions such as Lahaul and Spiti [ 26 ], Kashmir apple garden [ 27 ], Thajiwas glacier [ 28 ], Gangotri glacier [ 29 ], and Wular Lake [ 30 ]. Other sources used for isolation of cold-adapted protease producers include an underground water sample of abandoned silver and lead mine [ 31 ] and cold food products and storage facilities [ 32 ]. Table 1 shows some recently isolated cold-adapted protease-producing microorganisms.…”

“… Himalayan cold soil [ 26 ] Stenotrophomonas sp. Thajiwas glacier soil [ 28 ] Stenotrophomonas maltophilia Gangotri glacier soil [ 29 ] Fungi Geomyces pannorum Antarctica [ 15 ] Penicillium nalgiovense Moldy sausage [ 40 ] Yeast Cryptococcus victoriae Turkish soil [ 25 ] Glaciozyma antarctica Antarctic marine water [ 17 ] Goffeauzyma gilvescens Antarctic soil [ 14 ] Naganishia globosa Antarctic soil [ 14 ] Naganishia adeliensis Antarctic soil [ 14 ] Naganishia albida Antarctic soil [ 14 ] Sporobolomyces roseus Water of disused silver and lead mine [ 31 ] Yamadazyma spp. Water, leaf, and grass from mountain [ 24 ] …”

“…Fructose in presence of skim milk was the best carbon source (2.4 fold increase), and tryptone in presence of skim milk (1.7-fold increase) was the best nitrogen source for protease production by Pseudoalteromonas arctica [ 13 ]. For psychrotrophic Sporobolomyces roseus , glucose was the best carbon source, whereas other sources like yeast extract, beef extract, and BSA showed a lower effect on protease production [ 31 ]. The highest cold-adapted protease production by psychrotolerant Bacillus subtilis [ 30 ], Chryseobacterium sp.…”

“…The Colwellia sp.-derived cold-adapted protease showed a 3.0-fold enhancement in production via response surface methodology [ 59 ]. Recently, Białkowska et al [ 31 ] reported an approximately 4.0-fold increase in protease production by combining various sources via the regression model. In another study, Han et al [ 13 ] reported a 15-fold improvement in protease production by Pseudoalteromonas arctica in a mineral optimized medium using a statistical approach and fed-batch culture.…”

“… Serine protease PMSF and AEBSF Ca 2+ and Mn 2+ 35/9 [ 11 ] Pseudomonas aeruginosa Serine protease PMSF and Ag + Mg 2+ , K + , Ca 2+ , Ba 2+ , and Zn 2+ 25/10 [ 21 ] Pseudoalteromonas sp. Serine protease PMSF, SDS, and H 2 O 2 , Nm 25–35/8–9 [ 16 ] Pseudoalteromonas arctica Subtilisin-like protease Linear alkylbenzene sulfonate (LAS) and SDS Ca 2+ 30/9.0 [ 64 ] Pseudomonas lundensis Metalloprotease EDTA, EGTA, Cu 2+ , Fe 3+ , Al 3+ , Fe 2+ , Mn 2+ , Al 3+ , and Co 2+ Na + , K + , and Li + 30/10.4 [ 19 ] Serratia marcescens Metalloprotease EDTA, MnCl 2 , CaCl 2 , CoSo 4 , HgCl 2 , and Na 2 Nm 40/8 [ 27 ] Sporobolomyces roseus Aspartic protease 2-Mercaptoethanol, dithiothreitol, SDS, and Pepstatin A Nm 50/4 [ 31 ] Stenotrophomonas sp. Alkaline protease Zn 2+ , Cu 2+ , and Co 2+ Mg 2+ , Mn 2+ , and Ca 2+ 15/10 [ 28 ] Stenotrophomonas maltophilia Alkaline protease Co 2+ Cu 2+ , Cr 2+ ...…”