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For saccharifying starch in one step, a chimeric biocatalyst (Amy-Glu) was generated from engineered α-amylase (Ba-Gt-amy) of Bacillus acidicola and glucoamylase (Glu) gene of Aspergillus niger. In order to join two enzymes, a linker peptide of 25 amino acids was used. Chimeric Amy-Glu was expressed in E. coli. Glu is of 75kDa, while Amy-Glu is of 145kDa. Both Amy-Glu and Glu displayed similar pH profile with good activity in the acidic pH range like that of Ba-Gt-amy with optimum at pH 4.0. All three enzymes (Ba-Gt-amy, Amy-Glu and glucoamylase) exhibited activity in the temperature range between 40 and 70°C with optimum at 60°C. Amy-Glu and Glu have T 1/2 of 90 and 70min at 60 and 70°C, respectively. The K m , V max and K cat values of Glu (soluble starch) are 0.34mgmL -1 , 606μmolmg -1 min -1 and 727s -1 , while for Amy-Glu are 0.84mgmL -1 , 13,886μmolmg -1 min -1 and 4.2×10 4 s -1 , respectively. The end product analysis suggested that Amy-Glu retains the activity of both parental enzymes and forms maltodextrins along with glucose as the major products. Amy-Glu saccharifies wheat and corn starches more efficiently than the Ba-Gt-amy and glucoamylase., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Streptomyces badius DB-1 produces α-amylase extracellularly, and its production was enhanced 5.1-fold (from 9.47 ± 0.51 to 48.23 ± 1.45 U mL -1 ) due to optimization by one-variable-at-a-time and statistical approaches. Soluble starch emerged as the most influential factor that strongly affected enzyme production. The purified enzyme is a monomer with a molecular mass of ~57 kDa and optimally active at 50 °C and pH 6.0. The enzyme hydrolyzes soluble as well as raw starches into simpler sugars with a high proportion (>40.0 %) of maltotetraose. It is optimally active at moderate temperature and generates maltooligosaccharides from starch, thus, useful as an antistale in bread making. It also plays a role in increasing the formation of maltooligosaccharides due to transglycosylation activity, thus, finds application in functional foods. This is the first report on the production of raw starch-digesting α-amylase by S. badius with transglycosylation activity.

In order to understand the role of N1 domain (1-257 aa) in the amylopullulanase (gt-apu) of the extremely thermophilic bacterium Geobacillus thermoleovorans NP33, N1 deletion construct (gt-apuΔN) has been generated and expressed in Escherichia coli. The truncated amylopullulanase (gt-apuΔN) exhibits similar pH and temperature optima like gt-apu, but enhanced thermostability. The gt-apuΔN has greater hydrolytic action and specific activity on pullulan than gt-apu. The k cat (starch and pullulan) and K m (starch) values of gt-apuΔN increased, while K m (pullulan) decreased. The enzyme upon N1 deletion hydrolyzed maltotetraose as the smallest substrate in contrast to maltopentaose of gt-apu. The role of N1 domain of gt-apu in raw starch binding has been confirmed, for the first time, based on deletion and Langmuir-Hinshelwood kinetics. Furthermore, N1 domain appears to exert a negative influence on the thermostability of gt-apu because N1 truncation significantly improves thermostability.

The gene (1,542 bp) encoding thermostable Ca(2+)-independent and raw starch hydrolyzing α-amylase of the extremely thermophilic bacterium Geobacillus thermoleovorans encodes for a protein of 50 kDa (Gt-amyII) with 488 amino acids. The enzyme is optimally active at pH 7.0 and 60 °C with a t 1/2 of 19.4 h at 60 and 4 h at 70 °C. Gt-amyII hydrolyses corn and tapioca raw starches efficiently and therefore finds application in starch saccharification at industrial sub-gelatinisation temperatures. The starch hydrolysis is facilitated following adsorption of the enzyme to starch at the C-terminal domain, as confirmed by the truncation analysis. The adsorption rate constant of Gt-amyII to raw corn starch is 37.6-fold greater than that for the C-terminus truncated enzyme (Gt-amyII-T). Langmuir-Hinshelwood kinetic analysis in terms of equilibrium parameter (K R) suggested that the adsorption of Gt-amyII to corn starch is more favourable than that of Gt-amyII-T. Thermodynamics of temperature inactivation indicated a decrease in thermostabilisation of Gt-amyII upon truncation of its C-terminus. The addition of raw corn starch increased t 1/2 of Gt-amyII, but it has no such effect on Gt-amyII-T. It can, therefore, be stated that Gt-amyII binds to raw corn starch via C-terminal region that contributes to its thermostability. Phylogenetic analysis confirmed that starch binding region of Gt-amyII is, in fact, the non-catalytic domain C, and not the typical SBD of CBM families. The role of domain C in raw starch binding throws light on the evolutionary path of the known SBDs.

