Purificação e caracterização bioquímica de xilanase-I produzida por Penicillium crustosum e sua aplicação.

Abstract

The! xylanolytic! complex! is! formed! by! a! group! of! enzymes! capable! of! acting! on! multiple!binding!sites!in!the!chain!of!xylan,!hemicellulose!one!found!in!plant!cell!walls! and! to! degrade! it! into! Xylooligosaccharides,! xilotrioses,! xilobioses! and! xylose.! The! endoI1,4IβIxylanases! (1,4IβIDIxylan!xylanohydrolasen!EC!3.2.1.8),!which!hydrolyze! the! xylan! backbone! randomly,! producing! a! mixture! of! xylooligosaccharidesn! βI xylosidases!(EC!3.2.1.37)!which!hydrolyze!only!xylan!short!chains,!releasing!xylosen! Acetyl! xylan! esterases! (EC! 3.1.1.72)! which! hydrolyse! acetate! groups! of! the! main! chain!and!αILIarabinofuranosidases!(EC!3.2.1.55)!which!hydrolyse!arabinofuranose! side!chains.!In!recent!decades,!interest!in!microbial!xylanase!has!increased!because! of! its! potential! for! biotechnological! applications:! they! have! been! used! in! various! industrial!sectors,!including:!the!supplementation!of!feed!monogastric!animalsn!in!the! preIbleaching!of!pulp!and!paper!pulpn!the!saccharification!of!lignocellulosic!biomass,! in!the!textile!industry,!in!baking!and!clarification!of!juices!and!wines.!In!this!context,! this!study!aimed!to!purify!and!characterize!biochemically!the!xylanaseII!of!the!fungus! Penicillium#crustosum!using!corn!stover!as!inducer!source!in!liquid!culture,!and!their! biotechnological!applications.!The! xylanaseII! of!P.# crustosum!was!purified! from! the! crude!extract!obtained! from! stationary! liquid!culture!supplemented!with!corn! stover! 2%.! After! three! chromatographic! columns! (DEAE,! CMISephadex! and! filtration! column! Sephadex! GI75)! the! enzyme! exhibited! homogeneity! and! presented! a! molecular!mass!of!23.44!kDa!single!band.!The!xylanaseII!exhibited!optimum!pH!5.0! and! stability! in! the! pH! range! from! 4.0! to! 7.0.! The! optimum! temperature! of! the! xylanase!was!50!°C,!with!a!half!life!of!62!minutes!at!50!°C,!but!at!40!°C!and!45!°C! the!enzyme!showed!halfIlife!(T½)!of!120!minutes!and!74!minutes,!respectively.!The! kinetic! parameters! for! the! Beechwood! xylan! substrate! Km! was! 1.56! mg/mL! and! Vmax!1,000!mol/!min!/!mL.!EDTA!compounds!and!Mn2+!increased!xylanase!activity! by!47%!and!75%,!respectively,!while!Na+!and!Ca2+!inhibited!the!enzyme!activity!18%! and! 22%! at! a! concentration! of! 5! mM.! The! crude! extract! and! purified! xylanaseII! showed! no! cytotoxic! effect,! thus! they! can! be! applied! in! the! food! industry.! The! xylanaseII!was!effective! in! clarifying! the!guava! juice,!pear!and!mango,! resulting! in! turbidity! of! 91,! 87! e! 89%,! respectively.! Therefore,! the! xylanaseII! P.! crustosum! showed! relevant! properties! for! application! in! food! industry,! especially! for! clarifying! juices!process.!