Synergistic effects of multiple enzymes from industrial Aspergillus niger strain O1 on starch saccharification

Background Starch is one of the most important renewable polysaccharides in nature for production of bio-ethanol. The starch saccharification step facilitates the depolymerization of starch to yield glucose for biofuels production. The filamentous fungus Aspergillus niger (A. niger) is the most used microbial cell factory for production of the commercial glucoamylase. However, the role of each component in glucoamylases cocktail of A. niger O1 for starch saccharification remains unclear except glucoamylase. Results In this study, we identified the key enzymes contributing to the starch saccharification process are glucoamylase, α-amylase and acid α-amylase out of 29 glycoside hydrolases from the 6-day fermentation products of A. niger O1. Through the synergistic study of the multienzymes for the starch saccharification in vitro, we found that increasing the amount of α-amylase by 5-10 times enhanced the efficiency of starch saccharification by 14.2-23.2%. Overexpression of acid α-amylase in strain O1 in vivo increased the total glucoamylase activity of O1 cultures by 15.0%. Conclusions Our study clarifies the synergistic effects among the components of glucoamylases cocktail, and provides an effective approach to optimize the profile of saccharifying enzymes of strain O1 for improving the total glucoamylase activity.

Archaeal and bacterial thermostable amylopullulanases: characteristic features and biotechnological applications

Amylopullulanases are endoacting bifunctional enzymes capable of hydrolyzing α-1,4- and α-1,6-glycosidic linkages in starch, amylose, pullulan, amylopectin and related oligosaccharides. These enzymes possess single or dual active site(s) for cleaving α-1,4- and α-1,6-glycosidic bonds; the former are called amylopullulanases, and the latter, α-amylase-pullulanases. These are grouped into GH13 and GH57 families based on the architecture of the catalytic domain and the number of conserved sequence regions. The amylopullulanases/α-amylasepullulanases are produced by bacteria as well as archaea, and among them, thermophilic and hyperthermophilic species are the major producers. The thermostable amylopullulanases find application in one-step starch liquefaction-saccharification to form various sugar syrups and maltooligosaccharides. The starch saccharification process catalysed by amylopullulanases minimizes the use of other amylolytic enzymes, like α-amylase and glucoamylase, thereby reducing the cost of sugar syrups. The enzymes also find applications in bread making as an anti-stale and as a detergent additive.

Bioconversion of degraded husked sorghum grains to ethanol

Efficient starch saccharification is an essential step towards achieving improved ethanol yields by fermentation. Sorghum grains are important starch sources for bioconversion to ethanol. In the present study, disease degraded (spoilt) husked grains from Nigerian sorghum cultivars were obtained from field sites and subjected to bioprocessing to ethanol. The crude husked grains (comprising husks, spikelet, awn, rachis and pubescence materials) were hammer milled and each meal separately mashed with enzyme cocktails comprising amylase, glucanase and protease enzymes. The saccharified worts obtained were then fermented with the yeasts, Saccharomyces cerevisiae and Pichia stipitis (aka Scheffersomyces stipitis), without exogenous nutrient supplementation. Sugars liberated during mashing were determined and it was found that enzymatic hydrolysis of milled sorghum grains was effective in yielding favourable levels of fermentable sugars up to 70g sugar/100g substrate with one particular cultivar (KSV8). Ethanol and carbon dioxide production was measured from subsequent trial fermentations of the sorghum mash and it was found that S. cerevisiae produced ethanol levels equating to 420 L/t that compares very favourably with yields from wheat and barley. Our findings show that crude degraded sorghum grains represent favourable low-cost feedstocks for bioconversion to ethanol with reduced energy input and without additional costs for nutrient supplementation during fermentation. Consequently, our results suggest some economic benefits could be derived from spoilt or degraded sorghum grains.

Enhancing the Secretion Efficiency and Thermostability of a Bacillus deramificans Pullulanase Mutant (D437H/D503Y) by N-Terminal Domain Truncation

ABSTRACTPullulanase (EC 3.2.1.41), an important enzyme in the production of starch syrup, catalyzes the hydrolysis of α-1,6 glycosidic bonds in complex carbohydrates. A double mutant (DM; D437H/D503Y) form ofBacillusderamificanspullulanase was recently constructed to enhance the thermostability and catalytic efficiency of the enzyme (X. Duan, J. Chen, and J. Wu, Appl Environ Microbiol 79:4072–4077, 2013,http://dx.doi.org/10.1128/AEM.00457-13). In the present study, three N-terminally truncated variants of this DM that lack the CBM41 domain (DM-T1), the CBM41 and X25 domains (DM-T2), or the CBM41, X25, and X45 domains (DM-T3) were constructed. Upon expression, DM-T3 existed as inclusion bodies, while 72.8 and 74.8% of the total pullulanase activities of DM-T1 and DM-T2, respectively, were secreted into the medium. These activities are 2.8- and 2.9-fold that of the DM enzyme, respectively. The specific activities of DM-T1 and DM-T2 were 380.0 × 108and 449.3 × 108U · mol−1, respectively, which are 0.94- and 1.11-fold that of the DM enzyme. DM-T1 and DM-T2 retained 50% of their activity after incubation at 60°C for 203 and 160 h, respectively, which are 1.7- and 1.3-fold that of the DM enzyme. Kinetic studies showed that theKmvalues of DM-T1 and DM-T2 were 1.5- and 2.7-fold higher and theKcat/Kmvalues were 11 and 50% lower, respectively, than those of the DM enzyme. Furthermore, DM-T1 and DM-T2 producedd-glucose contents of 95.0 and 94.1%, respectively, in a starch saccharification reaction, which are essentially identical to that produced by the DM enzyme (95%). The enhanced secretion and improved thermostability of the truncation mutant enzymes make them more suitable than the DM enzyme for industrial processes.