A gene encoding amylopullulanase (gt-apu) of the extremely thermophilic Geobacillus thermoleovorans NP33 was cloned and expressed in Escherichia coli. The gene has an open reading frame of 4,965 bp that encodes a protein of 1,655 amino acids with molecular mass of 182 kDa. The six conserved regions, characteristic of GH13 family, have been detected in gt-apu. The recombinant enzyme has only one active site for α-amylase and pullulanase activities based on the enzyme kinetic analyses in a system that contains starch as well as pullulan as competing substrates and response to inhibitors. The end-product analysis confirmed that this is an endoacting enzyme. The specific enzyme activities for α-amylase and pullulanase of the truncated amylopullulanase (gt-apuT) are higher than gt-apu. Both enzymes exhibited similar temperature (60 °C) and pH (7.0) optima, although gt-apuT possessed a higher thermostability than gt-apu. The overall catalytic efficiency (K(cat)/K(m)) of gt-apuT is greater than that of gt-apu, with almost similar substrate specificities. The C-terminal region of gt-apu appeared to be non-essential, and furthermore, it negatively affects the substrate binding and stability of the enzyme.

The α-amylase encoding gene from acidophilic bacterium Bacillus acidicola was cloned into pET28a(+) vector and expressed in Escherichia coli BL21 (DE3). The recombinant E. coli produced a 15-fold higher α-amylase than B. acidicola strain. The recombinant α-amylase was purified to homogeneity by one-step nickel affinity chromatography using Ni(2+)-NTA resin with molecular mass of 62 KDa. It is active in the pH range between 3.0 and 7.0 and 30 and 100 °C with optimum at pH 4.0 and 60 °C. The enzyme is Ca(2+)-independent with K (m) and k (cat) values (on soluble starch) of 1.6 mg ml(-1) and 108.7 s(-1), respectively. The α-amylase of B. acidicola is acidstable, high maltose forming and Ca(2+)-independent, and therefore, is a suitable candidate for starch hydrolysis and baking.

Glucoamylase is one of the oldest and widely used biocatalysts in food industry. The major application of glucoamylase is the saccharification of partially processed starch/dextrin to glucose, which is an essential substrate for numerous fermentation processes and a range of food and beverage industries. Glucoamylase for commercial purposes has traditionally been produced employing filamentous fungi, although a diverse group of microorganisms is reported to produce glucoamylase, since they secrete large quantities of the enzyme extracellularly. The commercially used fungal glucoamylases have certain limitations such as moderate thermostability, acidic pH requirement, and slow catalytic activity that increase the process cost. Consequently, the search for newer glucoamylases and protein engineering to improve pH and temperature optima leading to amelioration in catalytic efficiency of existing enzymes have been the major areas of research over the years. The present review focuses attention on the recent advances in molecular biology and protein engineering of glucoamylase to improve its production and functional properties including the so far success achieved in isolating mutants with enhanced thermostability and selectivity, higher pH optimum and improved catalytic activity. A comprehensive account is included on the diversity, regulation of production, classification, purification and properties, and potential applications of microbial glucoamylases to provide an overview on all the important aspects of the enzyme.

Among physical and nutritional parameters optimized by "one variable at a time" approach, four cultural variables (sucrose, MgSO4 .7H2O, inoculum size, and incubation period) significantly affected glucoamylase production. These variables were, therefore, selected for optimization using response surface methodology. The p-values of the coefficients for linear effect of sucrose and inoculum size were less than 0.0001, suggesting them to be the key experimental variables in glucoamylase production. The enzyme production (34 U/ml) attained under optimized conditions (sucrose, 2%; MgSO4 .7H2O, 0.13%; yeast extract, 0.1%; inoculum size, 5 x 10(6) spores per 50 ml production medium; incubation time, 48 h; temperature, 40 degrees C; and pH 7.0) was comparable with the value predicted by polynomial model (34.2 U/ml). An over all 3.1-fold higher enzyme titers were attained due to response surface optimization. The experimental model was validated by carrying out glucoamylase production in shake flasks of increasing capacity (0.25-2.0 l) and 22-l laboratory bioreactors (stirred tank and airlift), where the enzyme production was sustainable. Furthermore, the fermentation time was reduced from 48 h in shake flasks to 32 h in bioreactors.

