**2. Glucoamylase-active heterogeneous biocatalysts for starch dextrin hydrolysis**

The glucoamylases (glucan 1,4-alpha-glucosidases, EC 3.2.1.3) hydrolyze the glycosidic bonds at the end of polymer (starch) or oligomer (dextrin) chains releasing glucose. The main areas of industrial application of these enzymes are as follows: (1) a large scale two-step hydrolysis of raw starch successively catalyzed by α-amylase then glucoamylase, for production of sweeteners such as treacle and glucose syrups used in food industry, and (2) large-scale processes of hydrolytic conversion of starch to fermentable sugars as feedstocks for the production of some commodity chemicals and the first-generation biofuel such as bioethanol. These industrial processes are conducted on an enormous scale. Although the enzymes involved are relatively inexpensive, they are used on a single-use, throw-away basis. As mentioned above, immobilization of enzymes ensures enzyme recycling that can provide significant saving in the cost of final products not less than 20%.

The glucoamylases are the main enzymes used in the key second stage of starch conversion—hydrolysis of dextrin (saccharification), following the stage of liquefication (dextrinization) of starch by amylases. It should be noted that the immobilization of glucoamylase (not amylase) is justified and appropriate because this enzyme converts relatively low molecular weight substrates such as dextrin (3– 5 kDa), and diffusion limitations can be overcome, in particular via design of employed reactor. The development of heterogeneous biocatalysts with high glucoamylase activity and operational stability is of great importance since they can serve as the basis for modern technology for deep processing of renewable vegetable raw materials into demandable sweeteners.

Back in 1970s, Corning Glass Co. carried out the fist pilot plant tests of a packedbed reactor filled with a heterogeneous biocatalyst prepared by covalent immobilization of glucoamylase on macroporous silica; the glucose productivity was 450 kg/ day. The main requirement for the commercial glucoamylase-active biocatalysts was a sufficiently high operational stability at pasteurization temperature of 60°C or higher. Inactivation half-times (t½) of the tested Corning Glass biocatalyst were 520, 150, and 75 h at 55, 60, and 70°C, respectively [4]. The best result described later in 2000 for a biocatalyst prepared by immobilization of glucoamylase on polystyrene was that t½ = 300 h at 50°C [5]. The best result described in the recent papers during 2008–2019 is that the glucoamylase immobilized by formation of cross-linked enzyme aggregates (CLEA) has "excellent recyclability, retaining over 45% of the relative activity after 24 runs" over a broad range of temperature (55–75°C) [6]. According to the opinion of the specialists working on the R&D projects of heterogeneous stage of saccharification the low thermal stability of the immobilized glucoamylase at elevated temperature (above 50°C) was the main reason why this process has not been commercialized so far.

Reputedly, the inexpensive and available carbonaceous materials with appropriate texture parameters are promising supports for adsorptive immobilization of enzymes, in particular of glucoamylase, for the preparing of commercially

of microbiology, molecular biology, biochemistry, chemical technology, and engineering sciences. Biocatalysis is a scientific field fully focused from the very beginning on practice, and the main task is to study and use only one selected enzymatic reaction for the purposeful target biotransformation of the initial reagent—substrate (S)—into the valuable product (P) demanded by the market [1]. Heteroge-

Despite unique catalytic properties of soluble enzymes, such as 100% selectivity

enzymes and ensures their multifold reusability. The heterogeneous biocatalysts are prepared by immobilizing the enzymatic active substances. These biocatalysts are undoubtedly of great practical interest for widespread implementation in industrial periodic and continuous bioconversion processes using batch stirred-tank, packedbed, and novel types of vortex reactors specially designed for the heterogeneous diffusion-controlled biocatalytic processes in order to overcome diffusion limita-

