Characterization of a Thermotolerant Phytase Produced by Rhizopus microsporus var. microsporus Biofilm on an Inert Support Using Sugarcane Bagasse as Carbon Source

Abstract

The Rhizopus microsporus var. microsporus biofilm was able to produce increased levels of an extracellular thermotolerant phytase using polyethylene and viscose as an inert support in both modified NBRIP medium and modified Khanna medium containing sugarcane bagasse as the carbon source. The enzyme production was strictly regulated by the phosphorus content with optimal production at 0.5 mM of sodium phytate and KH2PO4. The extracellular phytase, RMPhy1, was purified 4.18-fold with 4.78 % recovery using DEAE-cellulose and CM-cellulose. A single protein band with a molecular mass of 35.4 kDa was obtained when the samples were subjected to 10 % SDS-PAGE. The optimum temperature for activity was 55 °C and the optimum pH was 4.5. R. microsporus var. microsporus phytase exhibited high stability at 30 and 40 °C with a half-life of 115 min at 60 °C. The enzyme activity increased in the presence of Ca (2+) and was inhibited by Zn(2+), arsenate, and sodium phosphate. Phytase demonstrated high substrate specificity for sodium phytate with K m = 0.72 mM and V max = 94.55 U/mg of protein and for p-NPP with K m = 0.04 mM and V max = 106.38 U/mg of protein. The enzyme also hydrolyzed ATP, AMPc, glucose 6-phosphate, glucose 1-phosphate, and UDPG. This is the first report on phytase characterization delivered with biofilm technology. The properties of the enzyme account for its high potential for use in biotechnology and the possibility of application in different industrial sectors as feed in the future.

Extracellular Tannase from Aspergillus ochraceus: Influence of the Culture Conditions on Biofilm Formation, Enzyme Production, and Application

“…morphologic characteristics were also observed for the A. niger[34] and R. microsporus[14] biofilms. According toHarding et al (2009) …”

“…Another way to produce fungal enzymes is through the fungal biofilm technology. This method of production has been described previously in cellulase production by Aspergillus niger described by Gutierrez-Correa et al (2012) [13], phytase production from Rhizopus microspores described by Sato et al (2016) [14], and βfructofuranosidase production by A. japonicus described by Mussato et al (2009) [15]. Fungal biofilm fermentation combines both submerged and solid-state fermentation, allowing high production of biomolecules such as enzymes.…”

Purification and characterization of an iron-activated alkaline phosphatase produced by Rhizopus microsporus var. microsporus under submerged fermentation using rye flour

“…In spite of the use of agroindustrial byproducts, this is the first time that rye flour is used as carbon source targeting the production of a fungal alkaline phosphatase. Rye flour presents calcium, potassium, sodium and phosphorus as mineral constituents [27] and the presence of phosphorus was demonstrated to be positive for phosphatase production by R. microsporus [28]. Phosphorus impacts directly upon the production of phosphatases and for many filamentous fungi, the regulation of genes encoding The SbmF was performed using Khanna medium with initial pH adjusted to 6.3, with addition of 0.5% (w/v) rye flour as additional carbon source, for 76 hours at 32°C under orbital agitation (100 rpm).…”

Microorganisms: the secret agents of the biosphere, and their key roles in biotechnology

“…A lipase produced by the endophytic fungus Cercospora kikuchii was studied under the project "Bioprospecting in fungi: the search of lead compounds for drug design and enzymes for pharmaceutical and industrial applications" (Costa-Silva et al 2014) (grant 2004/07935-6). Although many projects aimed at the isolation and characterization of lipases, other enzymes with potential industrial applications were also isolated and characterized, including tannases, phytases, cellulases, among others (Sato et al 2016, Alves et al 2018, Cavalcanti et al 2018. The use of environmental fungal strains in the biotransformation of organic molecules is also an important addressed goal (de Carvalho et al 2010, Capel et al 2011, Severiano et al 2013, de Souza et al 2021.…”