**2. Amylase**

**1. Introduction**

226 Enzyme Inhibitors and Activators

Soil contains, among many others, enzymes that are constantly interacting (regulating, being regulated by) with soil constituents, for example, minerals, rhizosphere and numerous nutrients. Enzymes, in turn, catalyse important biochemical reactions for rhizobacteria and

The available enzymes inside soil could originate from plants, animals or microbes (bacteria or fungi). The enzymes that are produced from these organism could exhibit activities intracellular of the source organism, at its cell membrane, interacting therefore with soil and its constituents, or extracellularly (so freely available). Therefore, vis-à-vis to plant nutrition, or bioavailability of the macro- or micro-nutrients, the (extra or sub) cellular localization has a key role. Typical major enzymes available in soil can be listed as dehydrogenases, hydrogenases, oxidases, chief among those being glucose, aldehyde, urate, catechol, p-diphenol, ascorbate oxidases, catalases, peroxidases, phenol o-hydroxylase, dextransucrase, levan sucrase, aminotransferase, rhodanese, carboxylesterase, arylesterase, lipase, phosphatase, nuclease, nucleotidase, phytase, arylsulphatase, amylase, cellulase, laminarinase, inulase, xylanase, dextranase, levanase, poly-galacturonase, glucosidase, galactosidase, galactosidase, invertase, proteinase, peptidase, asparaginase, glutaminase, amidase, urease, inorganic pyrophosphatase, polymetaphosphatase, adenosine triphosphatase, aspartate decarboxylase, glu-

An interesting strategy for improving the nutritional quality of the soil would be either inoculating microorganism to soil while giving attention to mineral or other compounds that affect enzyme activity in soil. Since, some elements or compounds could show both activation and inhibitory effect, such as Fe, Na, etc., metals, the regulation of their bioavailability is crucial. Measurement of soil enzyme activity is important to determine soil characteristics, for further studies, such as, improving soil composition for plant growth using enzymes. A simple example can be given for proteases. Soil, when supplemented with proteases, would degrade proteins, thereby, increasing the amount of available nitrogen, which in turn is expected to improve plant nutrition. Similarly, soil supplemented with urease would increase bioavailable nitrogen level, and as such, this enzyme can be seen as a 'knob' for nitrogen regulation in soil and indirectly in plants. Finally, the use of enzymes, typically from microorganisms as plant growth promoting rhizobacteria (PGPR), is important not only from an economical perspective (improved crop

yield), but also environmental point-of-view (reduced use of chemical fertilizers).

production and application conditions.

Enzymes are, at industrial scale, typically produced using either fungi or bacteria, either technology having advantages and disadvantages. While cultivation of bacteria is easier to handle (from both process and genetics perspective) and to scale up, fungi has typically larger portfolio of enzymes and the latter is more resilient to stress conditions, a characteristic of the

Vis-à-vis plant nutrition, enzymes have crucial roles, tightly coupled to soil remediation as soil contains impurities in the form of heavy metals as well as polymers, for example, starch and cellulose residues, polyphosphate rocks, urea from N-cycle, oils and fats from either plants or animals that cannot be readily used by plants, in particular for nutrition. Enzymes

plants, stabilize the soil by degrading wastes and mediate nutrient recycling [1].

tamate decarboxylase and aromatic amino acid decarboxylase [1].

Amylases are enzymes hydrolyzing glycosidic bonds of polysaccharides. Usually these are classified into three sub-classes as α-amylase (E.C. 3.2.1.1), β-amylase (E.C. 3.2.1.2) and γ-amylase (E.C. 3.2.1.3). α-Amylase is responsible in endo-hydrolysis of (1-4)-α-D-glucosidic linkages, while β-amylase is responsible in the hydrolysis of (1-4)-α-D-glucosidic linkages in polysaccharides to remove maltose units from non-reducing ends. γ-Amylase, in contrast, is responsible in the hydrolysis of terminal (1-4)-α-D-glucose residues from non-reducing ends of the chains for releasing of β-D-glucose.

All the three versions of this enzyme are produced by bacteria and fungi. α-amylase have been reported by *Acinetobacter* spp., *Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus megaterium, Bacillus subtilis* and some thermophilic actinomycetes organisms as well, for example, *Thermomonospora curvata* and *Thermomonospora vulgaris* [2, 3], while β-amylase have been reported to be produced by *Bacillus cereus, Bacillus circulans, B. megaterium* and *Paenibacillus polymyxa* [4, 5]. Lastly,for γ-amylase, in addition to the *Bacillus* species, halophylic *Halolactibacillus* sp. and thermophilic organisms, for example, *Thermoactinomyces vulgaris* have been reported to produce this enzyme [6–8].

Amylases are reported to be active in a broad range of pH 1–13 [9, 10], yet β- and γ-amylases have narrower ranges. The optimum working pH range is reported to be from 2 [2] to 10.5 [11] for α-amylase, the other two being in a narrower range. As for the temperature, again α-amylases are active in a broad range or temperature from 20 [12] to 145°C [13]. Lastly, molecular weights range between 10 [14] and 240 kDa [15].

Despite the broad range of pH and temperature where the amylases are active, there is fairly long list of inhibitors for the microbiologically produced amylases: Ag+ , Ba2+, Ca2+, Co2+, Cu2+, Fe2+, Hg2+, Mg2+, Mn2+, Ni2+, Sr2+, Zn2+ [14]; Cd2+, iodoacetate [16]; ethylenediaminetetraacetic acid (EDTA), K<sup>+</sup> [17]; Na+ , Triton X-100, Tween 20 [18]; phenylmethylsulfonyl fluoride (PMSF), 4-bromophenacyl bromide [19]; Bi(NO<sup>3</sup> )3 , N-ethylmaleimide and sodium deoxycholate [20] are reported to be inhibitors. Interestingly, sodium dodecylsulfate (SDS), urea and 2-mercaptoethanol are reported to be both activating [21, 22] and inhibiting [17, 18] compounds.

The production of enzymes is typically performed in submerged fermentation, less often via solid state fermentation, typically under mesophilic conditions, moderate pH and temperature (30–50°C, mostly in 37°C; pH range of 3–9, mostly at 7) in chemically defined ((NH<sup>4</sup> ) 2 HPO4 as N-source, KH2 PO<sup>4</sup> as K-source) or complex media (yeast extract as N and K source), lactose, maltose glucose or starch as C-source, using chiefly *Bacillus* species [23–27]. Additionally, agro-wastes are also used as substrates and inducers as coconut oil cake, wheat/ rice bran, spent brewing grain, cassava bagasse, jackfruit or tamarind seed powder, palm kernel, olive oil or mustard oil cake and rice husk [27].