It is generally recognized that heterogeneous biocatalytic processes are more commercially attractive for large-scale implementation than homogeneous technologies due to considerable simplification and reduction (in 1.2–1.4 times) of the total production cost. Of course, the cost of the final product decreases with a decrease in the cost of the enzymatic active component of the biocatalyst, as well as the support and the method of immobilization. In order to reduce all expenses, both not purified enzymes but partially or fully disrupted, or whole nongrowing microbial cells, as well as inorganic support such as silica and carbon, and adsorptive immobilization

For successful commercialization of the heterogeneous biocatalytic processes, for example, the process of starch dextrin hydrolysis (saccharification), a biocatalyst has to convert 45% of the substrate during 15–20 min with the inactivation halftime (t½) of 30–120 days, which corresponds to 3–12 months operation of the reactor without reloading. A high value of t½ (50–100 days) is essential to increase productivity up to the recommended value from 100 kg to 10 tons of final product

Heterogeneous biocatalysts for two important bioconversion processes are briefly described here. One of the biocatalysts is glucoamylase immobilized by adsorption on mesoporous carbon support Sibunit™ type. This glucoamylase-active biocatalyst is used at the stage of starch saccharification, i.e., hydrolysis of dextrin to treacle and glucose syrups. The second of the biocatalysts is recombinant *T. lanuginosus* lipase immobilized on mesoporous silica KSK™ type and macroporous

–105 times). Immobilization prevents the inactivation of

and high reaction rates under very mild, usually ambient conditions, as well as chemo-, regio-, and stereo-specificity, their industrial applications are limited due to the main disadvantages, namely, homogeneous conditions of periodic processes and inability to reuse enzymes that fall into wastewater. Immobilization of enzymes on/in support may overcome this drawback. Immobilization is defined as the fixation of enzymatic active substances onto/inside water insoluble solid supports, accompanied by retaining their enzymatic activity at a high level and their signifi-

neous biocatalysis is a very important part of biocatalysis that is based on immobilized enzymatic active substances such as individual enzymes, whole microorganisms, and partially or completely disrupted microbial cells. Heterogeneous biocatalysis as an interdisciplinary sphere of professional activities, undoubtedly, has great scientific importance and commercial potential for industrial implementation, including the processing of renewable raw materials into valuable market products. In accordance with the 12 principles of green chemistry listed in [1, 2], biocatalytic processes satisfy all the requirements and provide environmentally friendly and energy-saving technologies that are a promising alternative to

traditional chemical processes.

*Molecular Biotechnology*

cant stabilization (up to 10<sup>3</sup>

per 1 kg of biocatalyst [3].

**4**

tions and enhance biocatalysts' productivity.

are preferable for preparing commercial biocatalysts.

attractive heterogeneous biocatalysts. Sibunit™-type supports from a new class of carbonaceous materials are porous carbon-carbon composites that combine the advantages of both graphite such as chemical stability and electrical conductivity, and active carbons in particular high specific surface area and adsorption capacity. These supports are characterized by a high volume of mesopores and a narrow controllable pore size distribution; some types have a large proportion of pores with a size of 5–20 nm or a bidisperse meso- and macroporous structure suitable for enzyme immobilization. Indeed, the sizes of globular molecules of most enzymes in aqueous solutions are approximately 10 nm. And it can be argued that the specific accessible surface area can be calculated using pore size distribution diagrams on the assumption that pores with a diameter more than 10–15 nm are available for immobilization of enzymes.

biocatalyst prepared by adsorption of glucoamylase on Sibunit granules of 0.2–

*Heterogeneous Biocatalysts for the Final Stages of Deep Processing of Renewable Resources…*

