Xylan, the second most prevalent polysaccharide in nature after cellulose, is hydrolyzed by xylanase. The primary enzymes of the microbial hemicellulolytic system or xylanases, randomly cut the xylan's-1,4 backbone. Most xylanases are grouped into glycoside hydrolase family (GH) 10 and 11 based on amino acid sequence homologies and hydrophobic cluster analysis. The (GH) families 5, 7, 8, 16, 26, 43, 52 and 62 are home to additional minorities [1,2].

In nature, β-galactosidase (EC 3.2.1.23), also known as lactase or -D-galactosidase galactohydrase, is mostly disregarded. Reaction mechanism [3,4] and three-dimensional structure [5] are two topics on which research has been done. E. coli produces the most completely studied β-galactosidase (pH optimum, pH 7), whereas yeasts and various Aspergillus species produce the most significant industrial β-galactosidase (pH optimum, between pH 4 and 5). In food technology, these fungal β-galactosidase are used to hydrolyze the lactose molecule's-(1,4) linkage to glucose. According to Gonzalez and Suarez [6], there are roughly 5.75 million metric tons of lactose produced annually worldwide.

Micoorganisim

The xylanase and β-galactosidase enzymes producing bacterial Bacillus sp. Strain (RL1) was isolated from soil.

Identification of the Isolates

The standard protocols [7] was used and identification was carried out according to Burges’s Manual (7th Ed.).

Secondary Screening

The method used by Teather and Wood [8] is used which indicates the formation of a clear zone from hydrolysis to decomposition of cellulose.

Xylanase and β-Galactosidase Enzymes Assay

As detailed by Bailey et al. [9], xylanase and β-galactosidase activity were assessed as the substrate and the amount of reducing sugars released was calculated using the dinitrosalicylic acid method [10].

pH Stability

The xylanase and β-galactosidase enzymes were pre-incubated at pH values ranging from pH 4 to 10 at 40°C for 30 min. to determine pH stability. Under typical enzyme assay conditions, the residual activity was measured.

Temperature Stability

The xylanase and β-galactosidase enzymes were pre-incubated at different temperatures between 30 and 90°C for 30 min at an ideal pH and the remaining activity was assessed under standard enzyme assay conditions to determine the temperature stability.

Stability of pH for Xylanase and β-Galactosidase Enzymes

Findings of Figure 1 demonstrate that the xylanase enzyme maintained some of its caustic activity between pH 6 and 8, while the enzyme lost more than 10-15% of its enzymatic activity at the acidic pH that ranged from 4-5, while the remaining enzymatic activity was 89.43 at a pH of 10.

Figure 1: Optimal pH Curve for the Stability of Xylanase Enzyme Produced from Bacillus sp. Strain RL1

The results shown in Figure 2 show that the β-galactosidase enzyme showed stability between pH 5-6 and retained an amount of its caustic activity, while the enzyme lost more than 15% of its enzymatic activity at the acidic pH that ranged from 4, while the remaining enzymatic activity was 64.13 at a pH of 10.

Figure 2: Optimal pH Curve for the Stability of β-Galactosidase Enzyme Produced From Bacillus sp. Strain RL1

The influence of the medium's acidity on the enzyme's composition and the ionization of the groups in the active site account for the enzyme's decreased activity at non-extreme pH levels, while the decrease in the stability of the enzyme at the extreme acidic and basic values occurs as a result of the irreversible denaturation of the enzyme molecule due to the large change that occurs in the charges of the ionizable side chains Which leads to the destruction of the triple structure of the enzyme molecule to create a more random structure instead and change the effective site and thus the effectiveness [11].

Seo etal. [12] when studying the stability of the xylanase enzyme produced from Bacilluslicheniformis JK7, ranged from 4-6, while Gowdhaman et al. [13], stated that the best stability of the xylanase enzyme produced from Bacillusaerophilus KG12 bacteria was in the pH range from 6-8. While Sarika et al. [14], stated that the best Stability in a range from pH 6-10, while the xylanase enzyme produced from Paenibacillus macquariensis gave stability in a range from pH 5.8-10 [15].

Crueger and Crueger [16], stated that the stability of the β-galactosidase enzyme produced from Aspergillus niger, Aspergillus oryzae, pH ranged (2.5-8) and (2.5-7), respectively. pH levels between 6 and 9 were used by Miyazaki [17] to examine the stability of the β-galactosidase enzyme generated by Bacillus macerals. According to Jensen and Olsen (1992), the pH range between 6 and 9 provided the highest stability for the β-galactosidase enzyme generated by Thermocycles lanuginose.

Figure 3: The Impact of Temperature on the Xylanase Enzyme Generated by Bacillus sp. RL1 During Incubation

Stability of temperature for xylanase and β-galactosidase enzymes

Findings of Figure 3 from incubating the xylanase enzyme at temperatures ranging from 30 to 90 C revealed that the enzyme was most effective at temperatures between 30 and 60°C and that as the temperature rose, the activity reduced and the xylanase enzyme did not lose its effectiveness and retained more than 70% of them at a temperature of 90°C, the remaining enzymatic activity reached 70.79%.

The results of incubating the β-galactosidase enzyme at temperatures ranging from 30-90°C, shown in Figure 4, showed that the β-galactosidase enzyme retained its full effectiveness at temperatures limited to between 30-50°C, after which the activity decreased with the increase in temperature and the β-galactosidase enzyme did not lose its effectiveness and retained more than 60% of them at a temperature of 90°C, the remaining enzymatic activity reached 63.55%.

Figure 4: The effect of temperature on the incubation stability on the β-galactosidase enzyme produced by Bacillus sp. RL1

The reason for the decrease in the activity of the enzymes at high temperatures is due to the denaturation of the enzyme, as the rapid change in the nature of the enzyme leads to the breaking of the weak hydrogen bonds in a way that leads to the enzyme losing its activity. The difference in the type of organism from which the enzyme is extracted and the different development and production conditions.

Pithadiya et al. [18] reported that the xylanase enzyme produced from Bacillus circulans gave stability in a range of temperatures ranging from 40-60°C and Kumar et al. [19] reported that the temperature of the stability of the xylanase enzyme was in the range of 60-80°C. Gowdhaman et al. [13] found that the best stability of the xylanase enzyme produced from Bacillus aerophile. While Guo et al. [20] reported that the stability of the xylsnase enzyme produced from Bacillus subtilis was in a range of temperatures ranging from 55-65°C, the product from basil spanned from 40 to 60°C.

According to Lind et al. [21], a Thermoanaerobacter sp.'s intracellular and remarkably thermostable β-galactosidse is. At 75°C. While Jensen and Olsen [22] reported that Thermomyces lanuginosus generated β-galactosidase with around 58% of its enzymatic activity at a temperature of 50°C.

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