Biochemical engineering books free pdf download

Ghose, V. Bisaria: Development of Biotechnology in India. Roehr: History of Biotechnology in Austria. Hollo, U. Fiechter: Biotechnology in Switzerland and a Glance at Germany. Score: 5. It is addressed to all scientists at universities and in industry who wish to keep abreast of advances in the topics covered. Thisvolume re? Along this vein, the focusof thisvolume istoprovide abiotechnology driven perspective on cell engineering fundamentals while highlighting their signi?

Our aim is to present an overview of the state of the art of a selection of these technologies, punctuated with current applications in the research and development of cell-based therapies for human disease.

To prepare this volume, we have solicited contributions from leaders and experts in their respective? Particular emphasis was placed on including reviews that discuss various aspects of the biochemical p- cesses underlying cell function, such as signaling, growth, differentiation, and communication. The reviews of research topics cover two main areas: cel- lar and non-cellular components and assembly; evaluation and optimization of tissue function; and integrated reactor or implant system development for research and clinical applications.

Many of the reviews illustrate how bioche- cal engineering methods are used to produce and characterize novel materials e. Process Biotechnology, fundamentals of microbiology, immobilization, enzymes, bioreactor sterilization, fermentation technology, aeration and agitation in bioprocess, separation process in product recovery, important topics of scale-up of operation, bioreactor instrumentation and control, principles of effluent treatment and bioprocess engineering and medical applications are covered.

This book will be ready reference to postgraduate students and also useful to practicing process engineers working in the biotechnology based industries. Salient Features Important aspects of Upstream and Downstream process of biotechnology have been covered with suitable illustrations Efforts are made to emphasis on application of basic biological principles to bioprocess engineering Various figures are provided at appropriate places along with photographs to aid students for comprehensive understanding of the subject Review questions have been added at the end of each chapter.

This volume stays abreast of recent developments in stem cell biology and the high expectations concerning the dev- opment of stem cell based regenerative therapies. Regenerative medicine is the focus of current biomedical research, with unique challenges related to scientific, technical and ethical issues of stem cell research, and the potential added value of connecting biomedicine with enabling techno- gies such as materials sciences, mechanical- and nano-engineering.

Research activities in regenerative medicine include strategies in endogenous regeneration of injured or degenerated tissues by means of gene therapy or cell transplantation, as well as complex approaches to replace or reconstruct lost or malformed tissue structures, by applying tissue engineering approaches.

In most cases, the speci- ized functional cell types of interest cannot be isolated from the diseased organ or expanded to a sufficient degree, and various stem and progenitor cell types rep- sent the only applicable cell source. In almost all cases, stem cells have to be engineered, sometimes for functional improvement, in many cases to produce large numbers of cells, and frequently to achieve efficient and specific differentiation in the cell type s of interest.

Popular Books. As an example Wiseman and Gould, , let's consider one acidic amino acid, glutamic acid, which is acidic in the lower pH range. On the other hand, an amino acid, lysine, is basic in the range of higher pH value. An enzyme is catalytically active when the amino acid residues at the active site each possess a particular charge. Therefore, the fraction of the catalytically active enzyme depends on the pH.

Let's suppose that one residue of each of these two ammo acids, glutamic acid and lysine, is present at the active site of an enzyme molecule and that, for example, the charged form of both amino acids must be present if that enzyme molecule is to function. Enzyme Kinetics 37 2. The temperature dependence of many enzyme-catalyzed reactions can be described by the Arrhenius equation.

An increase in the temperature increases the rate of reaction, since the atoms in the enzyme molecule have' greater energies and a greater tendency to move. However, the temperature is limited to the usual biological range.

As the temperature rises, denaturation processes progressively destroy the activity of enzyme molecules. This is due to the unfolding of the protein chain after' the breakage of weak for example, hydrogen bonds, so that the overall reaction velocity drops. Some enzymes are very resistant to denaturation by high temperature, especially the enzymes isolated from thermophilic organisms found in certain hot environments.

The mechanical force that an enzyme solution normally encounters is fluid shear, generated either by flowing fluid, the shaking of a vessel, or stirring with an agitator. The effect of shear on the stability of an enzyme is important for the consideration of enzyme reactor design, because the contents of the reactor need to be agitated or shook in order to minimize mass- transfer resistance.

However, conflicting results have been reported concerning the effect of shear on the activity of enzymes. Charm and Wong showed that the enzymes catalase, rennet, and carboxypeptidase were partially inactivated when subjected to shear in a coaxial cylinder viscometer.

The remaining activity could be correlated with a dimensionless group gammatheta, where gamma and theta are the shear rate and the time of exposure to shear, respectively. They found that there was no significant loss of enzyme activity due to shear force alone at shear rates up to sec-I. They reasoned that the deactivation observed by Charm and Wong was the result of a combination of shear, air-liquid interface, and some other effects which are not fully understood.

Charm and Wong did not seal their shear apparatus. This was further confirmed, as cellulase deactivation due to the interfacial effect combined with the shear effect was found to be far more severe and extensive than that due to the shear effect alone Jones and Lee, To give students an experience with enzyme reactions and assay procedures 2.

