Biochemical Engineering Fundamentals, Part 1 - Bailey J., Ollis D. 1989

Applications of Enzyme-Catalyzed Reactions
Applications of Hydrolytic Enzymes
Hydrolysis of Starch and Cellulose

Amylases, which find wide application, are Enzymes that hydrolyze glycosidic bonds in starch and related glucose polymers. To understand the difference between the two MAIN TYPES OF amylases, we must recall that starch consists of linear glucose polymers called amylose and a branched polymer (amylopectin). The latter is significantly more soluble than amylose and is capable of dramatically increasing the viscosity of starch solutions. When treated with α-amylase, the viscosity of the solutions decreases due to the random Cleavage of any α-1,4-glycosidic bonds; for this reason, α-amylase is often referred to as a starch-liquefying enzyme*. In contrast, β-amylase can attack α-1,4-bonds only at the non-reducing ends of the polymer chain, so the Hydrolysis of linear polymers by this enzyme always yields maltose. For this reason, β-amylase is also called a saccharifying enzyme. Treating starch with β-amylase ultimately produces a mixture of maltose and dextrins, which are fragments of starch molecules with terminal 1,6-bonds. These bonds are not hydrolyzed by β-amylase.

* Apparently, α-amylase non-specifically cleaves glycosidic bonds only in substrates with a sufficiently long polymer chain; in the case of shorter oligomers, a certain Specificity is observed.

Another saccharifying enzyme, amyloglucosidase (which has many other names, including glucoamylase), primarily attacks the non-reducing terminal α-1,4-bonds of starch, Glycogen, dextrins, and maltose; amyloglucosidase also hydrolyzes α-1,6-bonds, but at a significantly lower rate. If the goal of the process is to obtain glucose rather than maltose, a mixture of enzymes or sequential Treatment with α-amylase and glucoamylase is used; such processes are applied in distilleries (as opposed to breweries), in The production of concentrated glucose solutions (corn syrup), and crystalline glucose. It is estimated that in 1971, 0.61 billion kg of glucose was obtained¹ by this method in the USA. The Use of amylases in glucose production and other fields (Table 4.3) accounts for the immense Practical significance of these enzymes. The relative amounts of α- and β-amylases used in each specific case are determined by the ultimate goal of the process.

Amylases are obtained from A wide variety of sources. This is not surprising, since starch is a common storage carbohydrate for many different life forms. Amylases are produced by A number of Bacteria and Molds; a practically important enzyme is the amylase from Clostridium acetobutylicum, a microorganism involved in the microbiological conversion of Polysaccharides into butanol and acetone. Amylase preparations used by humans for food purposes are typically obtained from cereal crops, primarily barley, wheat, rye, oats, corn, sorghum, and rice. In grain amylases, The ratio of saccharifying to liquefying enzymatic activities depends not only on the plant type but also on the state of the grains, primarily whether they have germinated or not. In the production of malt (softened germinated barley grains) used in brewing, ungerminated barley grains are stored under conditions of high humidity and appropriate Temperature to promote rapid germination, during which the α-amylase content simultaneously increases. The germinated grains are then slowly kiln-dried; in the dry preparation, amylase activity is not expressed, but the enzymes do not undergo irreversible inactivation. Having immense hydrolytic activity, the dried and ground malt is then used to convert starch into sugars, which can subsequently be processed by Yeast Fermentation.

Class="center">Table 4.3. Areas of the most widespread application of amylase enzymes^a

^a From: Weiser H. H., Practical Food Microbiology and Technology, p. 37, Avi Publishing Co., Westport, Conn., 1962.

Other carbohydrases listed in Table 4.1 also cleave glycosidic bonds. For example, it has been reported that the combined use of pullulanase, which selectively cleaves 1,6-glycosidic side-chain bonds, and amylase increases the yield of glucose from starch. In ice cream production, lactase is commonly used to cleave lactose into the sweeter Monosaccharides glucose and galactose. A related enzyme, invertase, hydrolyzes sucrose and polysaccharides containing β-D-fructofuranosyl bonds. This enzyme received its name after it was established that the hydrolysis of sucrose in solutions and The formation of a glucose-fructose solution is accompanied by A change in the direction of Rotation of the plane of polarized light. A partially or fully hydrolyzed sucrose solution possesses two qualities valuable to the confectionery industry: it is somewhat sweeter than the starting solution and does not crystallize upon evaporation to significantly higher concentrations.

