Fiber Materials Enzymatic Mechanism Analysis And Application Of Strategy,

Cellulose is a biomass having the most potential in utilization. Its can be used to replace fossil energy. The main problem in its utilization is the conversion of cellulose to fermentable reducing sugars.Because of the insolubility and inhomogeneous structure of cellulose, and the complexity of cellulase system, it is difficult to design a rational quantitative assay using cellulose as substrate. However, The cornerstone of enzyme engineering is to achieve a direct correlation between the enzyme assays or screening approaches and the changes in enzyme functions in the desired application.Information of the catalysis mechanism and the effect of catalytic condition is very important in improving the cellulase enzyme performance. And the binding of enzyme and substrate is the prerequisite step of catalytic reaction. So far, the analysis in binding/ adsorption studies assumes a one-step reversible interaction between the ligand and receptor. However, as criticized in chemistry textbooks elsewhere, the above simple assumption only adapted for the elementary reaction, and is limited for multi-component systems. In addition, temperature is a very important factor of the enzymatic catalysis reaction. However, the method and the conclusion of studies about the effect of temperature on enzymatic reactions in kinetics, thermodynamics, and bioengineering are conflicting.The crude cellulase and the purified 1,4-β-D-glucan cellobiohydrolase I (CBHI) produced by a cellulolytic fungus Trichoderma pseudonkoningii S-38 were used as the material. A new approach to accurately estimating the potential capacity of a cellulase system for the hydrolysis of insoluble cellulosics is described, which can be used to evaluate the susceptibility of different cellulose. Based on the dynamic spectra of reaction, an assay method determining the activity of CBHI for p-nitrophenyl-D-cellobioside (PNPC) was established. The mechanism of CBHI binding to PNPC and the effect of temperature on catalysis in certain time courses were studied in detail. At last, the strategy in biomass utilization was discussed. New discoveries established during my Ph.D., were as follows:1. Establish a new approach to accurately estimating the potential saccharifying activity of a cellulase system for the hydrolysis of insoluble cellulosics, which can be used to evaluate the susceptibility of different cellulose.Because the classic cellulase assay methods do not take into account all of the effective factor, it prevents any accurate comparison to be made between the outcomes of different experiments.Specific Substrate conversion percentage (SSC) of cellulose is closely related to the saccharifying activity of cellulase. SSC was used to express the catalytic rate to overcome the influence cause by the concentration of enzyme and cellulose (Fig.1-A). The converting ability of cellulase for the cellulose can be expressed by the of d[SSC]/dt. For expressing the intrinsic saccharifying of cellulase, the total area under the curve (AUC) of d[SSC]/dt was used to reflect information of the total hydrolysis reaction (Fig.1-B). The Y-intercept of AUC-cellulase adding curve was linear related with the amount of cellulase added.Fig. 1 (A) Time courses of SSC, during hydrolysis for filter paper by different concentrations of crude cellulase; (B) The relationship between cellulase concentration and its AUC calculated as d[SSC]/dt.This method takes account into the influence of concentration of cellulase and cellulose added, and the hydrolysis time. Thus, it quantitatively reflects the saccharifying activity of cellulase, and reliable results were obtained that the method can also be used to determine the activity of cellulase for cotton fiber, Avicel PH101, and phosphate acid swollen cellulose (PASC). The comparison of morphology, structure, and the susceptibility of dilute and enzymatic hydrolysis between cellulose I and cellulose II showed that the susceptibility of cellulosics for enzymatic hydrolysis must be estimated by actual hydrolysis. The assay method can also be used for estimating the susceptibility of different cellulosics to evaluate the effect of cellulose treatment. The susceptibility of enzymatic catalysis of cotton fiber, Avicel PH101, and PASC was studied. The treatment of cellulose often increases the special area and the ratio of amorphous region, so they were estimated as well.2. Establish an assay method of CBHI by numerical differentiation of dynamic UV spectroscopyIn the widely used assay method of CBHI, the reaction conditions such as concentrations of enzyme and PNPC, and the reaction time are empirically selected, thus the dynamic properties of catalytic reaction will not be estimated.This method was more sensitive based on the analysis of numerical differentiation of dynamic UV spectroscopy, and the result of which is more comparable, furthermore it can be used to analyze the entire reaction process. The main step of the method is:(1) Smoothing experimental data by spline interpolation method;(2) Use of isosbestic points of derivative absorbance curves as an index to determine the concentration of PNP.