To determine the Michaelis-Menten kinetic parameters based on initial-rate reactions in a series of batch runs 3. To simulate batch and continuous runs based on the kinetic parameters obtained Materials: 1. Spectrophotometer 2. Glucose assay kit No. Louis, MO 4. Cellobiose 5.

Water bath to control the temperature of the jacketed vessel Calibration Curve for Glucose Assay: 1. Prepare glucose solutions of 0, 0. Enzyme Kinetics 39 2. Using these standards as samples, follow the assay procedure described in the brochure provided by Sigma Chemical Co.

Plot the resulting absorbances versus their corresponding glucose concentrations and draw a smooth curve through the points. Experiment Procedures: 1. Prepare a 0. Dilute the cellobiase-enzyme solution so that it contains approximately 20 units of enzyme per mL of solution. One unit of cellobiase is defined as the amount of enzyme needed to produce 1 Jl-mol of glucose per min.

Initiate the enzyme reaction by adding 1 mL of cellobiase solution to the reaction mixture and start to time. Take a 1 mL sample from the reactor after 5- and minutes and measure the glucose concentration in the sample. Data Analysis: 1. Calculate the initial rate of reaction based on the 5 and 10 minute data. Determine the Michaelis-Menten kinetic parameters as described in this chapter. Simulate the change of the substrate and product concentrations for batch and continuous reactors based on the kinetic parameters obtained.

For this run, take samples every 5 to 10 minutes for 1 to 2 hours. The reaction was initiated by adding 1 mL of enzyme beta-glucosidase solution which contained 0.

At 1, 5, 10, 15, and 30 minutes, O. A unit is defined as the enzyme activity which can produce Imumol of product per minute. What is the initial rate of reaction?

Derive the rate equation by making the Michaelis-Menten assumption. If the concentration of Sl is much higher than that of S2' how can the rate equation be simplified? Please note that the rate constants of the second equilibrium reaction are the same as those of the third reaction.

Assume that you obtained the Cs versus t curve you calculated in part a experimentally. Is this approach reliable? Chemostat continuously stirred-tank reactor runs with various flow rates were carried out.

Enzyme Kinetics 43 2. The initial substrate concentration is 3. Assume that the reaction can be expressed by the Michaelis- Menten kinetics. What is the maximum reaction rate? What is the concentration of the substrate after 15 minutes?

What should be the size of the reactor if you employ a plug- flow reactor instead of the CSTR in part a? The flow rate is 0. The inlet substrate concentration is 50 giL and the enzyme concentration in the two reactors is maintained at the same value all of the time. Is the two-reactor system more efficient than one reactor whose volume is equal to the sum of the two reactors? Explain how you can estimate the parameters of the rate equation in part c experimentally.

Derive the rate equation for the production of galactose by using Bnggs-Haldane approach. Does galactose inhibit the reaction competitively or noncompetitively? In this mechanism, glucose is released from the enzyme-substrate complex first, leaving the enzyme-galactose complex, which subsequently releases the galactose molecules.

How is the rate equation developed by this model simplified to that by Scott et al. Is prostigmine competitive or noncompetitive inhibitor? Evaluate the Michaelis-Menten kinetic parameters in the presence of inhibitor by employing the Langmuir plot. The following table shows the amount of sugar inverted in the first 10 minutes of reaction for various initial substrate concentrations.

The amount of invertase was set constant. Substrate Sugar Con. Derive the rate equation using the Michaelis-Menten approach. Determine the three kinetic parameters of the equation derived in part a using the preceding experimental data. What is the reaction rate in the reactor? You measured the steady-state outlet substrate concentration as a function of the inlet flow rate and found the following results.

Estimate Michaelis kinetic parameters by using the best plotting technique for the equal weight of all data points. Flow rate Outlet Substrate Cone. Calculate the Michaelis-Menten constants of the above reaction.

Justify your answer by showing the effect of the inhibitor graphically. Write the kinetic model for this enzyme reaction. Derive the rate equation. State your assumptions for any simplification of the rate equation.

Estimate the value of inhibition kinetic parameter. It has been found Frantz and Stephenson, J. Bailey, J. Ollis, Biochemical Engineering Fundamentals. Bohinski, R. C, Modern Concepts in Biochemistry, p. Briggs, G. Brown, A. Burden, R. Faires, Numerical Analysis. Carberry, J. Carnahan, B. Luther, and J.

Wilkes, Applied Numerical Methods. Chapra, S. Charm, S. Wong, "Enzyme Inactivation with Shearing," Biotechnol. Crueger, W. Eadie, G. Enzyme Kinetics 49 Eveleigh, D. Hofstee, B. Identification of Two Pancreatic Aliesterases," J. Gerald, C. Wheatley, Applied Numerical Analysis. Jones, E. Levenspiel, 0. Lineweaver, H. Michaelis, L.

