| Microbial esterases have attracted considerable attention in recent years due to their potential applications in food,pharmaceutical,environmental,textile and detergent industries.In this thesis,the gene cloning,high-level expression,characterization and application of a cutinase from the thermophilic fungus Thielavia terrestris,a cutinase and a lipase from the thermophilic fungus Malbranchea cinnamomea were investigated.The main results are as follows:(1)A cutinase gene from T.terrestris was codon optimized and efficiently expressed in Pichia pastoris.The codon adaption index was increased from 0.52 to 0.91,and the GC content was decreased from 66.7%to 45.2%after codon optimization.The recombinant cutinase(TtCutopt)was secreted into the medium at a high level of 5.3 mg mL-1 having an activity of 10200 U mL-1 in 5-L fermentor.The optimal pH and temperature of TtCutopt were 7.0 and 50 ?,respectively.It was highly stable in a broad pH range of 3.0-11.0 and up to 85 ?.TtCutopt displayed broad substrate specificity and showed the highest activity towards p-nitrophenyl butyrate and tributyrin,with specificity activity of 2322.4 U mg-1 and 1152.5 U mg-1,respectively.The enzyme was extremely stable in organic solvents and surfactants.TtCutopt efficiently synthesized butyl butyrate,hexyl butyrate,butyl hexanoate and hexyl hexanoate,with esterification efficiency of more than 95%.Furthermore,it efficiently degraded phthalate esters with short alkyl chains to their corresponding monoalkyl phthalates,with degradation rates of more than 90%for dimethyl phthalate,diethyl phthalate,dipropyl phthalate and dibutyl phthalate within 24 h.TtCutopt exhibited great potential application in flavor esters synthesis and phthalate esters biodegradation.(2)A novel cutinase gene(McCut)was cloned from M.cinnamomea for the first time and expressed in P.pastoris.It has an open reading frame(ORF)of 648 bp encoding 215 amino acids.The deduced amino acid sequence of McCut showed the highest identity of 65%with the cutinase from Aspergillus nidulans.The highest cutinase activity of 12536 U mL-1,with protein concentration of 10.8 mg mL-1 was achieved in 5-L fermentor,which is by far the highest production for a cutinase.McCut was purified by QSFF ion-exchange chromatography with a specific activity of 1181.6 U mg-1.It was optimally active at pH 8.0 and 45 ?.It exhibited excellent stability within pH 2.5-10.5 and up to 75 ?.The cutinase displayed broad substrate specificity with the highest activity towards p-nitrophenyl butyrate and tributyrin.Moreover,it was capable of hydrolyzing polycaprolactone and poly(butylene succinate).The crystal structure of McCut was resolved by X-ray crystallography.It is an ?/? hydrolase with a central ?-sheet of five parallel strands surrounded by nine a-helices.McCut efficiently synthesized butyl butyrate,hexyl butyrate,butyl hexanoate and hexyl hexanoate,with esterification efficiency of more than 95%at 4 h.High production and excellent features of McCut make it useful in polyesters biodegradation and flavor esters synthesis.(3)A novel lipase gene(McLip)was cloned from M.cinnamomea and expressed in P.pastoris.It has an ORF of 1698 bp encoding 565 amino acids.According to sequence analysis,the deduced amino acid sequence of McLip shares the highest identity of 46%with the Candida rugosa lipase LIP4.McLip belongs to the Candida rugosa lipase like superfamily.The secreted lipase activity of 4304 U mL-1 was achieved in 5-L fermentor.McLip was purified by SP ion-exchange chromatography with a specific activity of 543.2 U mg-1.The optimal pH and temperature of the purified McLip were 7.5 and 40 ?,respectively.It was highly stable in a broad pH range of 3.0-9.0 and up to 45 ?.The lipase showed the highest activity towards p-nitrophenyl hexanoate,p-nitrophenyl laurate and tributyrin.McLip degraded several phthalate esters to their corresponding monoakyl phthalates,with degradation rates of more than 90%for dipropyl phthalate,dibutyl phthalate and dihexyl phthalate. |
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