(3) From the plot of first derivative AUC_340-400nm versus concentrations of CBHI, evaluation of suitable reaction time, in which a linear relationship would be established between enzyme concentration and catalytic activity.(4) Finally, at the selected reaction time, using higher concentration of PNPC as substrate hydrolyzed by a series of different concentration of CBHI or a series of diluted crude cellulase samples. The V_max and [E]_t can be directly observed from the plot of instantaneous rate of CBH I activities versus different concentration of CBH I. Then K_cat will be obtained by simple calculate.The above assay procedure was very well not only for pure CBH I preparations but also for crude cellulase preparations. 3. Analyzed the mechanism of PNPC binding to CBHI in detail. The binding stoichiometry was determined base on the instantaneous rate by UV and fluorescence quenching spectra, and isothermal titration calorimetry (ITC) technique. And the structure changes of CBHI and PNPC were determined at SBP.The behavior of specific bonds between CBHI and PNPC was expressed by the binding stoichiometry — molar binding sites that can accurately determined by its instantaneous rate. Fig.2 combines the results of the instantaneous rate of UV spectra, fluorescence quenching, and the quantity of heat released (Fig.2), from which the SBP can be easily determined.The conformational changes in CBHI induced by binding of PNPC were analyzed by second-derivative fluorescence spectrometry at the saturation binding point (SBP). And the configurational changes of PNPC in binding process have been evidenced by UV spectra. The technique of isothermal titration calorimetry (ITC) was used to accurately determine the stoichiometry of binding (the molar binding sites) for CBHI to PNPC. All the results suggested that there are at lease two points on CBHI surface that can be bound by PNPC, and the configurationally changes in PNPC converted to PNP (p-nitrophenyl) were observed during binding process. Analysis by fluorescence quenching indicated that the hydrophobic interactions probably play an important role in the binding process, and two tryptophan residues located in catalysis domain contribute to the binding. The results also demonstrated that the binding process of PNPC to CBHI is not an equilibrium process controlled by binding and de-sorption, but an irreversible process controlled by the saturability — degree of fractional saturation. These new insights derived from the interaction between the CBHI and PNPC may provide a basis for further understandings of the binding mechanism for enzyme-substrate interaction.4. Propose that the effect of temperature on the catalysis and Arrhenius E_a must be analyzed combining with time course; the instantaneous rate (c-v_inst and dp_t,T was used as a basis for analyzing the effect of temperature and time course on the dynamics; both temperature and time course have the effect on the character of thermodynamics, and temperature was more affective; and estimating the effect of temperature on the structure of CBHI. Suggest that the conformation of enzyme continuously changes during the hydrolysis, further more an enzyme is a "machine" that translates the energy into work, and the enzyme take part in the catalysis process.Temperature is a important variable of catalytic reactions. However, the effect of temperature on enzyme differs greatly with the presence and absence of substrate. Thus, the result is more instructive for actual use when analyzed the temperature effect on the enzymatic catalysis. The time courses of PNPC hydrolyzed by CBHI under different temperatures were determined. The result showed that output of PNP is the compound function of hydrolysis time (t) and reaction temperature (T). Thus, the effect of temperature on the catalysis must be analyzed combining with time course. The instantaneous rate (V_Inst) obtained by compound function and instantaneous increment (dP_T,t) obtained from partial derivative are two different concepts.The variation of CBHI activity was controlled only by both combination of catalysis and inhibition effects and the affect by the irreversible denaturation can then be neglected. This is supported by the melting studies of Circular Dichroism. In the paper, compound function method was used to evaluate the combining effects of temperature and time course on V_inst , and the instantaneous increments (V_inc)(Fig. 3). Fig. 3 Dependence of temperature and time course on the c-V_inst(A) and dp_T,t (B).Several models can be used to describe the characteristics of those normal and asymmetric curves. However, because of the complexity of these curves (fronting, symmetric, and tailing etc), it is so difficult to construct a highest flexibility model best fitting to all those different curves, and in most cases no physical or chemical