In order to overcome external diffusion limitations, a novel type of reactor such as immersed vortex reactor (IVR) was designed and tested in lab scale [7, 8]. The reactor body was filled with biocatalyst granules and then was immersed in a substrate solution and rotated. Substrate solution of dextrin was sucked through a bottom hole upon the body rotation and then moved with various high speeds through the biocatalyst bed toward side holes. Thus, a significant intensifying mass transfer of a substrate toward immobilized enzymes and, as a result, overcoming diffusion limitations was achieved by rotating the reactor body immersed in a substrate solution. Another very important advantage of the vortex type of reactor is an absence of stagnant zones and jet streams inside the bed of biocatalyst. To prevent formation of stagnant zones, the profiled reactor body was designed (**Figure 2a**). Since the profile of each half of the prefabricated reactor body was made in accordance with hyperbolic equation, the annular cross-section area for a substrate solution stream was equal to *2πRh* and remained constant during circulation of substrate solution under operation. During this movement, the liquid was obviously affected by hydrodynamic, centrifugal, and inertial (Coriolis) forces, which resulted in a vortex flow of the substrate solution. The centrifugal forces were responsible for slight compression of the biocatalyst bed, narrowing channels for liquid flowing between granules, which results in the additional increase of mass transfer. Because of high mechanical strength of the Sibunit support, the granules were not distorted during the operation. Thus, the formation of stagnant zones was

A lab scale setup of the immersed vortex reactor filled by the glucoamylaseactive biocatalysts GlucoSib was studied in the heterogeneous process of starch dextrin hydrolysis [7]. To elucidate optimal conditions for the IVR operation, the effect of rotation rate of the IVR body on the activity was studied. The maximal activity of GlucoSib, 700–750 U/g, was measured at body rotation of 300–900 rpm (**Figure 2b**). At 1000–1200 rpm, the reaction rate decreased perhaps due to the formation of a funnel in the rotating reaction medium, and special profiled device

The stability of the glucoamylase-active biocatalysts determined during continuous operation in saccharification heterogeneous stage was sufficiently high. Thus, the half-life time (t½) exceeded 700 and 350 h at 50 and 60°C, respectively.

*Lengthwise cut of IVR body (a) and glucoamylase activity of the biocatalyst depending on rotation rate of reactor body (b). Photo of IVR body. Conditions of dextrin hydrolysis: 50°C, 0.05 M acetate buffer pH 4.6,*

0.7 mm in size was determined to be maximal, 530 U/g.

*DOI: http://dx.doi.org/10.5772/intechopen.89411*

minimized.

**Figure 2.**

**7**

was designed to remove this defect.

*10 w/v% solution of potato dextrin as substrate.*

Textural parameters of the Sibunit support are as follows: total specific surface area, S sp BET = 550 m<sup>2</sup> /g; total pore volume, V<sup>Σ</sup> = 0.86 mL/g; and average pore diameter, Dpore = 18 nm as follows from pore size distribution diagram (**Figure 1a**). Specific surface area accessible for enzyme immobilization was estimated to be 92 m<sup>2</sup> /g, that is 16% of Ssp BET. Surface of Sibunit formed by round coke deposits of pyrolytic carbon looks rough and porous on the scanning electron microscopic (SEM) images (**Figure 1b**).

The properties of the best glucoamylase-active biocatalyst (designated as GlucoSib) prepared by physical adsorption of commercial enzyme preparation GlucoLux™ type (produced by Sibbiopharm, Novosibirsk, Russia) on mesoporous carbon support Sibunit™ are described in [7] and briefly here.

It was found that the porous texture of the carbon supports plays a key role in stabilization of glucoamylase [7]. Predominant mesopores (10–20 nm in diameter) of Sibunit and bulk catalytic filamentous carbon are appropriate in size to hydrated enzyme molecules, whereas micropores of activated carbon (4 nm in diameter) are too small. Exactly, mesopores provide multipoint binding enzyme molecules inside the support and, as a result, ensure great stabilization of the activity. The thermal stability of glucoamylase adsorbed on mesoporous carbon supports, measured in dextrin solutions (32–53 w/v%) was found to be 105 times higher than for soluble enzyme [7].