Menten, "Die Kinetik der Invertinwirkung," Bio-chem. Scott, T. Hill, and C. Thomas, C. Wiseman, A. Gould, Enzymes, Their Nature and Role, pp. Yang, S. Therefore, it is very difficult or inlpractical to separate the enzyme for reuse in a batch process. Enzymes can be inlmobilized on the surface of or inside ofan insoluble matrix either by chemical or physical methods. They can be also immobilized in their soluble forms by retaining them zvith a serniperrneable men'lbrane.

A main advantage of inlmobilized enzyme is that it can be reused since it can be easily separated from the reaction solution and can be easily retained in a continuous-flow reactor. Furthermore, immobilized enzyme may show seLectively altered chemical or physical properties and it may sirnulate the realistic natural environment where the enzyme came from, the cell.

The former is covalent bond formation dependent and the latter is noncovalent bond fornlation dependent. It is called a functional group. Already active polymers such as maleic anhydride copolymers will be simply mixed with enzymes to produce immobilized enzymes.

Normally, natural or synthetic polymers need to be activated by treating them with reagents before adding the enzyme. The activation involves the chemical conversion of a functional group of the polymer. The enzyme's active site should not be involved in the attachment, in which case the enzyme would lose its activity upon immobilization. The following experiment illustrates the immobilization of glucose oxidase in agarose Gutcho, Activation of agarose: Eliminate excess water from the water-swollen, ball-shaped agarose particles Sepharose 2B by subjecting them to suction on a glass filter.

Add 3. Wash the activated product on a glass filter with 1 L of ice water. Finally wash the particles rapidly with 0. Binding with glucose oxidase: React the activated polymer for 10 hours with mild stirring with 24 mg of glucose oxidase dissolved in 1 mL of 0.

Cross-linking Using Multifunctional Reagents: Water-insoluble enzymes can be prepared by using multifunctional agents that are all bifunctional in nature and have low molecular weight, such as glutaraldehyde. Enzymes can be reacted with multifunctional reagent alone so that they are cross-linked intermolecularly by the reagent to form a water- insoluble derivative. Another method is to adsorb enzymes on a water-insoluble, surface-active support followed by intermolecular cross-linking with multifunctional reagents to strengthen the attachment.

Multifunctional reagents can be also used to introduce functional groups into water-insoluble polymers, which then react covalently with water-soluble enzymes. Enzymes can be adsorbed physically on a surface-active adsorbent by contacting an aqueolls solution of enzyme with an adsorbent.

Commonly employed adsorbents are Zaborsky, : alumina, amon-exchange resins, calcium carbonate, carbon, cation- exchange resins, celluloses, clays, collagen, colloid-ion, conditioned metal, glass plates, diatomaceous earth, and hydroxyapatite.

The advantages of adsorption techniques are as follows: I. The procedure of immobilization is simple. It is possible to separate and pllrify the enzymes while being immobilized. The enzymes are not usually deactivated by adsorption. The adsorption is a reversible process.

Immobilized Enzyme 53 However, adsorption techniques also have several disadvantages: 1. The bonding strength is weak. The state of immobilization is very sensitive to solution pH, ionic strength, and temperature. The amount of enzymes loaded on a unit amount of support is usually low. Entrapment: Enzymes can be entrapped within cross-linked polymers by forming a highly cross-linked network of polymer in the presence of an enzyme. This method has a major advantage in the fact that there is no chemical modification of the enzyme, therefore, the intrinsic properties of an enzyme are not altered.

However, the enzyme may be deactivated during the gel formation. Enzyme leakage is also a problem. The most commonly employed cross- linked polymer is the polyacrylamide gel system.

This has been used to immobilize alcohol dehydrogenase, glucose oxidase, amino acid oxidase, hexokmase, glucose isomerase, urease, and many other enzymes. Microencapsulation: Enzymes can be immobilized within semipermeable membrane microcapsules.

This can be done by the interfacial polymerization technique. Organic solvent containing one component of copolymer with surfactant is agitated in a vessel and aqueous enzyme solution is introduced. The polymer membrane is formed at the liquid-liquid interface while the aqueous phase is dispersed as small droplets. One example of this process is the polyamide nylon system, in which 1, 6-diaminohexane is the water- soluble diamme and 1,decanoyl chloride is the organic-soluble diacid halide.

The organic solvent for this system is a chloroform- cyclohexane mixture v Iv containing usually 1 percent v Iv Span85 surfactant Zaborsky, The immobilized enzyme produced by this technique provides an extremely large surface area. It is the mass-transfer resistance due to the large particle size of immobilized enzyme or due to the inclusion of enzymes in polymeric matrix. If we follow the hypothetical path of a substrate from the liquid to the reaction site in an immobilized enzyme, it can be divided into several steps Figure 3.

Step 3 is the intraparticle mass-transfer resistance. Bulk liquid Immobilized enzyme C. The rate of mass transfer is proportional to the driving force, the concentration difference, as 3. During the enzymatic reaction of an immobilized enzyme, the rate of substrate transfer is equal to that of substrate consumption. This equation shows the relationship between the substrate concentration in the bulk of the solution and that at the surface of an immobilized enzyme.

