Expression Of An Alkaline Feruloyl Esterases From Thermophilic Chaetomium Thermophilum And Its Application In Wheat Straw Pulp Bleaching

The chemical ester bond between lignin and hemicellulose in the three-dimensional network structure of lignocellulose seriously hinders the application of biological enzymes in the pulping and papermaking process and affects the removal efficiency of lignin.Feruloyl esterase（FAE,EC 3.1.1.73）can hydrolyze cross-linked ester bonds.However,the removal of lignin in the bleaching stage is performed under high temperature and strong alkaline conditions,in which the use of neutral or acidic FAE will lose its catalytic activity.It is difficult to obtain alkali-tolerant and high-temperature-resistance FAE by wild strain screening or protein engineering.Therefore,it is very meaningful to select alkali-tolerant thermophilic microorganisms to obtain both high-temperature-resistance and alkaline FAE directly.In this paper,feruloyl esterase B gene（Ctfae B）from Chaetomium thermophilus HY026 was cloned,and Pichia pastoris engineering strain CtFAEB1 was constructed.A new alkaline and heat-resistant recombinant feruloyl esterase（CtFAEB）was obtained,and its application in wheat straw pulp bleaching was studied.The main conclusions are as follows:（1）Cloning of alkaline Ctfae B gene from C.thermophilum and sequence analysis.With feruloyl esterases protein of Neurospora crassa as the target sequence,the whole genome of C.thermophilus was searched to obtain the potential feruloyl esterases sequence（Identity:73.3%）.The mature coding peptide Ctfae B gene was obtained by gene splicing by Gene splicing by overlap extension PCR（SOE-PCR）amplification.Sequencing indicates that the Ctfae B gene was a 870 bp gene,encoding 289 amino acids and containing a secretion signal peptide of 18 amino acids（MLLPSLFGLATMASSCLA）.The predicted molecular weight and isoelectric point of the mature FAE protein was 29,735 Da and 7.6,respectively.The protein structure of CtFAEB was successfully constructed by SWISS-MODEL structure prediction server.The CtFAEB belongs to theα/βhydrolase superfamily and esterase subfamily（cd00312）,and contains the typical catalytic triad structure of esterase（118Ser-254His-201Asp）.The FAE protein sequences from thermophilic fungi（C.thermophilu,Thielavia terrestris,Humicola insolens and Myceliophthora thermophila）and mesophilic bacteria（Chaetomium Globosum）were compared.The results indicates that the residues with strongα-helix formation ability replaced the residues with weak helix formation ability in the FAE protein sequence of thermophilic fungi,which led to the increase of proteinα-helix stiffness and improved the stability of protein structure.（2）Construction of engineering Pichia pastoris secreting recombinant CtFAEB.With the aim of increasing the production of recombinant CtFAEB,Ctfae B gene from C.thermophilus was optimized according to the codon bias of P.pastoris.The original ferulic acid esterase gene and codon optimized gene expression vector of Chaetomium thermophilus were successfully constructed.The codon-optimized yeast expression vector p PIC9K-Ctopfae B of feruloyl esterase gene was successfully constructed.The recombinant expression vector was transformed into Pichia pastoris GS115 by electrotransformation,and the results of transparent circle of ethyl ferulate plate showed that the expression level of optimized gene Ctopfae B was higher.Taking the transformant CtFAEB1 with the largest transparent circle diameter as the research object,when the temperature was 24℃,the inoculum size was 4%,and the methanol induction time was120 h,the yield of recombinant CtFAEB was the highest,reaching（7.83±0.61）U/m L,and the total protein content in fermentation broth was（227.4±1.1）μg/m L.The recombinant CtFAEB has an optimal temperature of 65℃ and p H of 7.0.Under the condition of p H 7.0,the enzyme has an excellent thermal stability at 50～65℃and after treated at 65℃for 1 h,CtFAEB can still retain 63.21%of its maximum activity.The relative enzyme activity remained above 60%in the range of p H 3.0～10.0,which indicated that the recombinant CtFAEB had good thermal stability and extensive p H stability.Except that Cu²⁺can inhibit the enzyme activity,other metal ions have different degrees of activation.Substrate specificity analysis showed that the enzyme belonged to the type B FAE family.（3）Bio-assisted bleaching of pulp with recombinant CtFAEB.When CtFAEB was10 U/g,the release of phenolic compounds,hydrophobic compounds and lignin compounds was the highest when xylanase bleached wheat straw pulp.Compared with xylanase alone,the addition of recombinant CtFAEB can greatly promote the release of phenolic compounds,hydrophobic compounds and lignin compounds from wheat straw pulp by xylanase.At 70℃,recombinant CtFAEB and xylanase could still reduce the Kappa number of the wheat straw pulp to（5.2±0.19）;At p H 9.0,the Kappa number of the pulp was reduced to（4.4±0.18）;The bleaching results showed that the recombinant CtFAEB had excellent heat and alkali resistance.Adding recombinant CtFAEB and xylanase-assisted bleaching before chemical reagent treatment can reduce the chemical test dose of 10～20%in the chemical bleaching section.The viscosity and scanning electron microscope（SEM）characterization of the pulp treated with recombinant CtFAEB combined with xylanase and treated with xylanase alone showed that the viscosity（EP）of the pulp treated with recombinant CtFAEB combined with xylanase was7650.24 Pa·S,which was 30.33%higher than that of the pulp treated with xylanase alone,which meant that the quality and yield of the paper were improved.SEM could observe that after CtFAEB treatment,the split fiber of pulp fiber was smaller,more grooves and cracks appeared,and the shedding of fiber sheet and the loss of compactness were found locally.The application results of pulp bleaching showed that the addition of recombinant CtFAEB could promote the hydrolysis of hemicellulose by xylanase and greatly improve the efficiency of xylanase treatment of wheat straw pulp.