A Thermostable Glucoamylase from Bispora sp. MEY-1 with Stability over a Broad pH Range and Significant Starch Hydrolysis Capacity

Abstract

BackgroundGlucoamylase is an exo-type enzyme that converts starch completely into glucose from the non-reducing ends. To meet the industrial requirements for starch processing, a glucoamylase with excellent thermostability, raw-starch degradation ability and high glucose yield is much needed. In the present study we selected the excellent Carbohydrate-Activity Enzyme (CAZyme) producer, Bispora sp. MEY-1, as the microbial source for glucoamylase gene exploitation.Methodology/Principal FindingsA glucoamylase gene (gla15) was cloned from Bispora sp. MEY-1 and successfully expressed in Pichia pastoris with a high yield of 34.1 U/ml. Deduced GLA15 exhibits the highest identity of 64.2% to the glucoamylase from Talaromyces (Rasamsonia) emersonii. Purified recombinant GLA15 was thermophilic and showed the maximum activity at 70°C. The enzyme was stable over a broad pH range (2.2–11.0) and at high temperature up to 70°C. It hydrolyzed the breakages of both α-1,4- and α-1,6-glycosidic linkages in amylopectin, soluble starch, amylose, and maltooligosaccharides, and had capacity to degrade raw starch. TLC and H1-NMR analysis showed that GLA15 is a typical glucoamylase of GH family 15 that releases glucose units from the non-reducing ends of α-glucans. The combination of Bacillus licheniformis amylase and GLA15 hydrolyzed 96.14% of gelatinized maize starch after 6 h incubation, which was about 9% higher than that of the combination with a commercial glucoamylase from Aspergillus niger.Conclusion/SignificanceGLA15 has a broad pH stability range, high-temperature thermostability, high starch hydrolysis capacity and high expression yield. In comparison with the commercial glucoamylase from A. niger, GLA15 represents a better candidate for application in the food industry including production of glucose, glucose syrups, and high-fructose corn syrups.

Heterologous, Expression, and Characterization of Thermostable Glucoamylase Derived fromAspergillus flavusNSH9 inPichia pastoris

“…The rGA from A. flavus NSH9 had a higher optimum temperature compared to recombinant glucoamylase from Chaetomium thermophilum [ 21 ] and A. awamori [ 28 ]. Meanwhile, pH stability of rGA reported in this study was almost similar to a number of researchers [ 9 , 18 , 19 , 39 , 42 ]. Glucoamylase that shows high stability over a wide pH range for relatively long period time indicates its suitability for industrial applications [ 9 , 19 ].…”

“…Our finding was in parallel with previous reported studies with molecular weights of GA in fungi to be within a range of 25–112 kDa [ 9 ]. Many researchers reported that the molecular weight of recombinant glucoamylase was higher than the estimated molecular weight and it depended on the glycosylation site within the sequence, which supported this study [ 21 , 37 – 39 ]. The study did not measure the degree of glycosylation of the recombinant glucoamylase and deglycosylation where this will give a clearer picture of the recombinant enzyme properties and become one of the limitations of this study.…”

“…are commonly used in starch industry for the saccharification process [ 39 , 43 ]. But the glucoamylase from fungal sources are active at acidic condition and also less thermostable, and the industrial saccharification takes a long time to produce the desired yield [ 4 ], for this it also required pH and temperature adjustments from liquefaction to saccharification steps [ 39 ]. The reported recombinant glucoamylase (rGA) from A. flavus NSH9 can be used directly after liquefaction process for a complete hydrolysis of the dextrin into β -D-glucose, due to its thermostability.…”

Computational design of noncanonical amino acid-based thioether staples at N/C-terminal domains of multi-modular pullulanase for thermostabilization in enzyme catalysis

“…Enzyme thermostabilization remains a topic of interest in biocatalysis field, because thermostability is a significant property of enzymes that can be used to evaluate their feasibility for industrial applications [1] . Enzymes with considerable thermostability exhibit remarkable advantages in accelerating reactions, increasing evolutionary potential, and reducing microbial contamination and production cost [2] , [3] . Presently, developing thermostable enzymes is a fundamental priority for green manufacturing processes, including pharmaceutical development, fine chemical production, and biofuel synthesis.…”

Purification and Characterization of a Thermostable Starch‐Saccharifying Alpha‐1,4‐Glucan‐Glucohydrolase Produced by Bacillus licheniformis

“…3.2.1.3] (glucoamylase) is an exo‐type enzyme that converts starch completely into glucose from the nonreducing ends. Therefore, in order to fulfill the requirement of starch processing industries, glucoamylase that have raw‐starch degradation ability with tremendous physicochemical properties and high glucose yield is desirable …”