Dependingupon the magnitude of N Oa' Eq. The rate that would be obtained with no mass-transfer resistance at the interface is the same as Eq. If X s is equal to 1, the concentration at the surface Cs is equal to the bulk concentration CSb.

On the other hand, if Xs approaches zero, 1] also approaches zero, which is the case when the rate of mass transfer is very slow compared to the reaction rate. In order to derive an equation that shows how the mass-transfer resistance affects the effectiveness of an immobilized enzyme, let's make a series of assumptions as follows: 1.

The reaction occurs at every position within the immobilized enzyme, and the kinetics of the reaction are of the same form as observed for free enzyme. Mass transfer through the immobilized enzyme occurs via molecular diffusion. There is no mass-transfer limitation at the outside surface of the immobilized enzyme. The immobilized enzyme is spherical. The model developed by these assumptions is known as the distributed model.

First we derive a differential equation which describes the relationship between the substrate concentration and the radial distance in an immobilized enzyme. The material balance for the spherical shell with thickness dr as shown in Figure 3. Let's solve the equation first for the simple cases or zero-order and first-order reactions, and for the Michaelis-Menten equation.

Immobilized Enzyme 59 In order to convert Eq. Figure 3. Therefore, differentiating Eq. It can be solved by various numerical techniques. We repeat this process until the solution of the initial value problem satisfies the boundary conditions.

When guessing the values of Rc and xs o' we can think of two different cases: 1. This is the case when the rate of enzyme reaction is slow compared to that of mass transfer which is represented by the low value of phi. As a result, the substrate reaches the center of sphere. As a result, the substrate is consumed before it reaches the center of sphere.

Table 3. The effectiveness factor when the reaction rate is expressed by the Michaelis-Menten equation can be calculated as A p D s CSb ax. The gel structure increases the path length for diffusion, and as a result decreases the diffusion rate. Various techniques are available for determining the effective diffusivity of solute in gel Itamunoala, One of the most reliable techniques is the thin-disk method which uses a diffusion cell with two compartments divided by a thin gel. Each compartment contains a well-stirred solution with different solute concentrations.

Effective diffusivity can be calculated from the mass flux verses time measurement Hannoun and Stephanopoulos, A few typical values of effective diffusivities are listed in Table 3. Derive the equation for the effectiveness of an immobilized enzyme for this diffusion limited case by employing the same assumptions as for the distributed model. The rate of substrate consumption can be expressed by the Michaelis-Menten equation.

Solution: Since all substrate is consumed in the thin layer, we can assume that the layer has a flat geometry. Consider a control volume defined by the element at r with thickness dr, as shown in Figure 3. However, in this problem we do not need to solve this equation. Assume that the external mass-transfer resistance for substrate is not negligible and that the Michaelis-Menten equation describes the intrinsic kinetics of enzyme reaction.

Derive an expression which shows the relationship between the substrate concentration at the surface and that in the bulk solution. If they are different, what will be the meaning of K:tfx and Ka'lJl? You can pack the immobilized enzyme in a cylindrical column pass through substrate solution, which is known as a packed-column immobilized reactor. What will be the effect of the substrate flow rate on KaPJ for the packed- column reactor?

Can you determine the intrinsic kinetic parameters r max and KM for free enzyme by using the packed-column immobilized reactor? The effectiveness of the immobilized enzyme was found to be 70 percent of the free enzyme when the concentration of the substrate was 0. The rate of substrate consumption can be expressed as a first-order reaction. S mm in diameter.

Initial concentration of substrate is 0. It has been found that both external and internal mass-transfer resistance are significant for this immobilized enzyme. The mass-transfer coefficient at the stagnant film around the particle is about 0. If the internal mass transfer resistance is negligible, what is the concentration of the substrate at the surface of the particle?

What is the effectiveness factor for this immobilized enzyme? What is the overall effectiveness factor considering both internal and external mass-transfer resistance? One side is in contact with substrate solution C Sb and the other side is in contact with a glass plate.

The mass transfer coefficient on the surface of the gelatin is ks. Derive the equation for the substrate concentration with respect to x when the substrate is catalyzed by zero-order reaction k o ' Assume that the substrate is transferred by molecular diffusion in the x direction only and the gelatin slab is thick enough to catalyze all the substrate while it diffuses into the slab. What is the critical thickness xc at which all substrate is consumed?

What is the substrate concentration at the surface of the slab Cs o? Fnedman, L. Gutcho, S. Hannoun, B.

Itamunoala, G. Tanaka, H. Matsumura, and 1. Zab orsky, 0. In this chapter, we review several industrial processes, utilizing industrial enzymes such as starch conversion and enzymatic hydrolysis of celluloses. Before we discuss the enzymatic hydrolysis of starch and cellulose, we review the organic chemistry ofcarbohydrates.

They are essential to the maintenance of plant and animal life. Carbohydrates are classified into three major groups:monosaccharides, oligo- saccharides, 1 and polysaccharides. Monosacchandes are the simplest carbohydrate units. Oligosaccharides contain two or more of these simple mono sacchande units, and polysaccharides contain hundreds or thousands of them.