The extensive efforts to screen thermophilic fungi and bacteria, isolated from various environmental samples, have resulted in the selection of Thermomucor indicae-seudaticae, Geobacillus thermoleovorans NP33 and G. thermoleovorans NP54 for the production of glucoamylase, amylopullulanase and alpha-amylase, respectively. Submerged and solid-state fermentation processes were optimized for maximizing the secretion of glucoamylase by T. indicae-seudaticae. The production of amylopullulanase and alpha-amylase by NP33 and NP54 in submerged fermentation was also optimized. Glucoamylase was optimally active at pH 7.0 and 60 degrees C and was shown to saccharify soluble as well as raw starches. Amylopullulanase and alpha-amylase exhibited optima at pH 7.0 and 100 degrees C and saccharified starch efficiently. Differential inhibition and action on mixed substrates clearly suggested that there are two separate active sites for alpha-amylase and pullulanase activities of amylopullulanase. Both alpha-amylase and amylopullulanase are high maltose-forming and Ca(2+)-independent. These amylolytic enzymes have been shown to be useful in starch saccharification alone and in combination.

The purified glucoamylase of the thermophilic mold Thermomucor indicae-seudaticaehad a molecular mass of 42 kDa with a pI of 8.2. It is a glycoprotein with 9-10.5% carbohydrate content, which acted optimally at 60 degrees C and pH 7.0, with a t(1/2) of 12 h at 60 degrees C and 7 h at 80 degrees C. Its experimental activation energy was 43 KJ mol(-1) with temperature quotient (Q(10)) of 1.35, while the values predicted by response surface methodology (RSM) were 43 KJ mol(-1) and 1.28, respectively. The enzyme hydrolyzed soluble starch at 50 degrees C (K(m) 0.50 mg mL(-1) and V(max) 109 micromol mg(-1) protein min(-1)) and at 60 degrees C (K(m) 0.40 and V(max) 143 micromol mg(-1) protein min(-1)). The experimental K(m) and V(max) values are in agreement with the predicted values at 50 degrees C (K(m) 0.45 mg mL(-1) and V(max) 111.11 micromol mg(-1) protein min(-1)) and at 60 degrees C (K(m) 0.36 mg mL(-1)and V(max) 142.85 micromol mg(-1) protein min(-1)). An Arrhenius plot indicated thermal activation up to 60 degrees C, and thereafter, inactivation. The enzyme was strongly stimulated by Co(2+), Fe(2+), Ag(2+), and Ca(2+), slightly stimulated by Cu(2+) and Mg(2+), and inhibited by Hg(2+), Zn(2+), Ni(2+), and Mn(2+). Among additives, dextran and trehalose slightly enhanced the activity. Glucoamylase activity was inhibited by EDTA, beta-mercaptoethanol, dithiothreitol, and n-bromosuccinimide, and n-ethylmaleimide inhibited its activity completely. This suggested the involvement of tryptophan and cysteine in catalytic activity and the critical role of disulfide linkages in maintaining the conformation of the enzyme. The enzyme hydrolyzed around 82% of soluble starch and 65% of raw starch (K(m) 2.4 mg mL(-1), V(max) 50 micromol mg(-1) protein min(-1)), and it was remarkably insensitive to glucose, suggesting its applicability in starch saccharification.

Abstract: A gene encoding acidic, thermostable and raw starch hydrolysing α-amylase was cloned from an extreme thermophile Geobacillus thermoleovorans and expressed. The ORF of 1650bp encodes a 515 amino acid protein (Gt-amy) with a signal peptide of 34 amino acids at the N-terminus. Seven conserved sequences of GH-13 family have been found in its sequence. The specific enzyme activity of recombinant Gt-amy is 1723Umg−1 protein with a molecular mass of 59kDa. It is optimally active at pH 5.0 and 80°C with t 1/2 values of 283, 184 and 56min at 70, 80 and 90°C, respectively. The activation energy required for its temperature deactivation is 84.96kJmol−1. Ca2+ strongly inhibits Gt-amy at 10mM concentration, and inhibition kinetics with Ca2+ reveals that inhibition occurs as a result of binding to a lower affinity secondary Ca2+ binding site in the active centre in a mixed-type inhibition manner. The K m and k cat of the Gt-amy are 0.315mgmL−1 and 2.62×103 s−1, respectively. Gt-amy is Ca2+-independent at the concentration used in industrial starch saccharification, and hydrolyses raw corn and wheat starches efficiently, and thus, is applicable in starch saccharification at the industrial sub-gelatinization temperatures. [Copyright &y& Elsevier]

Bacillus acidicola TSAS1 produced a novel acid-stable, thermostable, Ca-independent and high maltose-forming α-amylase with optimum activity at pH 4.0 and 60°C, and T of 27 min at 90°C. The enzyme saccharified raw as well as soluble starches, and ameliorated bread quality when the dough was supplemented with the enzyme. [ABSTRACT FROM AUTHOR]