Extracellular serine proteases by Acremonium sp. L1-4B isolated from Antarctica: Overproduction using cactus pear extract with response surface methodology

Abstract

Acremonium sp. L1-4B isolated from lichen in Antarctica was used to produce extracellular proteases through submerged fermentation using cactus pear extract (Opuntia ficus-indica Mill.). A 2 3 factorial design was applied to optimize the protease production using three independent variables, namely temperature, pH and concentration of yeast extract, was also used a Central Composite Design (CCD) under Response Surface Methodology (RSM). All variables and interactions analyzed in the factorial design were significant or marginally significant, a Central Composite Design was developed, and the Response Surface Methodology towards the highest point it was established. The experimental model was validated under 14°C, pH 7.54, and 0.55% yeast extract, showing a protease activity of 447.65 7 2.6 U/mL by a prediction model of 445.48 U/mL. The enzyme showed a molecular weight of 59 kDa; it was inhibited in the presence of PMSF (serine protease); it presented optimal conditions at pH 8.0 and 50°C; it remained stable at pH in the 3.0-9.0 range and between 10 and 40°C; it showed a tolerance to 3000 mM NaCl as well as to surfactants, hydrogen peroxide and urea at 5%. This paper presents a proposal for an economically attractive production methodology using cactus pear as a primary source of carbon. In addition, the protease secreted by Acremonium sp. L1-4B presented a combination of biochemical characteristics that grants a promising variability of biotechnological applications.

Biochemical and thermodynamic characteristics of a new serine protease from Mucor subtilissimus URM 4133

“…Moreover, that the best production conditions were found at 35 °C using 0.25 g of wheat bran, 0.2 g of soybean meal and 0.2 g of monobasic potassium phosphate (KH 2 PO 4 ), obtaining a maximum proteolytic activity of 8.943 ± 0.220 U.mL −1 . Nascimento et al [ 23 ], working with statistical planning for the optimization of extracellular serine protease production by Acremonium sp. L1−4B, used a factorial 2 3 as the first-order planning and obtained quite different results between assays, with proteolytic activities ranging from 0.07 ± 0.01–191.32 ± 0.01 U.mL −1 .…”

“…However, even after the 24 h incubation period, the enzyme remained above 50 % of activity at 40 °C. Proteases produced by mesophilic fungi are generally denatured at temperatures above 50 °C [ 23 , 35 ]. Similar results were obtained by Areces et al [ 36 ], for the protease produced by Mucor bacilliformes , a mesophilic species, with residual activity of 29 % at 55 °C and only 8% after 30 min.…”

Cold-adapted enzymes produced by fungi from terrestrial and marine Antarctic environments

“…In the study reported by Turkiewicz et al [107], the serine protease secreted by Glaciozyma antarctica 107 (formerly Leucosporidium antarcticum) was most active at 25.0 C and exhibited activity even at À10.0 C. On the other hand, the serine protease purified from Acremonium sp. L1-4B was active at temperatures ranging from 10.0 to 40.0 C [82]. In another study, the protease produced by Pseudogymnoascus pannorum (formerly Geomyces pannorum) exhibited the greatest activity at 4.0 C [7].…”

“…Antarctic fungi able to produce proteases were recovered from soil [7,12,80,99], marine invertebrates and macroalgae [18,34], melt water [12], and lichens [82]. Protease screening has been measured in solid media with skim milk, defatted milk, or casein.…”

“…Antarctic fungi reported as protease producers belong to the genera: Acremonium, Candida, Cryptococcus, Chrysosporium, Embellisia, Exophiala, Geomyces, Glaciozyma, Glomerella, Leuconeurospora, Leucosporidium, Mrakia, Phoma, Pseudogymnoascus, Rhodotorula, Trichoderma, Vanrija, and Wickerhamomyces [12,18,33,34,[80][81][82][83]99,105]. In most cases, the secretion of proteases was dependent on the medium composition, temperature, and the phase of growth [80][81][82]. In the study performed by Duarte et al [18], 14% of the Antarctic yeasts were able to produce proteases in solid and liquid media.…”