Studies On The Tobacco Pectin Biodegradation And Application Technology

Pectin is one component of the cell wall of tobacco and other plants, is a class of more complex polysaccharides, which is mainly linked by D-galacturonic acid and D- galacturonic acid methyl ester with α-1,4 glycosidic. Pectin content of tobacco products is 5%-13%.Pectin in tobacco content is too high will attribute to the incomplete combustion of tobacco,and its decomposition will produce more methanol, acetic acid and other substances which will adversely affect the smoke flavor in cigarette. This study take use of the pectinase produced by the selef-screening microorganism to the degradating the pectin of tabcco stem.First, microbial fermentation conditions and degradation conditions were optimized and respectively researched the physical and chemical properties of the pectin hydrolysis,the research on purifing the pectinase was also conducted. finally, establishing polygalacturonic acid genetically engineered enzyme via setting Pichia bacteria as expression vector. We hope this study lay a good foundation for in-depth study of microbial fermentation of pectin degradation stems.Research by transparent circle screening and shake flask method rescreening selected two high-yield pectinase fungus, named sw06 and sw09. Determination of ITS sequence and phylogenetic analysis identified as Aspergillus niger and Penicillium janthinellum. Fermentor determine the best training cycle was 48 h and 72 h. Optimization of Aspergillus niger(Aspergillus niger) sw06 with response surface method optimum fermentation parameters:fructose 48.90 g / L, peptone content of 5.00 g / L, fruit powder content of 6.00 g / L. Under these conditions, the enzyme activity theoretically up 4198.45 U / m L, validate assay value(4175.65±21) U / m L, compared to the initial fermentation medium, the enzyme activity increased by 1.6 times; orthogonal experimental design optimization janthinellum(Penicillium janthinellum) sw09 optimum fermentation parameters: glucose 50 g / L, yeast extract 3g / L, KH2PO4 1g / L, fruit powder 2g / L. Where the carbon substrate and induce a significant influence on the enzyme activity.Pectinase was separated and purified by ammonium sulfate precipitation, Sepharose CL-6B molecular sieve column chromatography, DEAE-Sepharose FF ion-exchange columnchromatography, the results indicate: black niger pectinase producing single-component molecular mass 66.2k D. Crude pectin enzyme optimum p H was 5.0, and the optimum reaction temperature is 55 ℃. The crude pectin enzyme remained above 90% of enzyme activity when p H range from 3.0 to 5.0. Thermal stability of the enzyme or less 50 ℃, 2h later still maintain 56.7% activity, the thermal stability of the enzyme decreased when the temperature increased. The kinetics of enzymatic reactions in line with the Michaelis-Menten hyperbolic equation, Michaelis constant Km =(0.50±0.01) mg/m L, the maximum reaction rate Vmax=(5000.00±0.02)μg/(m L·min). Janthinellum pectinase producing single-component molecular mass 66.2k D. The crude enzyme optimum p H value of 5.0, and the optimum reaction temperature was 45 ℃. stable p H range of 4.0 to 6.0, the thermal stability of the enzyme than 50 ℃. The kinetics of enzymatic reactions in line with the Michaelis-Mentenhy perbolic equation, Michaelis constant Km =(1.67±0.03) mg / m L, maximum velocity Vmax =(3333.33±0.02) μg /(m L · min).This experiments use the pyrolysis products of degraded tobacco stem, tobacco stem powder, high-concentrated mixed slurry of three pilot sites for the study, the pyrolysis products of degraded tobacco stem was digested by crude enzyme produced by janthinellum,experimental results show that solid-liquid ratio of 1: 5, the reaction temperature is 50 ℃,treatment time 2h, pectin degradation rate up to 41.35%,the dry weight decreased from 3.07%to 1.81%.Infrared spectroscopy shows that pectin polysaccharide molecules stems mainly consists of α-glycoside bond, before its basic structure enzymatic functional groups did not change significantly. By GC-MS analysis, the pectin degradation product mostly galacturonic acid monomer form, but significant changes in the composition and content of pectin has occurred.Pectic substance average relative molecular weight reduced to 55 KDa from 341 KDa.SEC-MALLS determination of pectin qualitative absolute molecular weight, molecular weight from RMS radius and heavy double logarithm curve in determining pectin molecular conformation in aqueous solution. Pectin molecules exist as spherical, after enzymolysis,molecular form straight-chain type, undermine its spherical conformation shows pathways open into straight-chain type; All molecular weight(Mw) from 3.227×105 g/mol reduce to7.526×104g/mol, this has the same result with gel method.The galacturonic acid in the original peduncle after enzymatic hydrolysis reduced to 19.71% and 28.08% from39.89%.TGA showed pectin enzyme residue after heavy fell from 24.46% to 9.84%,flammability been improved. Pyrolysis analysis shows that after stem volatile acid content and nitrogen heterocyclic substances greatly reduced, sensory evaluation showed that after stem enzyme is added to cigarettes to reduce irritation, but aroma and slightly less momentum.Polygalacturonic acid gene extracted from A.niger sw09, recombinant plasmid p PICZαA,transformed into Pichia pastoris to build a strain of genetically engineered bacteria X33 /p PICZαA-PG. Transformant genomic fragment was 1608 bp. This specific fragment recovered after PCR amplification, sequencing find PG gene expression has been successfully inserted into the plant body. Positive clones were detected by SDS protein having a molecular weight of about 60 KDa, probably due to glycosylation of the impact, so that the molecular weight of the protein of 41 KDa higher than the theoretical value. Recombinant strains culture period for36 h, crude enzyme activity of 2872.91 U/m L. Fusion protein was purified by Ni-Sepharose affinity chromatography, testing for a clear band, the recombinant protein concentration was determined to be 8.1μg / μL.Online research results show that, the PG degrade pectic of paper with pulp slurry substance effect is good, with 8 ‰ adding quantity, pectic in paper with pulp reduced to 3.01% from 3.65%, the degradation rate is 17.53%.