A vast amount of work conducted in various countries has been devoted to The Study of the Enzymatic hydrolysis of cellulose. Before proceeding to this topic, it should be emphasized that the enzyme preparations used for the depolymerization of cellulose, commonly referred to as cellulase, represent a complex mixture of many enzymes. Moreover, The Nature of the enzymes comprising cellulase and their relative amounts depend on the type of microorganism from which the cellulase was isolated, and in some cases, on the preparation process of the enzyme. As we already mentioned in Chapter 2 during the Structure/133.html">Discussion of lignocellulose structure, all biomass and industrial wastes from various processes differ in several properties, including the degree of crystallinity and specific surface area, as well as chemical composition. Some pretreatment Methods allow these substrates to be modified, thereby reducing their resistance to hydrolysis. Thus, The rate of hydrolysis of cellulosic Materials and the product yields in any given process depend on a combination of several factors: first, The Nature and Properties of the substrate; second, the results of the pretreatment; and third, the level of activity and Specificity of the individual enzymes that make up the cellulase.

Currently, the cellulase systems produced by Trichoderma Fungi are the most thoroughly studied and characterized. As shown in Fig. 4.1, these systems comprise Three types of enzymes acting on different substrates and forming different reaction products. The same figure indicates the feedback inhibition pathways of individual enzymes. The kinetics of each stage of this complex reaction system obey the Michaelis–Menten Equation with strictly competitive or non-competitive inhibition. Cellulases with various properties and activities are also produced by many other microorganisms, including the molds Fusarium solani, Aspergillus niger, Penicillium funiculosum, Sporotrichum pulverulentum, Cellulomonas sp., Clostridium thermocellum, and Clostridium thermosaccharolyticum.

Table 4.4 provides additional information on the cellulase system from T. viride. Note that various "standard" substrates are used to determine specific activities. The sizes of the enzyme molecules, which are comparable to the sizes of natural cellulose microfibrils, are also of no small importance. The rate of hydrolysis depends mainly on the degree of crystallinity of the cellulose. The corresponding expressions describing the kinetics of hydrolysis, taking into account the degree of crystallinity, will be described below in Example 4.1; for now, we will briefly discuss the chemical and Physical Methods of pretreatment of lignocellulosic materials.

FIG. 4.1. Schematic diagram of the interaction of substrates, cellulases, and reaction products during the biodegradation of cellulose (→ main reaction; → side reaction; → inhibition effects). [Reproduced from: Lee Y. H., Fan L. T., Properties and Mode of Action of Cellulase, in Advances in Biochemical Engineering, Fiechter A. (ed.), 17, 101 (1980).]

Studying chemical wood treatment methods provides insight into The Effect of Lignin on the resistance of cellulosic materials to enzymatic hydrolysis. In the paper industry, to dissolve lignins and Pectins, ground wood is subjected to sulfite pulping (treatment with an aqueous bisulfite solution containing free SO₂) or sulfate pulping (treatment with an aqueous solution of NaOH and Na₂S) at elevated temperature and pressure (kraft process). Through such treatment of four different wood species under various conditions, wood pulps with different lignin contents were obtained, which were then treated with sulfuric acid to hydrolyze cellulosic substances. The results of this experiment (Fig. 4.2) clearly demonstrate that the hydrolyzability of cellulose primarily depends on The amount of residual lignin. Other methods are also used to degrade lignin, including treatment with gaseous SO₂, mineral acids, and specific enzymes produced by several fungi, such as Sporotrichum pulverulentum and Pleurotus ostreatus.

The structure of cellulose, including characteristics such as the degree of crystallinity, specific surface area, and degree of polymerization, can be altered by pretreatment, for example, by ball or roller milling, γ-irradiation, pyrolysis, or treatment with acidic or alkaline Reagents. As an example, let us consider the kinetics of changes in cellulose crystallinity during the acid treatment of wood; information on changes in other cellulose properties can be found in the literature cited at the end of the chapter.

Table 4.4. Properties of various types of enzymes in the T. viride cellulase system

^a Commercially available crystalline cellulose that has undergone acid treatment.