All simple monosaccharides have the general empirical formula, CH 20 n, where n is the whole number ranging 3 to 8. All monosaccharides can be grouped into two general classes as: 1. As indicated in Table 4. The asymmetric carbon atom may have two possible alTangements of the groups around it. The two structures may be nonsuperimposable mirror images, and can be expected to differ in the rotation of plane- polarized light to an equal extent but in the opposite direction.

Industrial Applications of Enzymes 71 Table 4. Monosaccharides have asymmetric carbon atoms 2. The number of possible optical isomers for a compound can be determined by the formula 2n , where n stands for the number of asymmetric carbons.

As an example, aldohexose see Table 4. Glucose or dextrose is one of aldohexoses which has two isometric forms Figure 4. The D form predominates in tIle nature. Glucose is the most common and most important hexose and is found in most sweet fruits and in blood sugar.

In solution, very few sugar molecules exist with free aldehyde or ketone functional groups. The resulting structure has a ring form which is known as cyclic hemiacetal. There is an equilibrium between the ring and open forms. The open form allows the aldehyde or ketone group to react. The formation of a cyclic hemiacetal generates an additional asymmetric center at the original carbonyl atom. Pure fj-D-glucose equilibrates in water to give a mixture of 64 percent f3- and 36 percent a-D-glucose.

A more realistic representation for the hemiacetal ring structure is the Haworth projection formulas. The formulas for a-D-glucose are shown in Figure 4. The shorthand form of the Haworth projection eliminates the Hs and indicates OHs by dashes. Five- and six- membered cyclic sugars are called furanose and pyranose, respectively. Industrial Applications of Enzymes 73 Figure 4. However, despite the inaccuracy of the Haworth formulas, they are used more frequently than the chair conformation, because they are easier to draw and interpret.

Fructose is a keto sugar and is found in fruits and honey. Fructose sweeter than other natural sugar. If we take the relative sweetness of cane sugar as one, glucose is measured to be 0. Figure 4. Maltose and cellobiose are repeating units of polymeric starch and cellulose, respectively.

Disaccharides may hydrolyze to form two monosaccharide molecules. For this reason sucrose is termed a nonreducing sugar. Sucrose is the only nonreducing sugar among the four disaccharides.

Lactose, sugar present in milk, is a dimer of fi-D-galactose bonded 1' - 4' with D-glucose. The aldehyde group of the left ring of lactose is used for linkage. However, the right ring of the lactose can be opened to react because its aldehyde group is not used for linkage. As a result, lactose is a reducing sugar.

Further hydrolysis of maltose yields two molecules of glucose. Cellobiose, a stereoisomer of maltose, is obtained by the partial hydrolysis of cellulose. Maltose and cellobiose are both reducing sugars, since the right rings may open to react, as reducing agents.

One of the most important polysaccharides is starch, which is produced by plants for food storage. Animals produce a related material called glycogen. Starch comprises a large percentage of cereals, potatoes, corn, and rice. Complete hydrolysis of starch yields glucose, but partial hydrolysis gives maltose as well.

This shows that starch is a polymer of glucose units, joined by a-glycosidic linkage. Starch can be separated into two mam fractions by treatment with hot water. The glucosyl residues glucose minus water of amylose are linked by a 1' - 4' glycosidic bonds in a single chain that contains up to 4, glucose units Figure 4. The long linear molecule of amylose exists as a helix that contains six glucosyl residues per turn.

Amylopectin is a highly branched amylose. Various length of the linear chains, a 1' - 4' glucans containing 20 to 25 residues, are linked to a core chain by a 1' - 6' glycosidic bonds Figure 4.

Amylose and amylopectin are degraded by a- and f3-amylase, which are found in the pancreatic juice and saliva of animals, a-amylase is an endoglycosidase which attacks the amylose and amylopectin randomly along the a 1' - 4' bonds.

Neither enzyme can hydrolyze the a 1' - 6' branch points, which can be degraded by other enzymes, called debranching enzymes. Enzymatic hydrolysis of starch by sequential treatment with a-amylase and glucoamylase will produce glucose as the mam final product. They are used in infant food because they are easier to digest than starches. Dextrins are sticky when wet and are used as mucilage on postage stamps and envelopes. Cellulose is the most abundant organic compound of natural origin on the face of the earth.

Complete hydrolysis of cellulose gives glucose. The cellulose molecule is comprised of long chains of cellobiose molecules joined together by fi-I,4-glucosidic bonds as shown in Figure 4. The molecular weight of cellulose ranges from , to , 1, to 3, glucose units. The digestive systems of man and most other animals except ruminants do not contain the necessary enzymes cellulase for hydrolyzmg p-glucosidic linkages.

However, cellulases are found in ruminants, various insects, fungi, algae, and bacteria. High-fructose corn syrup HFCS is approximately twice as sweet as sucrose. It is used in soft drinks, canned fruits, lactic acid beverages, juice, bread, ice cream, frozen candies, and so on.

