Screening, isolating and characterizing acetyl xylan esterase enzymes from a novel ecological niche

Marais, Charl (2015-03)

Thesis (MSc)--Stellenbosch University, 2015.

Thesis

ENGLISH ABSTRACT: Hemicellulose, a plant cell wall polysaccharide composed amongst other 6-carbon sugars of xylose, a 5-carbon fermentable sugar, has various side-chains which hinder breakdown by the hydrolytic enzyme xylanase. We executed a screen of a metagenomic library established from heat treated saw dust to isolate novel genes for plant cell wall esterases and isolated two clones that potentially could be utilised as acetyl xylan esterases: HEL12 and HEL37 from Citrobacter farmeri and Bacillus vallismortis respectively are confirmed short-chain acetyl xylan esterases (C2-C4) with an optimal temperature of 30°C and 35°C and pH 8.0. HEL12 (33kDa) and HEL37 (25kDa) are small, dextrous acetyl xylan esterases with HEL37: Km of 1.621mM for p-nitrophenyl acetate and Km of 3.571mM for the substrate p-nitrophenyl palmitate with a Vmax of 2.462 mMol/min/mg protein and Vmax of 0.4363 mMol/min/mg protein respectively. In addition HEL12 was active on other substrates with a Km of 1.321mM protein for p-nitrophenyl acetate and Km of 1.692mM for the substrate p-nitrophenyl butyrate with a Vmax of 3.812 and Vmax of 1.523 mMol/min/mg protein respectively. Both enzymes were assayed on various acetylated polymers including acetylated xylan to indicate their ability to hydrolyse plant lignocellulosic polymers.

AFRIKAANSE OPSOMMING: Met die herwinde interisering in die energie krisis, is “herwinbare energie” vinnig besig om die nuwe modewoord te raak tussen verskeie industriële bedrywighede. Hemicellulose, ‘n plant sel wand polisakkaride opgemaak uit xilose, ‘n 5-koolstof fermenteerbare suiker, het verskillende sykettings wat ‘n hindernis veroorsaak in die hidrolase van die komponent deur xilanase ensieme. HEL12 en HEL37 van bakterieë Citrobacter farmeri en Bacillus vallismortis afsonderlik, is deel van die hidrolitiese groep van ensieme wat die hidrolisasie van ester sy-bindings op die xilaan suikerstring kataliseer. Die ensieme was primêr geassesseer vir aktiwiteit deur middel van sintetiese substraat 4-nitrofeniel asetaat. HEL12 en HEL37 was gevind om kort-ketting asetiel xilaan esterase (C2-C4) te wees, met ‘n optimale temperatuur van 30°C en 35°C afsonderlik, beide met ‘n optimale pH van 8.0. Met ‘n geskatte gewig van omtrent +- 30kDa is beide die ensieme van die kleiner meer behendige asetiel xilaan esterase, met HEL37: Km = 1.621 op 4-nitrofeniel asetaat en Km = 3.571 op substraat 4-nitrofeniel palmetaat, en Vmax = 2.462 met ‘n Vmax = 0.4363 afsonderlik, en HEL12: Km = 1.321 op 4-nitrofeniel asetaat en Km = 1.692 op sustraat 4-nitrofeniel buteraat met ‘n Vmax = 3.812 en Vmax = 1.523 afsonderlik.

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