Food and feed is possibly the area where processing anchored in biological agents has the deepest roots. Despite this, process improvement or design and implementation of novel approaches has been consistently performed, and more so in recent years, where significant advances in enzyme engineering and biocatalyst design have fastened the pace of such developments. This paper aims to provide an updated and succinct overview on the applications of enzymes in the food sector, and of progresses made, namely, within the scope of tapping for more efficient biocatalysts, through screening, structural modification, and immobilization of enzymes. Targeted improvements aim at enzymes with enhanced thermal and operational stability, improved specific activity, modification of pH-activity profiles, and increased product specificity, among others. This has been mostly achieved through protein engineering and enzyme immobilization, along with improvements in screening. The latter has been considerably improved due to the implementation of high-throughput techniques, and due to developments in protein expression and microbial cell culture. Expanding screening to relatively unexplored environments (marine, temperature extreme environments) has also contributed to the identification and development of more efficient biocatalysts. Technological aspects are considered, but economic aspects are also briefly addressed.

/pdf/enzymes-in-food-processing-a-condensed-overview-on-46ssg3fta5.pdf

Enzymes in Food Processing: A Condensed Overview on Strategies for Better Biocatalysts

Glucoamylase: structure/function relationships, and protein engineering.

http://imb.savba.sk/~janecek/papers/pdf/emt_00.pdf

Thermophilic archaeal amylolytic enzymes

One stop mycology

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.

Microbial glucoamylases: characteristics and applications

To decrease irreversible thermoinactivation of Aspergillus awamori glucoamylase, five Gly residues causing helix flexibility were replaced with Ala residues. Mutation of Gly57 did not affect thermostability. Mutation of Gly137 doubled it at pHs 3.5 and 4.5 but barely changed it at pH 5.5. The Gly139-->Ala mutation did not change thermostability at pH 3.5, improved it at pH 4.5 and worsened it at pH 5.5. The Gly 137/Gly139-->Ala/Ala mutation gave 1.5-2-fold increased thermostabilities at pHs 3.5-5.5. Mutations of Gly251 and Gly383 decreased it at all pHs. Gly137-->Ala and Gly137/Gly139-->Ala/Ala glucoamylases are the most stable yet produced by mutation. Guanidine treatment at pH 4.5 decreased the reversible stabilities of Gly137-->Ala, Gly139-->Ala and Gly137/Gly139-->Ala/Ala glucoamylases at infinite dilution while not changing those of Gly251-->Ala and Gly383-->Ala glucoamylases, which is, in general, opposite to what occurred with thermoinactivation. Mutation of Gly57 greatly improved the extracellular glucoamylase production by yeast, that of Gly137 barely affected it and those of Gly139 and of both Gly137 and Gly139 strongly impeded it. These observations suggest that alpha-helix rigidity can affect reversible and irreversible glucoamylase stability differently, that the effects of multiple mutations within one alpha-helix to improve stability are not always additive and that even single mutations can strongly affect extracellular enzyme production.

/pdf/effect-of-replacing-helical-glycine-residues-with-alanines-id58v0p38q.pdf

Effect of replacing helical glycine residues with alanines on reversible and irreversible stability and production of Aspergillus awamori glucoamylase

Two additional disulfide bonds and three combined thermostabilizing mutations were introduced into Aspergillus awamori glucoamylase to test their effects on enzyme thermostability and catalytic properties. The single cysteine mutations N20C, A27C, T72C and A471C were made and combined to produce the double cysteine mutations N20C/ A27C and T72C/A471C. The double cysteine mutants were expressed efficiently in Saccharomyces cerevisiae, and disulfide bonds formed spontaneously after fermentation. At 50 degrees C, the single mutants N20C and A27C had decreased specific activity, whereas the specific activity of the double mutants N20C/A27C and T72C/A471C were similar to wild-type glucoamylase. The N20C/A27C mutation increased thermostability, with an increased activation free energy of 1.5 kJ/mol at 65 degrees C, while the single mutation A27C only slightly increased thermostability and N20C decreased it. The other disulfide bond-forming mutation T72C/A471C did not affect thermostability at pH 4.5. The N20C/A27C mutation was separately combined with two other thermostabilizing mutations, G137A and S436P. Thermostabilities of all of the combined mutated glucoamylases were additive. N20C/A27C/G137A glucoamylase had higher specific activity than wild-type glucoamylase from 45 to 67.5 degrees C. The disulfide bond between positions 20 and 27 connects the C-terminus of helix 1 and the following beta-turn, suggesting that this region is important for glucoamylase thermostability.

