Construction And Immobilization Of The Novel Multifunctional Enzymes And The Mechanism Of Synergistic Degradation Of Lignocellulose And Improvement Of Tea Quality

Plant cell wall is the most abundant biomass resource on earth,and lignocellulose is the main component of plant cell wall.Lignocellulose consists of cellulose,hemicellulose and lignin,and its complete degradation requires the synergistic action of many enzymes.Lignocellulosic degrading enzymes have important application value in the fields of food processing such as tea and juice flavoring,brewing,baking,production of functional food raw materials,and disposal of food industrial wastes.Under natural conditions,bacterial and fungal communities secrete a range of lignocellulosic degrading enzymes and play an important role in the food industry.However,high production cost,low hydrolysis efficiency,poor stability and single function of enzymes are the main bottleneck restricting the effective utilization of lignocellulosic biomass.It is of great significance to develop efficient,stable and multifunctional lignocellulosic degrading enzymes and improve the methods of protein purity and immobilization.Compared with single functional enzymes,multifunctional enzymes can act on different substrates simultaneously,reducing the type and amount of enzymes required for hydrolysis reactions and greatly reducing the cost.Therefore,the research of multifunctional enzymes is more attractive than that of single functional enzymes,especially those involved in hemicellulose degradation.Firstly,the functional domain of bifunctional xylanase/feruloyl esterase Xyn-Fae of Prevoella ruminicola 23 was determined by the method of amino acid truncation at the end of protein,and a novel bifunctional enzyme was constructed.The synergistic effect of this enzyme and cellulase greatly improves the degradation efficiency of lignocellulose and releases ferulic acid,xylo-oligosaccharide,reducing sugar and other functional substances,which has important application value in the field of food processing.Subsequently,the ELP-intein purification strategy and spore surface display technology were applied to the purification and immobilization of a novel bifunctional enzyme,which simplified the process of protein purification,reduced the cost of purification and improved the activity and stability of enzymes,broke through the technical bottleneck of bifunctional enzyme application,and expanded the application range of the above new technologies.Finally,a new multifunctional fusion enzyme GXF was successfully constructed,and the multifunctional enzyme was first applied to improve the quality of Longjing tea.GXF has a variety of activities includingβ-glucosidase,xylanase,feruloyl esterase and acetyl xylan esterase.Through the synergistic effect of multifunctional enzyme,GXF can significantly improve the aroma of Longjing tea,improve the biological active substances in Longjing tea soup,and provide a new idea for the improvement of tea quality.In addition,the construction of the new multifunctional enzyme has important reference value for the research of enzyme engineering and food enzymatic processing.The main results were as follows:（1）The functional domain of Xyn-Fae was revealed,and a novel bifunctional xylanase/feruloyl esterase Xyn Ⅱ-Fae was constructed,which could cooperate with cellulase to degrade lignocellulose efficiently.There was a significant synergistic hydrolysis between Xyn Ⅱ-Fae and commercial cellulase.When Xyn Ⅱ-Fae and commercial cellulase were added in a ratio of 4:6,the release of reducing sugar was increased by 65%compared with cellulase alone,and the conversion rate of xylan and glucan was increased by 125.1%and 54.3%,respectively.（2）The efficient expression and simple purification of Xyn Ⅱ-Fae were realized based on ELP-intein and His-ELP-intein tags.In order to simplify the process of protein purification and save purification time and cost,the protein purification system without affinity chromatography based on ELP-intein tag and the protein purification system without imidazole and high concentration salt based on His-ELP-intein tag were constructed,and the bifunctional enzyme Xyn Ⅱ-Fae was purified.The optimal purification conditions of ELP-intein-Xyn Ⅱ-Fae are as follows:the concentration of（NH₄）₂SO₄is 0.5 mol·L^-1,the phase transition temperature is 31.2℃,and most of the proteins can be precipitated at 42℃.The optimal purification conditions of His-ELP-intein-Xyn Ⅱ-Fae were as follows:（NH₄）₂SO₄concentration 0.4 mol·L^-1and phase transition temperature 34.1℃.The protein concentration and enzyme activity of Xyn Ⅱ-Fae purified by His-ELP-intein tag were better than that of ELP-intein.（3）The Bacillus subtilis surface display system was established to improve the stability and reuse performance of Xyn Ⅱ-Fae.In order to improve the stability,easy separation and reuse ability of Xyn Ⅱ-Fae,a display system based on the surface adsorption and covalent binding of B.subtilis was constructed using the spores as the immobilized substrate.The detection methods of Western blot and different proteases on the surface of spores proved that both adsorption and covalent binding methods can successfully display and express Xyn Ⅱ-Fae on the surface of spores.The covalent binding efficiency of Xyn Ⅱ-Fae on the surface of spores was 69.96%,which was significantly higher than the immobilization efficiency of surface adsorption（36.66%）,and the covalent binding of Xyn Ⅱ-Fae showed higher activity and stability than free enzymes in a wide range of p H and temperature.（4）A multifunctional fusion enzyme withβ-glucosidase,xylanase,feruloyl esterase and acetyl xylan esterase activities was constructed,which had synergistic degradation effect on lignocellulose.In order to increase the activity and function of the enzyme,β-glucosidase gene from Thermobifida fusca was fused with flexible ligand peptide（GGGGS）₃on the basis of Xyn-Fae to constructβ-glucosidase/xylanase/feruloyl esterase multifunctional fusion enzyme（β-Glu-Xyn-Fae）.The affinity and catalytic efficiency of the N-terminal of GXF were better than those of the parental enzyme,however,the catalytic efficiency of C-terminal fusion enzyme XFG was better than that of parental enzyme,and the catalytic efficiency of xylanase,feruloyl esterase,acetyl xylan esterase were lower than that of parental enzymes.The optimal conditions and stability of GXF enzymes are very similar,and there is a strong synergistic effect among all parts,which can effectively promote the hydrolysis of corn straw,wheat straw,bagasse and other agricultural residues.（5）Multifunctional fusion enzyme GXF can enrich tea endoplasm and significantly improve tea quality through the synergistic action of multiple enzymes.Headspace solid phase microextraction combined with GC-MS,LC-MS/MS,HPLC and other detection techniques were used to explore the effects of multifunctional enzyme on the improvement of aroma and quality of Longjing tea.The results showed that the activity ofβ-glucosidase,xylanase,feruloyl esterase and acetyl xylan esterase of the multifunctional enzyme GXF had high synergistic effect.GXF can significantly promote the aroma of Longjing tea,increase the contents of ferulic acid,xylo-oligosaccharide and reducing sugar,promote the transformation of flavonoid glycosides,increase the bioactive substances and improve the content and quality of Longjing tea soup.