HFCS can be obtained from a variety of cereals and vegetables, such as corn, wheat, rice, potatoes, and cassava. Corn is the most important source of HFCS because of low costs and excellent utilities of its by-products, corn meal, oil, gluten, germ, and fiber. The steeping lasts about 40 hours. The sulfur dioxide inhibits fermentation and helps softening of the kernel.

The steeped corn kernels are torn apart in a degerminating mill to free the germ containing corn oil and to loosen the hull. The germ is separated in a continuous liquid cyclone, washed, and dried for oil recovery. Starch and hull are ground and screened to eliminate the hull.

The resulting mill starch contains 5 to 8 percent protein which is separated in a centrifuge. The separated-out starch is further purified in a hydroclone to reduce the protein content to a minimum level of 0.

Industrial Applications of Enzymes 77 fi!. The syrup produced has D. The enzyme used for this step, a-amylase, is thermostable and splits the starch at the interior of the molecule Joglekar et al. After liquefaction, the syrup is further hydrolyzed to glucose by the action of fungal glucoamylase, which acts on starch by splitting glucose units from the nonreducing end.

After saccharification, the liquor 95 percent free glucose is filtered, and passed throug. It is then concentrated in an evaporator to 60 percent solids. The glucose syrup obtained is isomerized to fructose by passing through an immobilized isomerase colum. The glucose isomerase is immobilized by an inert carrier, such as glass beads or DEAE- cellulose.

A typical residence time is 30 minutes. The isomerization reaction is reversible with an equilibrium constant of about 1. Therefore, the expected final concentration of fructose will be less than half of the inlet glucose concentration.

The finished product leaving the reactor contains 42 percent fructose, 50 percent glucose, and other saccharides. After isomerization, the syrup is purified by passing through a filter and ion-exchanger and is concentrated in an evaporator.

Cellulose is a renewable resource that is inexpensive, widely available and present in ample quantities. Large amounts of waste cellulose products are generated by commercial and agricultural processes. In addition, municipal facilities must treat or dispose of tremendous quantities of cellulosic solid waste. Cellulose provides strength and flexibility, while lignin supports and protects the cellulose from biological and chemical attack. Hemicellulose bonds lignin to cellulose.

Native cellulose is basically composed of microfibrils, which are bundles of lamellae containing an indefinite number of fibrillar units. Their schematic representation is shown in Figure 4. Cellulose molecules, hydrophilic linear polymers, are linked together to form elenzentary fibrils or photofibrils , about 40A wide, 30A thick, and A long.

The linear polymers in an elementary fibril are oriented in a parallel alignment and are bounded by hydrogen bonds to form a crystalline region, which is surrounded by a disordered layer of cellulose molecules, an amorphous region or paracrystalline region Ranby, Microfibrils in cell wall components are again surrounded by hemicelluose layer and lignin.

Lignin is a complex aromatic biopolymer of high molecular weight and is formed by the polymerization of oxidatively formed radicals of p-hydroxycinnamyl alcohols Hira et aI, It should be noted that the term lignin cannot be regarded as one individually defined compound, but rather, as a collective term for a whole series of similar, large polymeric molecules which are closely related structurally to one another.

The complexity of the chemical structure of lignin makes it very difficult to utilize except as a fuel. Since isolated lignin is a by-product of the pulp industry, its economical utilization has been actively sought. Because of its relatively high calorific value 12, BTU lib , lnost of waste lignin is being used as fuel in the chemical recovery processes of the pulp plants. Only a small part of lignin is utilized in adhesives, structural polymers, coating, dispersants, soil conditioner, pesticide carrier, and so on.

Several processes for the conversion of polymeric lignin to simple chemical feedstock have been developed. However, y vanillin, dimethyl sulfide, and methyl mercaptan are produced in commercially significant quantities Drew et al. Hemicellulose or polyose is primarily composed of xylan, a branched polymer composed of five-carbon sugar, xylose.

Typical polymerization degree of hemicellulose is 50 - , which is shorter than the cellulose molecules. These obstacles necessitate a costly pretreatment step in which elementary cellulosic fibrils are exposed and separated. Many pretreatments have been employed to enhance the degradation of lignocellulosic materials to glucose. The treatments fall into two general areas Ryu and Lee, : 1. Ball milling is the most commonly used pretreatment. It reduces crystallmity and particle sizes, while it increases surface area, bulk density, and the water soluble fraction.

The major drawbacks of the milling are cost and the fact that noncellulosic substances are not removed. Common chemical pretreatments of alkali and acid contacting improve hydrolysis by breaking down the lignin, hemicellulose, and cellulose. However, chemical hydrolysis is not specific and a variety of products are formed.

