Degradation Characteristic And Mechanism Of Cellulose By Penicillium Melinii

The microbe degradation would be most potential and ideal way for application of cellulose. Because the degradation activity of microbe is finite, mechanism of cellulose biodegradation is focus on the single and model microbe, so far application of cellulose is relatively deferrable. In order to solving of present environmental and energy crisis, the high degradation activity microbe screened and research of mechanism of cellulose biodegradation would have important theory and practice value.The 16 strains were screened from 69 cellulose degradation strains from 35 samples through primary screening of CMC-Na congo red gel plate, addition of filter paper culture, the 5 more higher cellulase activity strains were gained after 16 strains were cultured in liquid state fermentation based on CMC and wheat bran as carbon resources, respectively, among strain was identified as P. melinii Thom in the pre-trail. The unknown field of P. melinii Thom Z18 was further investigated as target because of no literature reported.The effect of culture condition on cellulase production of P. melinii in liquid state fermentation was investigated by stepwise, and the result shows that the wheat bran in induction cellulase was better than that of straw, maize stalk and Leymus chinensis, BG Activity was significantly depressed by urea, and the 28℃and pH5.0 were the optimal fermenting temperature and acid value. The activity of CMCase and BG were better in high oxygen dissolution, whereas the FPA was unstable under the increased shear force with rotated rate. The cellulase attained activity highest value when the culture time reached to about 11 d in the Switzerland bioengineering co NLF19 bioreactor. The characteristic of P. melinii cellulase are investigated through degradation of carboxymethylcellulose sodium, microcrystalline and Sigmacell type 50, the results show that activity of cellulase was evidently depressed when the concentrations of substrates beyond 5%. The optimal reaction systems are the temperature of 50-60℃and acidity of pH5.5.The slow rate mechanism of cellulose degraded was presented with reaction time extended through monitor the change of cellulase activity in the total course of hydrolysis when the Sigmacell type 50 was used as research target. The activity of cellobihydrolases (CBH) rapidly descended in the total course of cellulose hydrolysis so as to reduce the sugar yield, at the same time the token on most low thermostable, and the activity of cellobihydrolases declined up to 53.71% in the preservation 45℃and 20.94% in the degradation of Sigmacell type 50 at 12 d. So the cellulase losing activity especially was important reason to inefficient rate of cellulose degraded, another reason was the structure change of cellulose super molecular. The result of X-ray diffraction and infrared spectrum to cellulose under cellulase action showed that cellulase could degrade cellulose amorphous and crystalline fractions synchronously; hydrolysis of amorphous region cellulose was more preferential than that of crystalline region only on primary hydrolysis stage. The crystalline index shows reduced trend in the extended course of cellulose hydrolysis. The CBH adsorption capability to micro fiber suggests descend with amorphogenesis of cellulose micro fiber arrangement. Super molecular structure of hydrolysis cellulose by cellulase presents transform trend from celluloseⅠto celluloseⅡ, but not transform trend fromⅠαcrystal phase toⅠβcrystal phase, observation suggests that the hydrolysis process results in a gradual increase at the relative intensity of the hydrogen bond network.The TBA reaction was unusually used to detect HO˙ radical. Multi index detected results suggest that P. melinii can produce HO˙ radical in culture based on cellulose induction. Observations of X-ray diffraction and infrared spectrum of cellulose in treatment of hyper filter liquid suggested that cellulose crystalline index and infrared index reduce with time. The hydroxyl of cellulose glucose has already been transformed carboxyl in term of infrared spectrum analysis, and the result showed that the oxidation mechanism was perhaps universal in the degradation of cellulose by P. melinii. The digestibility of pretreated cellulose after Fenton reagents could be declined in terms of reduce sugar yield, because lots of the reducing ends was produced after pretreatment, although the CrI of pretreated cellulose was lower than that of original cellulose. The mechanism of HO˙ radical reaction in P. melinii is more intricacy than that of Fenton system. Cellulase was extracted from P. melinii cultured in Bioengineering NLF19 bioreactor, and then was separated by ammonium sulfate precipitation, and finally enzymes purified by sephadexG-100 chromatography. The BG was purified by sephadexG-100, EGⅠand EGⅡwere purified by sephadexG-100 and DEAE Sephadex A-50. The molecular weight of BG was 129kD, the optimal acid value and temperature are pH 6.0 and 60℃, respectively, the dynamic constants Vm of BG shows strong hydrolysis capability ofβ-glucan bond, and reveals an extraordinary upstanding application prospect.The molecular weight of EGⅠwas 29kD, the optimal acid value and temperature are pH 3.5 and 40℃, respectively. The dynamic constants Vm of EGⅠ, anti-acid, enduring low temperature and obviously rapid hydrolysis of whatman filter paper show that it was an important component and holds an outstanding value of research and application in pulp industrial etc. The characteristic of EGⅡwere investigated. The molecular weight of EGⅡwas 38kD, the optimal acid value and temperature are pH 6.0 and 60℃, respectively. Although relative slow the Vm and hydrolysis rate of EGⅠ, relative high activity to xylan reveals a nicer application in amylose chemical field etc.The application of foundation of P. melinii in the practice was created, it was well showed that cellobihydrolases (CBH) will be as a future important orientation of molecular modification, mechanism of the degradation of cellulose reveals combination of cellulase hydrolysis and radical oxidation through the above studies. So the paper has great theoretical and practical significance for the efficient utilization of cellulose resources.