Enzymes for food and brewing research

Enzymes for mixed-linkage glucan (β-glucan) analysis and degradation

Present in a number of grains, β-glucan is a homopolymer of glucose with predominantly β-1,4 linkages interspersed with β-1,3 linkages. The molecular weight of barley β-glucan has been reported as 175,000 daltons and 250,000 daltons. The molecular weight of oat β-glucan has been reported to be much higher, with molecular weights of soluble and insoluble forms at 1,100,000 and 1,600,000 daltons, though another report gives a value of 88,000. Cellotriose and cellotetraose units make up approximately 90% of the β-glucan in barley flour; these cellotriose and cellotetraose units are linked to each other by a single β-1,3-linkage.  The beta-glucan content in whole grain oats ranges from 2 to 8% of dry weight; the beta-glucan content of oat bran concentrate is higher, between 15 and 35% of dry weight. Other grains, especially barley, also possess significant amounts of beta-glucan (Figure 1).

Figure 1

beta-glucan graph

β-Glucanases cleave either β-1,4 linkages in mixed-linkage glucans containing  β-1,4 and β-1,3 linkages.  β-Glucanases cleave β-1,4 linkages in mixed-linkage glucans but not in crystalline cellulose, while endocellulases β-1,4 linkages in mixed-linkage glucans and crystalline cellulose.   (This also explains why both cellulase and β-glucanase activities are measured using β-glucan as substrate.)  β-Glucanases cannot cleave the β-1,3 linkages in mixed-linkage glucans, these can only be cleaved by curdlanases or lichenases (Figure 2).

Figure 2  

GLucan

 

C5-6 supplies a complete set of mixed-linkage glucan-degrading activities to advance your research. β-glucanases and endocellulases from C. thermocellumD. turgidumA. cellulolyticus, and Geobacillus sp. all possess the stability to withstand the high temperatures encountered in feed production.   All these enzymes can be easily scaled-up for pilot and production testing. Other β-glucanases, endocellulases, and curdlanases are available from a diverse range of organisms including B. cellulosilyticusF. succinogenes, Cellulomonas species, and T. reesei.  β-glucosidases are available from all these organisms, allowing you to match activity profiles of endo-activities and exo-activities.

Enzymes for xylan analysis and degradation

Xylan contains a backbone of  β-1,4 linked xylose residues.  Depending on the source, the backbone can be substituted with α-1,2 linked glucuronic acid (glucuronoxylan, GX), α-1,2 and  α-1,3 linked arabinose (arabinoxylan, AX) or α-1,2 linked glucuronic acid and  α-1,3 linked arabinose (glucuronoarabinoxylan, GAX) (Figure 3).  Additional substitutions such as acetyl and methyl groups can be present, and the material can be crosslinked to components of lignin such as ferrulic acid.  Xylanases (technically endoxylanases) cleave β-1,4 xylose linkages in the backbone.  β-xylosidases cleave xylose from the nonreducing end of the xylan chain.  α-Glucuronidases cleave the α-1,2 linked glucuronic acid from the backbone and α-arabinosidases (arabinofuranosidases) cleave the α-1,2 and  α-1,3 linked arabinose from the backbone.

Figure 3  

Xylan

 C5-6 supplies a complete set of xylan-degrading activities to advance your research.  Xylanases from C. thermocellumD. turgidum, and Geobacillus sp. all possess the stability to work under extreme conditions. A wide range of thermostable bacterial β-xylosidases,  α-glucuronidases, and α-arabinosidases are also available that complement these bacterial xylanases.  Many of these enzymes are available only through C-6.  A number of these thermostable bacterial enzymes have been shown to be superior to their fungal counterparts in producing both xylose and glucose from pretreated biomass (Gao D, Chundawat S, Liu T, Hermanson S, Gowda K, Brumm P, Dale BE, Balan V. Strategy for identification of novel fungal and bacterial glycosyl hydrolase hybrid mixtures that can efficiently saccharify pretreated lignocellulosic biomass. Bioenergy Research. 2010. 3(1):67-8).   All these enzymes can be easily scaled-up for pilot and production testing. C5-6 is also the only source for the full set of T. reesei xylan-degrading enzymes (xylanases, β-xylosidase,  α-glucuronidase, and α-arabinosidase), as well as the only source of  xylanases from Cellulomonas species, B. cellulosilyticus and F. succinogenes .

Enzymes for starch analysis and degradation

Starch is a complex polymer of alpha-(1,4) and alpha-(1,6)-linked glucose used by plants to store energy.  Amylose is a linear polymer of only alpha-(1,4)-linked glucose.  When treated with iodine, this polymer gives the typical blue starch reaction.  Amylopectin is a highly branched polymer of short and medium length alpha-(1,4)-linked glucose chains connected by single alpha-(1,6)-bonds, with an overall structure resembling a tree (Figure 4).

Figure 4

Slide1

In many applications, starch hydrolysis is carried out in two steps.  The first step, liquefaction, converts the amylose and amylopectin in a gelatinized starch into soluble, short chain oligosaccharides containing one to twenty glucose residues (dextrins) using an α-amylase (AA). The second step, saccharification, converts these dextrins into glucose using an amyloglucosidase (GA, also known as glucoamylase). In beer brewing, a single stage liquefaction and saccharification process is used.  In this step, the barley α-amylase and β-amylase partially degrade the starch to form a mixture of maltose, maltotriose and higher dextrins, called the wort.  

Optimum starch hydrolysis for ethanol or glucose production requires the following:

1.     Complete hydrolysis of both alpha-(1,4) and alpha-(1,6)-linked glucose residues. Incomplete hydrolysis results in formation of short oligosaccharides that are not converted by GA during the saccharification and fermentation steps.  The incomplete hydrolysis results in lower ethanol yield.

2.     Prevention of recrystallization of linear amylose chains. Recrystallization of amylose chains occurs rapidly when the temperature is lowered from 92°C to 60°C.  The result is a white precipitate or haze that is not attacked by the GA during saccharification or fermentation, resulting in decreased ethanol yield.
 

C5-6 supplies a number of starch-degrading activities to advance your research.  Thermostable Geobacillus stearothermophilus α-amylase (BstAA) is widely used in industrial application, with activity up to 107°C.  We also are the only supplier of two novel thermostable α-amylases, one from D. turgidum and one from A. acidocaldarius.  These enzymes have not been extensively characterized by our lab, but both show thermostability similar to BstAA.  Additional fungal and bacterial α-amylases, fungal glucoamylase,  and bacterial β-amylases can be produced upon request.