Industrial Application of Enzymes on Carbohydrate ... - ACS Publications

Chapter 6

Overcoming Practical Problems of Enzyme Applications in Industrial Processes: Dextranases in the Sugar Industry 1

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Gillian Eggleston , Adrian Monge , Belisario Montes , and David Stewart

Downloaded by UNIV LAVAL on May 1, 2016 | http://pubs.acs.org Publication Date: August 30, 2007 | doi: 10.1021/bk-2007-0972.ch006

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Commodity Utilization Research Unit, Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, L A 70124 Cora Texas factory, P.O. Box 280, White Castle, LA 70788 Alma Plantation L L C , 4612 Alma Road, Lakeland, LA 70752 2

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Dextranases only have a small market and low volume sales compared to many other industrial enzymes. Consequently, research and development efforts to engineer properties of dextranases to specific conditions of industrial processes have not occurred and are not expected soon. This book chapter highlights the difficulties associated with the practical application of dextranases, that are sometimes applied to hydrolyze dextran in sugar manufacture when bacterial deterioration of sugarcane or sugarbeet has occurred. Less than optimum application existed because of confusion about where to add the dextranase in the factory/refinery and which commercial dextranase to use. The wide variation in activity of commercially available dextranases in the U.S., and a standardized titration method to measure activities at the factory are discussed. Optimization by applying "concentrated" dextranase as a working solution to heated juice is described. Promising short-term technologies to further improve industrial dextranase applications are discussed, as well as the long-term outlook.

© 2007 American Chemical Society

Eggleston and Vercellotti; Industrial Application of Enzymes on Carbohydrate-Based Material ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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Introduction The major contributor to sugarcane and sugarbeet deterioration in the U.S. is Leuconostoc mesenteroides infections (7-2). Factors affecting infection in the sugarcane plant and extracted juice are ambient temperature and humidity, level of rainfall and mud, length of sugarcane billet, degree of burning, billet damage, delays between burning and cutting and subsequent processing, and mill hygiene. L. mesenteroides produce dextrans (a-(l—•6)-a-D-glucans), that in moderate and severe cases can interrupt normal processing operations. Dextran causes expensive microbial losses of sucrose and losses of sucrose to molasses. Moreover, the factory is penalized by refineries for dextran in the raw sugar. Dextrans are polydisperse by nature, i.e., they exist as a wide range of molecular weights. The high viscosity associated with the H M W portions (> 1000 KDa) of dextran affects boiling house operations, often reducing rates of evaporation and crystallization. Dextrans isolated from sugarcane products possess a largely linear structure (5) comprised of - 9 5 % glucose units linked by (1—»6) glycosidic bonds, but also containing - 5 % branching through (1—>4), (1—>3) and some (1—>2) linkages (Figure 1).

H

OH

Figure 1. Basic chemical structure of dextran. From Eggleston et al. (4).

Commercial dextranases (1—>6-a-glucan hydrolases, E C 3.2.1.11) have been used in sugarcane and sugarbeet (2) factories/refineries to break down dextran by hydrolyzing a-(l—>6) linkages at random endogenous sites (5). The


75 hydrolysis of dextran by dextranase is not an "all or nothing" mechanism. Instead, there is a gradual decrease in the average molecular weight of the various dextran fragments produced from the original H M W dextran, and these fragments in turn are continuously hydrolyzed (Figure 2). Dextranase is mostly added to improve boiling house operations. High mol. weight dextran (>1000 KDa)

H 0 I dextranase 2


f

Med mol. weight dextran (-100-1000 KDa)

H 0 I dextranase 2

• llllltll

I

>IM

MIMIM

Lower mol. weight dextran (-45-100 KDa) •-• IMM

6) glycoside linkages in random sites of HMW dextran. Dots and connecting lines represent chains of glucose molecules linked by a-(l^>6) bonds in the dextran molecule. Reaction time is not represented. * In dextranase application in the sugar industry, glucose as the end product rarely or never occurs. From Eggleston et al. (4).