As shown in Fig. 4.3a, the effect of pretreatment on crystallinity is best monitored by X-Ray Diffraction Analysis. The latter allows for the determination of a parameter called the crystallinity index (CrI), calculated using empirical formulas; in our case (Fig. 4.3a), this formula is as follows:

where I(am) (am — amorphous) and I(002) are the diffraction intensities at 2θ = 18.5 and 22.5°, respectively. The CrI values are not an exact measure of the crystalline fraction content; nevertheless, the CrI index is useful for assessing Changes in the degree of crystallinity and average crystallite sizes in various cellulosic materials. The experimental data presented in Fig. 4.3a clearly show that repeated roller milling reduces the crystallinity of cellulose.

FIG. 4.2. Results of an experimental study on the effect of the degree of delignification on the hydrolyzability of cellulose from four different wood species. [Reproduced with permission from: Baker A. J., Effect of Lignin on the in vitro Digestibility of Wood Pulp, J. Anim. Sci., 37, 768 (1973).]

FIG. 4.3. Changes in the X-ray diffraction pattern of Avicel cellulose after multiple passes through a two-roller mill (the curves indicate the number of passes) (a); after treatment with cellulase from T. reesei (the curves indicate the incubation time in hours) (b). To calculate the crystallinity index CrI, intensities at diffraction angles of 2θ = 18.5 and 22.5° were used. [Reproduced with permission: a) from Ryu D. D. Y., Lee S. B., Tassinari T., Macy C., Effect of Compression Milling on Cellulose Structure and on Enzymatic Hydrolysis Kinetics; Biotech. Bioeng., 24, 1047 (1982); b) from Lee S. B., Kim I. H., Ryu D. D. Y., Taguchi H., Structural Properties of Cellulose and Cellulase Reaction Mechanism; Biotech. Bioeng., 25, 33 (1983).]

Earlier, when discussing the structure of cellulose and The activity of cellulase enzyme systems, we mentioned that the rate of hydrolysis of amorphous and paracrystalline regions of cellulose should be higher than the rate of hydrolysis of crystalline cellulose. It follows that a decrease in the crystallinity of cellulose should be accompanied by an increase in the rate of its enzymatic hydrolysis. Indeed, such a relationship was found, in particular, in studying the hydrolysis of the same cellulosic materials investigated in the experiment shown in Fig. 4.3, a, under the action of a total cellulase preparation from Trichoderma reesei MCG-77 (Table 4.5). Table 4.5 lists the apparent average parameter values in the conventional Michaelis-Menten Equation; these were determined assuming that cellobiose was absent from the mixture when measuring the initial reaction rate.

Calculations of The change in the Rate of Enzymatic hydrolysis of cellulosic materials over time are complicated by the fact that during such processes, the relative content of the crystalline and amorphous fractions of the substrate also changes (Fig. 4.3, b). In the example below, we will consider the Modification of the modeling principles outlined in Chapter 3 as applied to systems of this kind.

Table 4.5. Effect of the number of passes through a roller mill on the crystalline structure of Avicel-type cellulose and the average kinetic parameters of its hydrolysis by cellulase from T. reesei^a

^a Data from: Ryu D. D. Y., Lee S. B., Tassinari T., Macy C., Effect of Compression Milling on Cellulose Structure and on Enzymatic Hydrolysis Kinetics, Biotech. Bioeng., 24, 1047 (1982).

Example 4.1. Effect of cellulose crystallinity on the rate of its enzymatic hydrolysis*. Several Mathematical models of the kinetics of these processes have been developed to study the hydrolysis of cellulose by cellulase-type enzyme systems. In one of them, the greatest attention is paid to the effect of crystallinity; in this approach, it is assumed that cellulase enzymes (their sum is denoted here by the symbol E) are adsorbed on cellulose in the E* state:

Assuming that enzyme adsorption is of an equilibrium nature and that the Enzyme Concentration in solution is sufficiently low (in practice, e₀ < 0.5 mg of protein per 1 ml of reaction mixture), we obtain

* Ryu D. D. Y., Lee S. B., Tassinari T., Macy C., Effect of Compression Milling on Cellulose Structure and on Enzymatic Hydrolysis Kinetics, Biotech. Bioeng., 24, 1047 (1982).

