Dextransucrase: A Microbial Enzyme with Wide Industrial Applications

Introduction

Dextransucrase is the important extra-cellular enzyme typically produced from lactic acid bacteria (LABs) such as Lactobacillus, Leuconostoc, oral Streptococcus, Weissella and Lactococcus species, respectively (Sidebotham 1974). Based on sequence similarities, this hydrolase enzyme has been categorized as protein belonging to family 70 glycoside hydrolase group (GH70) as per the CAZy (carbohydrate active enzymes) classification system and is related to GH13 and GH77 family (Lombard et al. 2014). Collectively, they form the GH-H clan of glycoside hydrolases with enzyme commission number E.C. 2.4.1.5 (Lombard et al. 2014). The distinguishing feature of GH-H clan enzymes includes cleavage of glycosidic bond between glucose and other sugars in presence of a catalytic domain (p/a)s. Based on the linking pattern, the enzyme produces three types of exo-polysaccharides such as alteman, dextran, and mutan designated by a general formula of C„(H20) wherein “n” implies any number varying from 200 to 2500. Dextran is a homo-polysaccharide containing repeating units of D-glucose joined by linear a-( 1 ->6) bonds, whereas the branches are due to a-(l->2), a-(l->3) or a-(l->4) bonds (Sidebotham 1974). The prebiotic effects of dextran produced by Weissella species, Lactobacillus plantarum A3 and Pediococcus pentosaceus 5S4 have been reviewed in recent times (Tingirikari et al. 2014). Several genes encoding dextransucrase are cloned and expressed from LAB strains belonging to Leuconostoc, Lactobacillus and Weissella genera (Kang et al. 2009). Moulis et al. (2006) expressed the curtailed “C” and N-terminal dextransucrase of Leuconostoc mesenteroides B-512F in E. coli, which produced a small polymer. The reducing end of the polymer chain was added with glucose (Robyt et al. 2008).

Several three-dimensional structures of glycoside hydrolase GH (70) dextransucrase (DS) have been elucidated in recent times, like recombinant glucosyl transferase (GTF) 180-AN from L. reuteri 180 (PDB:3KLK), GTF-SI from Streptococcus mutcms (PDB: 3AIE) and the glucan-binding domain AN123-GBD-CD2 of the a (l->2) branching GS DSR-E from L. mesenteroides NRRL B-1299 (PDB: 3TTQ), etc. (Ito et al. 2011). GTF180-AN complexes with sucrose and the acceptor maltose support the double-displacement mechanism and reveal that acceptor-binding sites have three domains (A, В and C), a feature which resembles that of GH 13-amylases (Pijning et al. 2008). The key amino acids involved at the catalytic site of dextransucrase were identified by active site mapping studies by targeting amino acids with specific inhibitors. The presence of lysine at the active site was confirmed by using specific inhibitor such as 2, 4, 6-trinitro benzene sulphonic acid, and Pyridoxal 5’ Phosphate forms e-NH, lysine derivative with dextransucrase. The presence of cysteine residues was confirmed by using 5, 5’-dithiobis (2-nitrobenzoic acid) to form thio-nitrobenzoate complex, and iodoacetic acid forms thioacetate complex (Tingirikari and Goyal 2014). The presence of lysine and cysteine at the active site of dextransucrase was confirmed by o-phthaladehyde, a bi-functional inhibitor, specific for the detection of both lysine and cysteine, which forms isoindole derivative between sulfhydryl group of cysteine and e-amino group of lysine if the distance between the two functional groups are between 2.6 A and 3.4 A apart (Tingirikari and Goyal 2013a).

Sources of Dextransucrase

Dextransucrase is produced by microorganisms belonging to genera Lactobacillus, Leuconostoc, and Streptococcus (Jeanes et al. 1954; Kandler et al. 1983). The most recent is Weissella species isolated from fermented vegetables and fruits (Hammes and Vogel 1995; Dols et al. 1998). Among these, dextrans produced by L. mesenteroides have been documented extensively in the past (Jeanes et al. 1954;

TABLE 9.1

Different Sources of Lactic Acid Bacteria That Produces Dextransucrase

Microorganism

Source

References

L. mesenteroides NRRL B-512F

Root beer

Jeanes et al. (1954)

L mesenteroides NRRL B-1431

Sugarcane juice

Jeanes et al. (1954)

L. mesenteroides NRRL B-1402

Orange concentrate

Jeanes et al. (1954)

L. mesenteroides NRRL B-1397

Sugar cane

Dols et al. (1998)

