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A study of selected xylanolytic and chitinolytic enzymes from Rasamsonia emersonii, and their potential application in the valorisation of mushroom-production waste streams.

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dc.contributor.advisor Walsh, Gary
dc.contributor.author Dwyer, Kelly
dc.date.accessioned 2020-02-05T12:53:25Z
dc.date.available 2020-02-05T12:53:25Z
dc.date.issued 2019
dc.identifier.uri http://hdl.handle.net/10344/8484
dc.description peer-reviewed en_US
dc.description.abstract The Irish mushroom production industry continues to grow rapidly. The two main waste streams from this are spent mushroom substrate (SMS) and mushroom stalks. This study focuses on identifying and recombinantly-producing novel thermostable glycosyl hydrolase enzymes with potential application in the valorisation of these waste streams. Two GH10 xylanases and three GH18 chitinases were expressed in P. pastoris. One xylanase (Xyn1) comprises of only a GH10 catalytic domain, the other (Xyn12) contains a GH10 catalytic domain and a CBM1 domain at the C’ terminus. The chitinases comprise of a GH18 catalytic domain only (Chit1), a GH18 catalytic domain and a ChitBM domain (Chit 2), and a GH18 catalytic domain and a substrate insertion domain (Chit3). The enzymes originate from the genome of the thermophilic filamentous fungus R. emersonii, which is known to produce thermostable commercially interesting enzymes. The xylanases display low pH (4-4.5) and high temperature (70-80°C) optima. They are thermostable, with over 50% relative activity remaining after 48h incubation at 65°C. To further understand the role of the CBM1, both enzymes underwent protein engineering whereby the CBM1 and linker region were removed from Xyn12, and added to the C’terminus of Xyn1. This affected the stability of the enzymes; the removal saw a drop in relative activity from 84% to 46% and the addition increased the thermostability from 65% to over 90% after 48h incubation at 65°C. All chitinases displayed endo-chitinase and chitobiase activity. Chit3 also displayed some N-acetyl-glucosamidase activity, likely due to the substrate insertion domain causing catalytic-cleft deepening. The chitinases displayed temperature optima of 50 – 55 °C, and low pH optima (pH 4.5 or lower). In particular, Chit3 indicated a pH optimum of pH 2.8. As far as we are aware, this is the lowest pH optima of a fungal chitinase to date. Chit2 displayed the highest chitin-degrading ability at 3456 μmol/mg/mL on 4-NP -triacetylchitotriose, with Chit1 displaying 403 μmol/mg/mL, and Chit3 displaying 268 μmol/mg/mL (on 4-NP-chitobioside). The chitinases displayed thermostability; Chit1 was markedly thermostable with over 70% relative activity remaining after 48h incubation at 50 °C. Chit2 displayed the lowest thermostability with 14 % remaining after 48h incubation at 50 °C. During preliminary industrial testing, Xyn1 yielded a notable reduction in viscosity of WE-SAX of 79.3% in 15 mins in comparison to controls. Xyn1 produced short-chain xylooligosaccharides from the same substrate. Chit1 and Chit3 displayed efficacy in hydrolysing shrimp and fungal chitin. These features, coupled with the thermostability of the enzymes, indicate suitability of Xyn1 for application in wheat-starch processing and animal feed, and suitability of the chitinases in valorisation of waste streams of industrial shrimp and mushroom production, and in the healthcare industry namely in the production of glucosamine supplements and designed human milk oligosaccharides. en_US
dc.language.iso eng en_US
dc.publisher University of Limerick en_US
dc.subject Irish mushroom production industry en_US
dc.subject xylanolytic and chitinolytic enzymes en_US
dc.subject industrial testing en_US
dc.title A study of selected xylanolytic and chitinolytic enzymes from Rasamsonia emersonii, and their potential application in the valorisation of mushroom-production waste streams.
dc.type info:eu-repo/semantics/doctoralThesis en_US
dc.type.supercollection all_ul_research en_US
dc.type.supercollection ul_published_reviewed en_US
dc.type.supercollection ul_theses_dissertations en_US
dc.contributor.sponsor IRC en_US
dc.rights.accessrights info:eu-repo/semantics/openAccess en_US


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