Effect on thermostability and catalytic activity of introducing disulfide bonds into Aspergillus awamori glucoamylase.

In Aspergillus awamori glucoamylase, Ala27, Ala393, Ala435, Ser436 and Ser460 were replaced with proline residues, in order to stabilize the enzyme by forming more rigid peptide backbones. Specific activities were unaffected except for a decrease in Ser460-->Pro glucoamylase. Thermostability was increased in Ser436-->Pro glucoamylase, unchanged in Ala435-->Pro glucoamylase and decreased in Ala27-->Pro, Ala393-->Pro glucoamylases. As measured by circular dichroism, mutant glucoamylases Ala435-->Pro and Ser436-->Pro resisted unfolding caused by guanidine hydrochloride at pH 4.5 and 25 degrees C better than wild-type glucoamylase, whereas mutant glucoamylases Ala27-->Pro, Ala393-->Pro and Ser460-->Pro were more susceptible to unfolding than wild-type glucoamylase, reaching a level of 50% unfolded enzyme at guanidine hydrochloride concentrations 0.50-0.75 M lower than that of the wild-type enzyme. Mutations Ala435-->Pro and Ser436-->Pro are located in a non-regular structure, which is assumed to be stabilized by these mutations. The Ala27-->Pro residue is partially buried, which may result in unfavorable steric contact and/or regional strains; mutation Ala393-->Pro results in loss of a hydrogen bond, since the N of the proline residue does not have an extra hydrogen to act as donor; and mutation Ser480-->Pro eliminates an O-glycosylation site, which could explain how these mutations destabilized glucoamylase.

Effect of introducing proline residues on the stability of Aspergillus awamori.

A fungal glucoamylase including a mutation pair Asn20Cys coupled with Ala27Cys forming a disulfide bond between the two members of the pair. The mutation provides increased thermal stability and reduced isomaltose formation to the enzyme. A fungal glucoamylase including a 311-314Loop mutation wherein reduced isomaltose formation is provided by the mutation is also provided. A fungal glucoamylase including a mutation Ser411Ala wherein increased pH optimum and reduced isomaltose formation is provided by the mutation is also provided. Combinations of the mutations in engineered glucoamylases are also provided as are combinations with other glucoamylase mutations that provide increased thermal stability, increased pH optimum and reduced isomaltose formation for cumulative improvements in the engineered glucoamylases. Also provided is a fungal glucoamylase including a mutation of Ser30Pro coupled with at least two stabilizing mutations forming a disulfide bond between the two stabilizing members. A fungal glucoamylase including a Ser30Pro/Gly137Ala/311-314 Loop is provided. A fungal glucoamylase including a mutation Ser30Pro/Glu137Ala/Ser411Ala is also provided.

Protein engineering of glucoamylase to increase pH optimum, substrate specificity and thermostability

Cette invention concerne une glucoamylase fongique qui comprend une paire de mutation Asn20Cys couple a Ala27Cys, et dans laquelle une liaison disulfure est formee entre les deux elements de la paire. Cette mutation permet d'accroitre la stabilite thermique et de reduire la formation d'isomaltose en ce qui concerne l'enzyme. Cette invention concerne egalement une glucoamylase fongique contenant une mutation 311-314Loop, et dans laquelle la formation d'isomaltose est egalement reduite grâce a la mutation. Cette invention concerne en outre une glucoamylase fongique contenant un Ser411Ala de mutation, et dans laquelle il est possible d'obtenir un pH optimal et de reduire la formation d'isomaltose grâce a la mutation. Cette invention concerne enfin des combinaisons des mutations dans les glucoamylases obtenues par genie genetique, ainsi que des combinaisons avec d'autres mutations de glucoamylase qui permettent d'accroitre la stabilite thermique, d'obtenir un pH optimal, et de reduire la formation d'isomaltose en vue d'ameliorations cumulees dans les glucoamylases ainsi obtenues.

Fabrication par genie genetique et a l'aide de proteines d'une glucoamylase permettant d'obtenir un ph optimal et d'accroitre la specificite d'un substrat ainsi que la stabilite thermique

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