Improvement and estimation of enzymic starch saccharification process

Linko, Yu‐Yen; Wu, Xiao Y.

doi:10.1007/bf00156327

Cited by 10 publications

(9 citation statements)

References 11 publications

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“…Aliquots were drawn at regular time intervals, and the amount of glucose liberated was determined by high-performance liquid chromatography (HPLC). The DX value (D-glucose content) was defined as the percentage of glucose on a dry-weight basis (29). All of the values presented in the graphs and tables are mean results of three replicates.…”

Section: Methodsmentioning

confidence: 99%

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

Duan

2015

Appl Environ Microbiol

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Pullulanase (EC 3.2.1.41), an important enzyme in the production of starch syrup, catalyzes the hydrolysis of ␣-1,6 glycosidic bonds in complex carbohydrates. A double mutant (DM; D437H/D503Y) form of Bacillus deramificans pullulanase 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 ؋ 10 8 and 449.3 ؋ 10 8 U · 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 the K m values of DM-T1 and DM-T2 were 1.5-and 2.7-fold higher and the K cat /K m values were 11 and 50% lower, respectively, than those of the DM enzyme. Furthermore, DM-T1 and DM-T2 produced D-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. P ullulanase (EC 3.2.1.41), which catalyzes the hydrolysis of ␣-1,6-glucosidic linkages in pullulan, amylopectin, and the dextrins of amylopectin, is a well-known starch-debranching enzyme (1). It belongs to the ␣-amylase family, which is identified as glycoside hydrolase family 13 in the CAZy database. When combined with ␣-amylase, ␤-amylase, glucoamylase, or cyclodextrin glucosyltransferase, pullulanase can be used in the production of glucose, fructose, maltose, cyclodextrins, and amylose (2, 3). The addition of pullulanase allows the reaction time to be reduced, the substrate concentration to be increased, and the conversion rate to be improved (4, 5). Recently, pullulanase has been used as a biocatalyst for the production of fuel ethanol, low-calorie beers, resistant starch, maltotriose, and other important products (6, 7). With this plethora of potential industrial applications, pullulanase production has attracted a great deal of attention in recent years (8,9).A large number of microorganisms, including mesophiles, thermophiles, and hyperthermophiles, have been shown to produce pullulanase (10, 11). Although numerous pullulanases have been identified and heterologously expressed (12)(13)(14), reports have shown that the secretion ratio of recombinant pullulanase is low. Low extracellular prod...

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Section: Methodsmentioning

confidence: 99%

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

Duan

2015

Appl Environ Microbiol

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“…Low quality grains and starch containing waste materials which are surplus agricultural commodities, could be used to produce value added chemicals like fuel ethanol, methane and lactic acid (Lee 1991a;Linko and Wu 1993). Countries like Brazil, Canada, South Africa and the United States have a large agricultural surplus annually and studies are being conducted on the production of ethanol from carbohydrate-rich plant biomass.…”

Section: Starch and Whole Grain Hydrolysis For Alcohol Productionmentioning

confidence: 99%

“…The first step in the process is the liquefaction of starch with a-amylase, followed by saccharification with glucoamylase to form a high yield of glucose. Debranching enzymes like isoamylase and pullulanase could be added to improve glucose yields (Linko and Wu 1993). In the typical fermentation process, glucose syrup is mixed with corn steep liquor, a by-product of wet milling.…”

Section: Starch and Whole Grain Hydrolysis For Alcohol Productionmentioning

confidence: 99%

Glucoamylases: Microbial Sources, Industrial Applications and Molecular Biology ? A Review

James

Lee

1997

J Food Biochemistry

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Glucoamylase (EC 3.2.1.3) hydrolyzes polysaccharides from the nonreducing chain ends by cleaving α‐1,4 and α‐1,6 glycosidic bonds consecutively. Glucoamylases are used mainly in the production glucose syrup, high fructose corn syrup, and in whole grain and starch hydrolysis for alcohol production. This paper reviews the status of glucoamylases with respect to microbial sources, biochemical and physical properties. Methods used to assay glucoamylase activity are also compared, with reference being made to the specificity of spectrophotometric methods in detecting end products of the enzyme action. Commercial glucoamylases and development of immobilization techniques are discussed. Also, structural analysis of glucoamylase and the main amino acids involved in catalysis and starch binding are emphasized. The cloning of the glucoamylase gene in Saccharomyces cerevisiae for the brewing of low calorie beer is presented. This review highlights the use of glucoamylase in the food industry.

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“…For the saccharification of sorghum starch to produce glucose, the optimised parameters were reported as sorghum syrup concentration 30% (w/v), glucoamylase 6 U mL −1 , temperature 55 °C and pH 4.5; then, the glucose syrup with DE value of 90% was achieved finally (Shewale & Pandit, ). Although previous studies have addressed the investigations on enzymatic saccharification (Linko & Wu, ; Lóapez et al ., ; Li et al ., ), most of them merely focused on the type of raw material and the general process and conditions. Development of a highly efficient saccharification process using mixed enzymes involving pullulanase is yet to be discussed in detail.…”

Section: Introductionmentioning

confidence: 99%

Enhancement of glucose production from maltodextrin hydrolysis by optimisation of saccharification process using mixed enzymes involving novel pullulanase

Zhao

Nie

et al. 2015

Int J of Food Sci Tech

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Summary Pullulanase is important to enhance the production and quality of glucose from starch hydrolysis by mixed enzymatic saccharification. To improve glucose production, it would be necessary to manipulate the enzymatic saccharification process and conditions. For enzymatic saccharification involving the newly identified recombinant pullulanase, glucose, maltose and isomaltose were detected, and glucose was the main product from maltodextrin hydrolysis, with the dextrose equivalent value over 99.5%. During the saccharification process, there would be a balance between glucose and disaccharides due to enzymatic hydrolysis and its reverse reaction. With the optimised ratio of glucoamylase to pullulanase (50:1) and dry maltodextrin concentration (20%, w/w), the glucose content (DX) around 99% was obtained with the space‐time yield of 6 mg g−1·h. After optimisation by orthogonal design and response surface methodology, the DX value of 99.35% was achieved with enzyme loading 75 U g−1 at 55 °C and pH 4.5 for 36 h.

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Improvement and estimation of enzymic starch saccharification process

Cited by 10 publications

References 11 publications

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

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

Glucoamylases: Microbial Sources, Industrial Applications and Molecular Biology ? A Review

Enhancement of glucose production from maltodextrin hydrolysis by optimisation of saccharification process using mixed enzymes involving novel pullulanase

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