L. mesenteroides IBT-PQ

“Pulque” prepared from Agave juice

Chellapandian et al. (1998)

L. mesenteroides PCSIR-3

Cabbage and Carrot

Ul-Qader et al. (2001)

L. mesenteroides species

Fermented Idli batter

Sawale and Lele (2009)

L. mesenteroides FT 045B

Alcohol and Sugar Mill plant

Vettori et al. (2012)

Weissella minor

Sludge of milking machines

Kandler et al. (1983)

Weissella confusa

Sugar cane, Carrot juice, Raw milk and Sewage

Hammes and Vogel (1995)

Weissella confusa - Cab3

Fermented Cabbage

Shukla and Goyal (2011)

Weissella cibaria - JAG8

Apple

Tingirikari and Goyal (2013a)

P. pentosaceus

Pork meat

Anastasiadou et al. (2008)

P. pentosaceus - SPA

Sugar cane filed

Patel and Goyal (2010)

Chellapandian et al. 1998; Sawale and Lele 2009). It was observed that LABs are predominantly found in fermented foods, which included vegetables, meat, pickles and juices (Ul-Qader et al. 2001; Shukla and Goyal 2011; Tingirikari and Goyal 2013b). Some of the bacteria that produces dextransucrase have been listed in Table 9.1.

Reaction Mechanism of Dextransucrase

Dextransucrase follows an acceptor reaction mechanism which involves (1) the transfer of D-glucose as initiator for D-glucosyl branch linkages, and the allocation of glucan chain to give dextranyl branched glucan chains (Tingirikari and Goyal 2013b) as well as (2) D-glucose attachment to an acceptor sugar which can be a mono- or oligo-saccharide (Tingirikari et al. 2017) as shown in Figure 9.1. (3) Sometimes it shows an acceptor response in which the D-glucose moiety of sucrose is moved to water to covalent glucosyl-enzyme intermediate followed by polymerization or chain growth to give dextran (Tingirikari and Goyal 2013b). (4) The allocation of glucan chain to water (or) to an acceptor sugar (D-glucose, D-fructose, sucrose or maltose) is followed by chain growth and termination of polymerization to cleave the dextran as shown in Figure 9.2 (Tingirikari et al. 2017).

Reaction mechanism for the synthesis of dextran by dextransucrase

FIGURE 9.1 Reaction mechanism for the synthesis of dextran by dextransucrase.

Dextransucrase-mediated synthesis of prebiotic oligosaccharides

FIGURE 9.2 Dextransucrase-mediated synthesis of prebiotic oligosaccharides.

Production of Dextransucrase

Among the nutrients, sucrose was chosen as the major carbon source, as it not only induces the dextransucrase production, but also acts as substrate for dextran production (Shukla and Goyal 2012). It was reported that different nitrogen sources significantly affect the yield or production levels of dextransucrase (Patel et al. 2011). The use of dipotassium hydrogen phosphate in the production medium to buffer the rising levels of lactate released has been illustrated (Patel et al. 2011). Literature review also suggests that the application of Tween 80 changes the characteristics of the membrane, thus making it easier to release the dextransucrase enzyme (Tingirikari and Goyal 2013c). Tween 80 also affects the uniformity of the broth and facilitates the nutrient and oxygen accessibility to the microorganism.

Santos et al. (2000) reported that at 20°C under static conditions enzyme activity of L. mesenteroides NRRL B-512F decreased gradually with an increase in temperature although the production levels were high. It was reported that 25°C and agitation were optimum for enzyme production in L. mesenteroides NRRL B-640, while in L. dextranicum NRRL B-1146, 28°C and static conditions were optimum for dextransucrase production (Patel et al. 2011). Surprisingly, the control of pH and shaking displayed no change on the enzyme synthesis in L. mesenteroides NRRL B-1299 (Dols et al. 1997).

Barker et al. (1993) displayed that if the pH of the medium was in the range of 5.0-5.5, dextransucrase could convert more amount of sucrose to dextran. Recombinant dextransucrase from Weissella cibaria CMU expressed in E. coli displayed maximum production of enzyme at pH 5.4 and at 20°C (Kang et al. 2009). Further, Shukla and Goyal (2011) proved that synthesis of enzyme from W. confusa Cab3 was optimum at 25°C, 180 rpm and pH range of 5.2-5.6. In contrast, the dextransucrase isolated from W. cibaria JAG8 was optimally produced at 24°C, pH 6.0 and stationary environment (Tingirikari and Goyal 2013b). For Weissella sp. TN610, the enzyme production was found to be maximum at pH 5.0 and 37°C (Tingirikari and Goyal 2013b). So overall, we can say that the optimal conditions for this enzyme vary in the temperature range of 20°C-28°C and pH 5.0-6.0. A brief outline of bioreactor level production of dextransucrase under statistically optimized conditions is given in Table 9.2.