A balance must be met between the enhanced hydrolysis and production. Deligmfication treatment, such as Kraft and sulfite pulping used in the pulp-and-paper industry, is too expensive to be considered as an economical pretreatment. The rate and extent of enzymatic hydrolysis was found to be increased significantly by combining the pretreatment and reaction steps into one process Kelsey and Shafizadeh, ; Ryu and Lee, ; Deeble and Lee, ; Jones and Lee, The separate processes can be combined by using an attrition bioreactor, which is a stirred reactor with stainless-steel balls Ryu and Lee, ; Deeble and Lee, ; Jones and Lee, By using this reactor, the amount of time required for the hydrolysis of newsprint or sawdust can be reduced to hours as compared to days which are necessary in a regular stirred reactor.

Enhanced conversion of cellulose in the attrition bioreactor is due to a combination of fact ors, including a reduction in crystallmity, an increase in pore volume and surface area, and an increase in the accessibility of glucosidic bond sites to the cellulase complex. It was also found that enzyme deactivation in the attrition bioreactor is not significant, since interfacial forces, not shear forces, cause the most deactivation.

Elimination of the air- liquid interface by covering the reactor substantially increased the enzyme stability. Industrial Applications of Enzymes 81 4. They are Trichoderma T reesei, T. Bacterial species such as Cellulomonas along with Clostridium thermocellum can also produce cellulases Marsden and Gray, The cellulase enzymes produced by submerged fermentation of the cellulolytic microfungus Trichoderma are usually classified into three general categories: endo-fj,I,4 glucanases endoglucanases , cellobiohydrolases, and ,4 glucosidases cellobiase.

Typical proportions of the endoglucanases and cellobiohydrolases in the extracellular protein from Trichoderma reesei grown in cellulose are 15 percent to 20 percent and 35 percent to 85 percent, respectively. The cellobiase makes up less than 1 percent of the protein Marsden and Gray, The endoglucanases hydrolyze cellulose molecules randomly along the molecule and are more effective in combination with cellobiohydrolases.

Cellobiohydrolases cleave cellobiose and glucose from the nonreducmg end of the cellulose molecule. Endoglucanases and cellobiohydrolases must be absorbed by the cellulose before a reaction can occur, producing soluble sugars and oligomers. Various enzymes have been reported to be susceptible to deactivation upon shearing due possibly to the disturbance of their tertiary structure.

Several investigators have studied the interfacial deactivation of T. The addition of a surfactarlt has been found to substantially reduce enzyme deactivation. The surfactant impedes the migration of enzyme to the air-liquid interface. Consequently, less enzyme reaches the interface, where it will deactivate by unfolding when subjected to surface tension forces.

The cellulase deactivation due to this interfacial effect combined with the shear effect was found to be far more severe and extensive than that due to the shear effect alone Kirn et aI. The basic assumptions for the derivation of models are summarized in Table 4. Cellulose materials are composed of a highly ordered crystalline phase and a permeable amorphous phase. Some considered this multiplicity of cellulose structure and others ignored it and assumed that cellulose is a homogeneous material.

Concerning the enzyme acted on the substrate, some researchers considered all three components of the cellulase enzyme system endoglucanases, cellobiohydrolases, and cellobiase. Since the kinetic behavior of the three enzyme system are different and not fully understood, simplified models were also suggested by assuming the cellulase system can be represented quantitatively by a single enzyme.

To illustrate the procedure for the development of kinetic models of the cellulose hydrolysis, let's examine the model proposed by Ryu et al.

A kinetic model for the enzymatic hydrolysis of cellulose "vas derived based on the following assumptions: 1. The cellulosic material So is composed of amorphous matter Sal crystalline matter SCI and nonhydrolyzable merts Sx' and their rates of enzymatic degradation are different. The products glucose and cellobiose in11ibit the cellulase enzyme competitively. The reaction scheme based on the preceding assumption can be written as follows: E At low enzyme concentration KeC E « 1 , Eq.

This is known as pseudo-steady-state assumption. The change of the intermediate concentrations are: 4. NaOH 3. Rochelle salts sodium potassium tartrate 5. Dissolve the Titrate 3 mL sample with phenolphthalein with 0.

It should take mL of 0. Dilute the standard glucose solution to make 0. Use these known gilicose solutions as samples for step 3 and 4. Prepare a calibration curve which shows the absorbance versus glucose concentration.

Dilute a sample so that the expected concentration is in the range of the calibration curve. Place in boiling water for 5 minutes. Cool to room temperature. Read light absorbance at nm with a water blank. Read the value of glucose concentration corresponding to the absorbance from the calibration curve. In describing the activity of the cellulase, one IV is equivalent to the strength to release 1JLmoies of glucose per minute, because the moleclliar weight of the substrate, cellulose polymer, is not well defined.

Frequently we use filter paper as a substrate for the measurement of cellulase activity because it is well defined and yields reproducible results. The experimental procedure to measure the FI-'U of cellulase Mandels et al.

Whatman No. DNS reagent Assay Procedures: 1. Dissolve 0. You are going to measure the enzyme activity of this solution. Coil the filter paper strip and put it in a small test tube. Add 0. Stop the reaction by immersing the test tube into an ice bath. Take 0. If the sample amount is 0. Add 3 mL of DNS reagent to stop the reaction. Cool to room temperature and read light absorbance at nm using water asa blank.