meaning can be associated with the values taken by the parameters. We therefore tried to consider a new way to overcome this difficulty.Based on the position of two turning points and one zero point on curve of c-v_sec, the change of instantaneous rate, the c-V_inst during entire curve can be separated into four phases:I. Accelerating increase phase. The c-V_inst accelerating increases from the initial up to themaximum point on curve of c-v_sec at which d²V/dT² reached the maximum value it as about the 1/2 maximum of the c-V_inst ,II. Decelerating increase phase. The c-V_inst increases with decelerating rate from the maximum point to the zero point on curve of c-v_sec at which d²V/dT² reaches zero value.III. Declining phase. The c-V_inst continuously decreases from zero point to about the 1/2 maximum of the decline step at which d²V/dT² reached the minimum point.IV. Accelerating declining phase. The c-V_inst accelerating decreases from 1/2 maximum point to the end.Mathematically, the corresponding point of the maximum value of c-v_inst on temperature-axis indicates the optimum temperature, T_opt-inst And the minimum point c-v_sec curve is corresponding to the T_inac-99-the catalysis activity has lost 99% at this temperature. They showed even the assays under same temperature, however, the T_opt-inst and T_inac-99 are all varied with the assay time course and increase of temperature can decreased the time course for reached the T_opt-inst and T_inac-99, while those effect is more forT_inac-99 than T_opt-inst(Fig. 4).Fig. 4 Estimation of the four phases of v_inst curve based on the relationship between v_inst and v_sec and there corresponding T_acc , T_opt-inst and T_inac-99Following the method proposed by Zhang and Wang for quasi-parabola model, and gives some modified, a rational approximation to modeling the curve of c-V_ins versus temperature to get the R_net . And R_net was used as independent variable to calculate the apparent activation energy by Arrhenius plot. As suggested, the Arrhemius activation energy (E_a) is not a constant while is a compound function of temperature and assay time course dependence. The positive slope is equivalent to negative activation energy, which only appeared as the temperature is beyond certain point.There are four experimental results performed in our Lab, for estimation of apparent activation energy, two of which belong to β-Endoglucanase, one is the β-Galactosidase and the another one is also CBHI but for hydrolysis of Microcrystalline cellulose powder. A negative Ea are always observed as the temperature beyond the T_opt.The studies in enzyme-catalysis, performed with Arrhenius plot to estimate the apparent activation energy, E_a that does not provide favor- able information for explain the reaction mechanism. And the existence of negative E_a during enzyme-catalysis reaction also suggested that there is no possibility for identification of the Ea as a criterion to express the temperature effect on enzyme-catalysis reaction. Thus in this paper, we suggest using the extent of reaction of enzyme-catalysis can be reflected by the net reaction rate.According to the Chemical affinity theory, based on the calculation of net reaction rates, a method was proposed for expressing the negative free energy, and examined by the hydrolysis result of CBHI, EGm and EGt (Fig. 5).The quantity of heat released, the Q, during the binding of PNPC to CBHI at 277, 279 and 281 K and hydrolysis at 309, 311 and 313 K were measured by ITC, and explored a new way for establishing the connection between catalysis and thermodynamics ?affinity analysis. And point out that there are irreversible and non-equilibrium processes in the whole catalysis process, and this character must be considered in the analysis so that we can obtain the more correct result. The above result suggested that the irreversible configurational changes of substrate has occurred during the binding process; while in this part, we found that the conformation of enzyme continuously changes during the hydrolysis, and furthermore, an enzyme is a machine that translate the energy in the circumstance into work, and it takes part in the catalysis process.5. Discussed the strategy in biomass utilizationThe main problem of biomass utilization is the high cost in conversion. The separation of cornstalk core and its further conversion was chosen as an example for discussing the feasibility of the production of alcohol, amylase, and protein feed. Propose that the utilization of agricultural waste should follow the fundamental of "make the best use of everything". While the transport and storage cost of industrial waste was very low compared to agricultural waste, on the contrary, its utilization is beneficial to reduce the pollution. Thus, developing the new use and increase the utilized ratio of industrial waste is main object. The industrial waste of kelp was selected as material in alcohol production, and the result suggested that it is more suitable than lignocellolose such as cornstalk.