Macrokinetics of dextrin hydrolysis by immobilized glucoamylase was investigated. Internal diffusion of dextrin inside the porous space of the biocatalysts toward adsorbed glucoamylase was found to be a rate-limiting stage of the saccharification process. Indeed, the rate of dextrin hydrolysis significantly reduced if the granule size was larger than 1 mm; for example, from 400 to 180 U/g for 1.2- and 3 mm granules, respectively [7]. The GlucoSib-type biocatalysts were prepared using support granules with diameter less than 1 mm; for example, the activity of the

**Figure 1.** *(a) Pore size distribution diagram and (b) SEM image of Sibunit surface.*

### *Heterogeneous Biocatalysts for the Final Stages of Deep Processing of Renewable Resources… DOI: http://dx.doi.org/10.5772/intechopen.89411*

biocatalyst prepared by adsorption of glucoamylase on Sibunit granules of 0.2– 0.7 mm in size was determined to be maximal, 530 U/g.

In order to overcome external diffusion limitations, a novel type of reactor such as immersed vortex reactor (IVR) was designed and tested in lab scale [7, 8]. The reactor body was filled with biocatalyst granules and then was immersed in a substrate solution and rotated. Substrate solution of dextrin was sucked through a bottom hole upon the body rotation and then moved with various high speeds through the biocatalyst bed toward side holes. Thus, a significant intensifying mass transfer of a substrate toward immobilized enzymes and, as a result, overcoming diffusion limitations was achieved by rotating the reactor body immersed in a substrate solution. Another very important advantage of the vortex type of reactor is an absence of stagnant zones and jet streams inside the bed of biocatalyst. To prevent formation of stagnant zones, the profiled reactor body was designed (**Figure 2a**). Since the profile of each half of the prefabricated reactor body was made in accordance with hyperbolic equation, the annular cross-section area for a substrate solution stream was equal to *2πRh* and remained constant during circulation of substrate solution under operation. During this movement, the liquid was obviously affected by hydrodynamic, centrifugal, and inertial (Coriolis) forces, which resulted in a vortex flow of the substrate solution. The centrifugal forces were responsible for slight compression of the biocatalyst bed, narrowing channels for liquid flowing between granules, which results in the additional increase of mass transfer. Because of high mechanical strength of the Sibunit support, the granules were not distorted during the operation. Thus, the formation of stagnant zones was minimized.

A lab scale setup of the immersed vortex reactor filled by the glucoamylaseactive biocatalysts GlucoSib was studied in the heterogeneous process of starch dextrin hydrolysis [7]. To elucidate optimal conditions for the IVR operation, the effect of rotation rate of the IVR body on the activity was studied. The maximal activity of GlucoSib, 700–750 U/g, was measured at body rotation of 300–900 rpm (**Figure 2b**). At 1000–1200 rpm, the reaction rate decreased perhaps due to the formation of a funnel in the rotating reaction medium, and special profiled device was designed to remove this defect.

The stability of the glucoamylase-active biocatalysts determined during continuous operation in saccharification heterogeneous stage was sufficiently high. Thus, the half-life time (t½) exceeded 700 and 350 h at 50 and 60°C, respectively.

#### **Figure 2.**

*Lengthwise cut of IVR body (a) and glucoamylase activity of the biocatalyst depending on rotation rate of reactor body (b). Photo of IVR body. Conditions of dextrin hydrolysis: 50°C, 0.05 M acetate buffer pH 4.6, 10 w/v% solution of potato dextrin as substrate.*

attractive heterogeneous biocatalysts. Sibunit™-type supports from a new class of carbonaceous materials are porous carbon-carbon composites that combine the advantages of both graphite such as chemical stability and electrical conductivity, and active carbons in particular high specific surface area and adsorption capacity. These supports are characterized by a high volume of mesopores and a narrow controllable pore size distribution; some types have a large proportion of pores with a size of 5–20 nm or a bidisperse meso- and macroporous structure suitable for enzyme immobilization. Indeed, the sizes of globular molecules of most enzymes in aqueous solutions are approximately 10 nm. And it can be argued that the specific accessible surface area can be calculated using pore size distribution diagrams on the assumption that pores with a diameter more than 10–15 nm are available for