Read the glucose concentration corresponding with the absorbance from the calibration curve. Louis, MO 3. Pour mL O. Start the hydrolysis reaction by adding 6 g of cellulose into the vessel. Take 3 mL sample once every hour and continue the experiment several hours.

DetermiIte the total reducing sugar content in the sample by using the DNS method and prepare a curve to show the change of the reducing sugar concentration with time. Industrial Applications of Enzymes 89 4. The substrate is pure cellulose, which is assumed to be a material with a uniform quality. The products glucose and cellobiose inhibit the cellulase enzyme competitively. The cellulosic material So is composed of amorphous Sa' crystalline Sc' and nonhydrolyzable inert part Sx' and their rates of enzymatic degradation are different.

The cellulose is first hydrolyzed at its surface to cellobiose by the synergistic action of enzymes, endoglucanases and cellobiohydrolases, which are denoted as E Cheng, Y.

Deeble, M. Drew, S. Kadam, S. Shoemaker, W. Glasser, and P. Sere 74, Fan, L. Fengel, D. Polymer Sci. Part C 36 Hira, A. Barnett, C. Shieh, and J. Montecalvo, Jr. Sere 74, : Howell, J. Huang, A.

Joglekar, R. Clerman, R. Ouellette, and P. Cheremisinoff, Biotechnology in Industry, pp. Industrial Applications of Enzymes 91 Jones, E. Kelsey, R. Kirn, M. Lee, D. Ryu, and E. Mandels, M. Andreotti, and C. Roche, "Measurement of Saccharifying Cellulase,"Biotechnol. Marsden, W. Miller, G. Okazaki, M. Peitersen, N. Ranby, B. Sere 95 : Reese, E. Ryu, D. Lee, T. Tassman, and C. Ryu, S. It covers only what is necessary for understanding the terminologies and procedures introduced in this book.

Today, the use of microorganisms is even more widespread than before. They are not only used for the traditional microbial processes but also for new processes such as the production of pharmaceuticals, industrial chemicals, enzymes, agricultural chemicals, waste water treatments, mineral leaching, and recombinant DNA technologies.

The protists refer relatively simple biological organisms compared to plants and animals and include algae, protozoa, fungi, and bacteria. The development of the electro microscope allowed the scientists to recognize that unit structure of all living organisms are divided into two category, prokaryotes and eukaryotes. The prokaryotic cell is the unit of structure in two microbial'groups: bacteria and blue-green algae.

The prokaryotic cell is small and simple, as shown in Figure 5. The cell has only two structurally distinguishable internal regions: cytoplasm and nuclear region or nucleoplasm.

The cytoplasm has grainy dark spots as a result of its 1 It is recommended that readers obtain any college-level textbooks in biology and micro- biology for further reading and reference as the need occurs. Cell Cultivations 93 content of ribosomes, which are composed of protein and ribonucleic acid RNA. The ribosome is the site of important biochemical reactions for protein synthesis. The nuclear region is of irregular shape, sharply segregated even though it is not bounded by membrane.

The nuclear region contains deoxyribonucleic acid DNA , which contains genetic information that determines the production of proteins and other cellular substances and structures. Table 5. The prokaryotic cell is surrounded with a cell wall and a cell membrane. The cell wall, considerably thicker than the cell membrane, protects the cell from external influences.

The cell membrane or cytoplasmic membrane is a selective barrier between the interior of the cell and the external environment.

The largest molecules known to cross this membrane are DNA fragments and low-molecular-weight proteins. The cell membrane can be folded and extended into the cytoplasm or internal membranes. The cell membrane serves as the surface onto which other cell substances attach and upon which many important cell functions take place.

The more complex eukaryotic cell is the unit structure in plants, animals, protozoa, fungi, and algae. The eukaryotic cell has internal unit membrane systems that segregate many of the functional components of the cell, as shown in Figure 5.

They are 1, to 10, times larger and more complex than prokaryotic cells. The nucleus is surrounded by a double membrane with pores 40 to 70 mJ.! The nucleus controls hereditary properties and all vital activities of the cell.

The chromosomes are long and threadlike bodies and are found in the nuclei of cells, which contain the genes arranged in linear sequence in nucleoproteins proteins plus nucleic acid. The cytoplasm contains large numbers of granules called ribosome, which are involved in continuous reactions to synthesize cell materials.

The ribosome is especially concentrated along the rough surface of the endoplasmic reticulum, an irregular network of interconnected membrane-delimited channels. The mitochondria contain the electron transport enzymes that utilize oxygen in the process of energy generation.

Vacuole and lysosome are organelles that serve to isolate various chemical reactions in a cell. Microbial Nomenclature: Microbiologists use the binomial system, in which each organism has two names, for example, Bacillus subtilis. Proper names of organisms are always italicized. The first word is the name of the genus plural, genera and is capitalized. The genus name is a Latin or Greek word. Salmon, Latinized Saccharomyces: sugar fungus The second word in the name of a microorganism is the species name and is not capitalized.