The Description of the model is completed by two more assumptions, according to which the adsorbed enzyme catalyzes the hydrolysis of amorphous (S_a) and crystalline (S_c) cellulose in accordance with the following equations of parallel reactions:

whereby the adsorbed and free cellulases bind to the inert material of the cellulosic substrate, as well as to the reaction product, respectively:

Applying the quasi-steady-state approximation to all enzyme-substrate complexes and summing the concentrations of all enzyme forms, we obtain the following expression for the initial rate of hydrolysis of pure cellulose (s_x = 0) at a low enzyme concentration and in the absence of the reaction product (p = 0):

Here, s (s = s_a + s_c) is the total cellulose concentration. The dependence of the apparent maximum rate and Michaelis constant on the initial crystallinity index CrI₀ [CrI₀ = s_c0/(s_a0 + s_c0)] and the rate constants of the elementary reactions can be expressed as follows:

where

Equations (4П1.8) and (4П1.9) indicate the dependence of the initial reaction rate on the initial crystallinity of the substrate CrI₀, with the pseudo-first-order reaction rate constant v_max^app/Km^app and the reciprocal of the apparent Michaelis constant, 1/Km^app, depending linearly on CrI₀; this makes it possible to verify the consistency of the described model with experimental data. Plotted on The basis of experimental results,

FIG. 4П1.1. Experimental Determination of the dependence of the apparent kinetic parameters v_max^app and Km^app on the initial crystallinity of the substrate CrI₀ for Avicel (A, O), cotton (X, O), and Solka-Floc cellulose (C, □). The numbers indicate the number of preliminary passes of the substrate through a roller mill. The X-ray diffraction patterns of the substrates are shown in Fig. 4.3, a. [Reproduced from: Ryu D. D. Y., Lee S. B., Tassinari T., Macy C., Effect of Compression Milling on Cellulose Structure and on Enzyme Hydrolysis Kinetics, Biotech. Bioeng., 24, 1047

Application of Enzyme-Catalyzed Reactions, the plots of these kinetic parameters versus the initial crystallinity of the substrate for several cellulose samples are shown in Fig. 4П1.1. Most of the data agree well with the described model; the greatest deviation is found in the case of the pseudo-first-order reaction rate constant for Solka-Floc cellulose.

These results are highly instructive in that they serve as an example of deriving useful mathematical expressions for interpreting kinetic data based on a sound hypothesis about the Nature of the transformations occurring in the system. The expression for the initial rate given above is somewhat cumbersome; however, it is already obvious from the process mechanism that the initial rate of hydrolysis must depend on all the parameters included in this equation. Moreover, the corresponding expression for the time dependence of the cellulose hydrolysis rate in a batch Reactor will obviously be even more complex, since it must also account for product inhibition [see Eq. (4.П1.6)]. Finally, when calculating the change in Substrate Concentration over time, one must also consider that the substrate is a mixture of crystalline and amorphous forms hydrolyzing at different rates. Consequently, the expression describing the kinetics of hydrolysis must reflect two parallel processes with two different substrates, and these processes are interrelated because the relative amounts of amorphous and crystalline cellulose are also not constant over time, as evidenced by the change in the crystallinity index.

This example illustrates only a few of the numerous challenges encountered when modeling biological substrates and catalysts. As we will see repeatedly in subsequent chapters and sections, the general principle of modeling such complex systems lies in choosing a biochemical or mechanistic scheme and corresponding mathematical expressions that reflect changes only in the key variables of primary interest in a specific analysis. Thus, in our example, we considered an idealized cellulosic substrate. This assumption allows us to evaluate the effect of crystallinity on the course of hydrolysis, determine the relevant kinetic parameters, and calculate the pattern of change in substrate crystallinity in a batch reactor. All the experimental data presented were obtained using the same cellulase enzyme complex; therefore, the described model does not account for METABOLISM/18.html">The Influence of individual enzymes within the complex or their relative Abundance. Clearly, when comparing these results with data obtained from cellulases of other origins, it will be necessary to consider the relative content of different enzymes in the complex and their respective activities. Ideally, through a series of rigorous experimental studies and The Development of mathematical models similar to the one discussed above, we would eventually obtain comprehensive information on the transformations occurring in the cellulose–cellulase system. This, in turn, would allow us to develop a general model that more fully reflects The properties of the substrates and enzymes in this system.

To conclude this review on Cellulose Utilization, It is worth mentioning that other Physical and Chemical methods of cellulose degradation also exist. For instance, advanced processes for the acid hydrolysis of cellulose have been developed in recent years, featuring higher temperatures and shorter reaction times, which yield the desired degradation products with minimal byproduct formation. Another promising method of cellulose Processing is based on its pyrolytic degradation. Other methods for lignocellulose processing currently being studied, developed, or already in use include screw extrusion, heterogeneous catalytic treatment, fluidized-bed gasification, and the oldest processing method, combustion.