Textural parameters of the Sibunit support are as follows: total specific surface

diameter, Dpore = 18 nm as follows from pore size distribution diagram (**Figure 1a**). Specific surface area accessible for enzyme immobilization was estimated to be

pyrolytic carbon looks rough and porous on the scanning electron microscopic

The properties of the best glucoamylase-active biocatalyst (designated as GlucoSib) prepared by physical adsorption of commercial enzyme preparation GlucoLux™ type (produced by Sibbiopharm, Novosibirsk, Russia) on mesoporous

It was found that the porous texture of the carbon supports plays a key role in stabilization of glucoamylase [7]. Predominant mesopores (10–20 nm in diameter) of Sibunit and bulk catalytic filamentous carbon are appropriate in size to hydrated enzyme molecules, whereas micropores of activated carbon (4 nm in diameter) are too small. Exactly, mesopores provide multipoint binding enzyme molecules inside the support and, as a result, ensure great stabilization of the activity. The thermal stability of glucoamylase adsorbed on mesoporous carbon supports, measured in dextrin solutions (32–53 w/v%) was found to be 105 times higher than for soluble

Macrokinetics of dextrin hydrolysis by immobilized glucoamylase was investi-

gated. Internal diffusion of dextrin inside the porous space of the biocatalysts toward adsorbed glucoamylase was found to be a rate-limiting stage of the saccharification process. Indeed, the rate of dextrin hydrolysis significantly reduced if the granule size was larger than 1 mm; for example, from 400 to 180 U/g for 1.2- and 3 mm granules, respectively [7]. The GlucoSib-type biocatalysts were prepared using support granules with diameter less than 1 mm; for example, the activity of the

carbon support Sibunit™ are described in [7] and briefly here.

*(a) Pore size distribution diagram and (b) SEM image of Sibunit surface.*

/g, that is 16% of Ssp BET. Surface of Sibunit formed by round coke deposits of

/g; total pore volume, V<sup>Σ</sup> = 0.86 mL/g; and average pore

immobilization of enzymes.

(SEM) images (**Figure 1b**).

area, S sp BET = 550 m<sup>2</sup>

*Molecular Biotechnology*

92 m<sup>2</sup>

enzyme [7].

**Figure 1.**

**6**

Comparing these data with the Corning's results in [4], the t½ of GlucoSib biocatalysts was estimated to be higher, 350 h vs. 150 h at 60°C, respectively. Longterm stability was sufficient also; the biocatalysts retained initial activity for 10 months upon storage at ambient temperature (18–22°C).

One of the best results of oils' methanolysis by commercial biocatalyst Novozym® is that conversion of triglycerides into methyl esters of fatty acids (biodiesel) was 99% for 50 h at 45°C [10]. In 2007, the first biocatalytic process of the biodiesel production was implemented by methanolysis of edible oils' waste with the productivity of 10,000 tons; the biocatalyst was prepared by immobilization of lipase

*Heterogeneous Biocatalysts for the Final Stages of Deep Processing of Renewable Resources…*

NOVO (NOVOZYMES) Company is a leader in the production and sale of heterogeneous biocatalysts prepared by immobilizing the recombinant lipases on various supports. The Lipozyme® TL IM biocatalyst is prepared by immobilization of recombinant thermostable 1,3-specific *T. lanuginosus* lipase on silica. This biocatalyst is widely used in the industrial interesterification of fat-oil blends in order to produce valuable products, such as specialized fats and spreads without undesirable *trans*-isomers of fatty acids, as well as substitutes of cocoa butter and dairy fat. The Novozym®435 biocatalyst is prepared by immobilization of nonspecific *Candida*