Although the application of dextranases in the sugar industry was pioneered by Australians in the 1970s (5-7), their application in U.S. sugar manufacture is still not optimized. This is partly because of confusion about which dextranase to use, and how and where to add the dextranase. The problem has been exacerbated by the lack of comprehensive U.S. dextranase studies, and the few studies that have been undertaken most never stating the dextranase activity (810). This has led to enormous confusion concerning what is the appropriate dosage to apply for each commercial dextranase. Also, dextranases have a small market and low volume sales compared to other industrial enzymes such as ctamylases in the large detergent and food industries. Thus, they have not been subjected to much research and development by large enzyme companies and their properties have not been tailored to sugar industry conditions. As a


76 consequence, at the request of the Louisiana raw sugar industry, studies were undertaken to optimize the application of dextranases in U.S. factories.


Standard Method to Measure Dextranase Activity at Factories or Refineries Until recently, the optimization of dextranase applications in sugar factories/refineries was hindered by the lack of a standard or uniform method to measure the activity of commercial dextranases and, unfortunately, there is no regulatory body in the U . S . to issue or regulate standard activity methods and units for commercial enzymes (77-72). Marquardt and Bedford (13) reported that a similar problem occurred in the animal feeds industry. The lack of a standard method meant that the activity of commercial dextranases were being quoted by suppliers/vendors in many units, which only confused the factory buyer/user, and direct comparison of activities at the factory was not possible. Furthermore, the commercial dextranase market is very dynamic and activities and prices change regularly. To solve this, Eggleston (14) identified and modified a simple titration method (Figure 3) to measure the activity of dextranases in D U / m L units, which is now being successfully used by several U.S. factories.

Figure 3. Photograph of the use of the Eggleston (14) titration method to measure the activity of commercial dextranases at the factory (See page 2 of color inserts.) The titration method (11J 4) is easy to use, and there are no requirements for sophisticated equipment or a standard curve. Although the titration method



77 measures the. activity of dextranases under more optimal conditions than factory juice applications, it is highly correlated to a spectrophotmetric method (7), and higher industrial temperatures made no relative difference (7). Moreover, Eggleston and Monge (7) reported that IC-IPAD (ion chromatography with integrated pulsed amperometric detection) profiles of dextran/dextranase mixtures, used as a reference method, confirmed the accuracy of the relative activities of commercial dextranases by the titration method. The urgent need for a standard method to measure the activity of dextranases at the factory or refinery was highlighted by the measurement of wide variations (up to 20-fold) in activity of commercial dextranases in the U.S. sugar industry that do not always reflect the costs of the enzymes (11). In some factories this has greatly reduced the dextranase efficiency because the users unknowingly added an dextranase of low activity. This is discussed further in the next section.

Differences in the Activities of Commercial Dextranases Most commercial dextranases in the U.S. are produced from Chaetomium gracile or erraticum fungi, generally recognized as safe (GRAS), and formulated as liquids. Table I lists the activities, measured using the Eggleston factory titration method (14% of some dextranases commercially available in the U.S. and used in U.S. sugarcane factories.

Table I. Relative Activities of Dextranases in 2003 and 2004. From Eggleston et al. (15) Commercial Dextranase A B C D a

b

Dextranase activity DU/mL 2003° 2004 52,000 51,920 57,687 ND 5,499 3,500 4,786 2,750 b

Classification

"concentrated" "concentrated" "non-concentrated" "non-concentrated"

From Eggelston and Monge (7) ND=not determined

U.S. dextranases occur in a wide range of activity, that Eggleston et al. (1, 11, 15) classified into "non-concentrated" (1,000 ppm/Brix) in juice. Applications of 2- or 5-fold working solutions (higher dosages than in laboratory studies were required at 6 ppm, that were normalized to the original enzyme activity) of "concentrated"dextranase (52,000 DU/mL) were successful in consistently hydrolyzing 70-94% dextran across a 5 min R tank. In strong comparison, the addition of 4.2 ppm (typical dosage applied previously by the factory staff) of "non-concentrated" dextranase (2,750 DU/mL) had neglibible effect on dextran hydrolysis in juice across the same 5 min R tank (75). At a second Louisiana factory, with lower antibody dextran concentrations (

Industrial Application of Enzymes on Carbohydrate ... - ACS Publications

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