Comparative Level Production of Dextransucrase by Different Lactic Acid Bacteria Under Optimized Conditions

TABLE 9.2

Microorganism

Method of Production

Substrate (gram/Litre)

Enzyme

Activity

(U/mL)

References

L. mesenteroides- NRRL B-1299

Batch

fermentation

Sucrose 40. Yeast extract 20, K,HP04 0.2

5.5

Dols et al. (1997)

L. mesenteroides- IBT-PQ

Batch

fermentation

Sucrose 30, Yeast extract 20, K,HP04 25

2.21

Chellapandian etal. (1998)

L. mesenteroides- NRRL B-512F

Batch

fermentation

Sucrose 40, Yeast extract 20, K,HP04 8

5.23

Santos et al. (2000)

L. mesentemides- B/l 10-1-1

Fed Batch fermentation

Sucrose 20, Yeast extract 15, Na.HPO, 20

6.19

Michelena et al. (2003)

L. dextranicum- NRRL B-1146

Batch

fermentation

Sucrose 59.8, Yeast extract 17.5, Beef extract 17.5, Peptone 17.5. K;HP04 10. Tween-80 5.4

6.75

Majumdar and Goyal (2008)

L. mesenteroides- NRRL B-640

Batch

fermentation

Sucrose 30, Yeast extract 30, K,HP04 30, Beef extract 15, Tween-80 2

10.7

Purama and Goyal (2008)

Leuconostoc species

Batch

fermentation

Sucrose 130.75, Yeast extract 5.3, Beef extract 5.3, Sodium acetate 15.1

23.9

Sawale and Lele (2009)

P. pentosaceus- SPAml

Batch

fermentation

Sucrose 55.2, Beef extract 2.3, Tween-80 8.3

15.6

Patel et al. (2011)

L. mesenteroides- FT045B

Batch

fermentation

Sugarcane Molasses 40, K,HP04 20, Corn steep liquor 20

4.0

Vettori et al. (2012)

Weissella confusa- Cab3

Batch

fermentation

Sucrose 50, Yeast extract 20, K:HP04 10, Tween-80 5

17.9

Shukla and Goyal (2012)

Acetobacter tropicalis

Batch

fermentation

Sucrose 20, Peptone 10, MnS04 0.25, Sodium acetate 5

15.8

Chauhan et al. (2013)

Purification and Characterization of Dextransucrase

Several purification methods for dextransucrase have been reported in the past, which include polyethylene glycol (PEG) or ethanol precipitation, phase partitioning, ultra-filtration and chromatographic techniques (Majumder et al. 2007; Pijning et al. 2008). Fractionation of proteins or enzymes by PEG is a very popular and low-cost technique employed in separation of extra-cellular dextransucrase enzyme. PEGs are non-ionic, hydrophilic cleansing agents possessing the ability to separate proteins of high molecular weights in accumulated forms (Tingirikari and Goyal 2013c). However, the PEG fractionation method has limitations as it is inefficient in completely removing the associated polysaccharides from dextransucrase. Thus, creates problems or interferes with biochemical and functional characterization of such enzymes as one requires protein of high purity for the above mentioned exploration. Therefore, this shortcoming was addressed by using a combination of dex- tranase treatment followed by ion-exchange and affinity chromatography after PEG precipitation (Majumder et al. 2007).

There are other reports also wherein dextransucrase was purified by dialysing the supernatant followed by adding dextranase and gel filtration chromatography (Bio-Gel A-5m), to substantially increase purification fold by 240 times with a specific activity of 53 U/mg (Robyt and Walseth 1979). Kang et al. (2009) purified recombinant dextransucrase rDSRWC (recombinant dextransucrase Weissella cibaria) from W. cibaria CMU in presence of immobilized metal ion affinity chromatography (Ni-NTA super flow IMAC column), followed by use of concentrators like Centricon Ultracel YM-100 column to achieve a five-fold purification with specific activity of 12 U/mg. In contrast, dextransucrase from IK cibaria JAG8 purified by gel filtration chromatography showed an activity of 37 U/mg (Tingirikari and Goyal 2013c). The molecular weight of dextransucrase from lactic acid bacteria ranged from 160 to 200 kDa (Bounaix et al. 2010).

 
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