*antarctica* lipase on a macroporous polyacrylic polymer. The commercial

that this reagent did not reduce the activity of the commercial biocatalyst

For example, mono- and triacyl glycerol are employed as fuel additives. Triacylglycerol (triacetin) is widely used in the food industry due to its good moisture-retaining properties. If the linseed oil is used in biocatalytic interesterification with ethyl acetate, the produced mixture of ethyl esters of ω3-, ω6-unsaturated fatty acids (vitamin F) is a valuable product for cosmetics industry and fodder

Novozym® even at a molar ratio of oil to methyl acetate equal to 1:12; and under optimal operating conditions, the yield of methyl esters was equal to 96% and the biocatalyst's half-life time (t½) increased 20-fold in comparison with t½ in reaction with methanol [12]. Acyl derivatives of glycerol produced in interesterification of vegetable oils with methyl or ethyl acetate are valuable commercial products also.

Nowadays, enzymatic esterification is considered as a competitive alternative to the chemical organic synthesis of various esters that are valuable commercial products commonly used in manufacturing flavors, fragrances, emollients, lubricants, antimicrobial agents, and nontoxic surfactants. The requirement of consumers for such natural products is constantly increasing. Compared with organic synthesis using strong liquid and solid acids as catalysts and temperature above 100°C (usually, 120–150°C), the enzymatic esterification is currently of great commercial interest since this method of esters' production proceeds efficiently at a low temperature (usually, at 20–40°C) without the formation of any by-products and with high specificity toward substrates. The heterogeneous lipase-active biocatalysts for the low-temperature esters'synthesis are prepared, as mentioned above, by immobilizing lipases on solid supports by various chemical origins and texture. These heterogeneous biocatalytic processes realized in periodic or continuous modes in anhydrous reaction media fully satisfy the requirements of "green" chemistry and they are promising for implementation into the organic synthesis

The authors and their collaborators have developed and researched systematically the heterogeneous biocatalysts in which the enzymatic active component was a

Novozym® biocatalysts type are used in biodiesel production by methanolysis of vegetable oil (rapeseed and soya been) and waste oils of cooking. Nowadays, the NOVO biocatalysts are intensively studied for application in various processes,

It is well-known that methanol and ethanol inactivate enzymes rapidly. Therefore, in order to reduce the biocatalysts' inactivation, methanol was added stepwise in small portions during the reaction cycle of the biodiesel production. Another acylating reagent—methyl or ethyl acetate—was examined to be used. It was found

from *Candida* sp. [11].

*DOI: http://dx.doi.org/10.5772/intechopen.89411*

including organic synthesis.

additives production.

industry [2].

**9**

A technological scheme has been proposed and tested on a laboratory scale using the GlucoSib biocatalyst and immersed vortex reactor IVR for production of starch treacle and glucose syrups by heterogeneous dextrin hydrolysis. The advantages of this technological scheme are as follows: (1) significant acceleration of dextrin hydrolysis; (2) energy and resource saving in comparison with traditional starch processing; (3) a high quality of the final products due to the lack of protein impurities; and also, very importantly, (4) easily regulated carbohydrate composition of the treacle by simply stopping rotation of reactor body. It should be noted that when comparing the efficiency of the process of dextrin hydrolysis in vortex reactor with parameters of traditional packed-bed reactor the productivity of the IVR was higher by 1.2–1.5 times. The productivity in a novel proposed technology was calculated to be 5.3 tons of glucose per 1 kg of biocatalysts GlucoSib that is quite commercially attractive.

#### **2.1 Conclusion for the part 2**

The highly active and stable heterogeneous biocatalysts for dextrin hydrolysis were prepared by adsorption of glucoamylase on mesoporous carbon support Sibunit™. Under technological conditions (32 w/v% dextrin, 60°C, pH 5), the maximal activity was observed to be equal to 750 U/g, and inactivation half-life time (t½) was 350 h. The immersed vortex reactor designed specially for the biocatalytic diffusion-controlled heterogeneous processes was used to carry out starch saccharification with enhanced productivity roughly estimated as 5 tons of glucose per 1 kg of biocatalysts.
