Production optimization using Plackett-Burman and Box-Behnken designs with partial characterization of amylase from marine actinomycetes

Amylase is an industrial enzyme that is used in the food and biofuel industries. We screened four actinomycetes strains for amylase biosynthesis. The Streptomyces rochei strain had a larger hydrolytic zone (24 mm) on starch agar plates, than the other isolates. Plackett-Burman’s experimental design was implemented to optimize the conditions for amylase production by the selected strains. Growth under optimized culture conditions led to 1.7, 9.8, 7.7, and 3.12 -fold increases for the isolates S. griseorubens, S. rochei, S. parvus, and Streptomyces sp., respectively, in the specic activity measurement in comparison with growth under primary conditions. When applying the Box-Behnken design on S. rochei using the most signicant parameters starch, K 2 HPO 4 , pH, and temperature, there was a 12.22-fold increase in the specic activity measurement: 7.37 U/mg. The optimal fermentation medium formula was kept at 30.6°C for seven days. The amylase from S. rochei was partially puried, and its molecular weight was determined using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight was found to be 45, 43, and 53 kDa. Amylase was particularly active at pH 6 and 65°C. The puried enzyme was most active at 65°C and a pH of 6, thermal stability of 70°C for 40 min and salt concentration of 1 M with a Km and Vmax of 6.58 mg/ml and 21.93 mg/ml/min, respectively. The amylase improved by adding Cu + 2, Zn + 2, and Fe + 2 (152.21%, 207.24%, and 111.89%). Increased production of amylase enzyme by Streptomyces rochei KR108310 attracts the production of industrially signicant products.

row No. 9. High (+1) and low (−1) values were evaluated for each component. Each experiment was replicated twice, and the mean of these values was used as the response. Temperature °C T 32 34 30 Both trials were conducted in triplicate. The main effect of each variable was estimated using the following equation: When Exi is the variable main effect, the amylase production radiuses for the tests were Mi+ and Mi-, where the independent variables were present in high and low concentrations, respectively, and N was used to measure the statistical t-values of the equal unpaired samples to determine the variable meaning by dividing by two using Microsoft Excel 2019.  Veri cation tests were conducted with double standards using the predicted optimized medium to validate the Plackett-Burman design statistical analysis results. The production of amylase enzyme was measured by dividing the activity into protein to determine the speci c activity.
Optimization of the culture conditions using the Box-Behnken design After assessing the relative importance of separate variables, the four most important variables were chosen to determine the optimum levels for amylase enzyme development. The Box-Behnken design (BBD) was used because it is a surface response methodology (Agrawal et al 2020 ; Box and Behnken 1960). This optimization method includes three key steps: coe cient estimation of a mathematical model, response prediction, and model adequacy veri cation. The four signi cant variables elucidated using the Plackett-Burman experimental design for the selected best amylase enzyme produced by the isolate of the Streptomyces rochei strain (HMM 13) were starch (X 1 ), K 2 HPO 4 (X 2 ), pH (X 3 ), and temperature (X 4 ). The moo, center and tall levels of each variable were assigned as −1, 0, and +1, individually. A framework was built for the 27 trials, alongside the normal values for the four variables. The experiments were performed in triplicate and the mean values were determined for speci c activities. The connection between the free factors and response functions was correlated using a second-order polynomial to actuate the optimal point. The condition for the four components was calculated using the formula below: Y=B 0 +B 1 X 1 +B 2 X 2 +B 3 X 3 +B 4 X 4 +B 12 X 1 X 2 +B 13 X 1 X 3 +B 14 X 1 X 4 +B 23 X 2 X 3 +B 24 X 2 X 4 +B 34 X 3 X 4 +B 11 X 11 +B 22 X 22 +B 33 X 33 +B 44 +X 44 Where Y is the anticipated response, β 0 is the show constant, X 1 , X 2 , X 3 , and X 4 are the free factors, β 1 , β 2 , β 3 , and β 4 are the direct coe cients, β 12 , β 13 , β 23 , and β 24 are the cross-product coe cients, and β 11 , β 22 , β 33 , and β 44 are the quadratic coe cients. Microsoft Excel 2019 was used to examination of the experimental data collected using regression analysis. 2 x3 x4 x1*x2 x1*x3 x1*x4 x2*x3 x2*x4 x3*x4 x1*x1 x2*x2 x3*x3 x4*x4

Statistical analysis
Numerous straight relaps were made utilizing Microsoft Excel predictions to determine the signi cance of the amylase protein (presented in speci c activity) in terms of t-value, P-value, and con dence level. The level of signi cance (Pvalue) was resolved using the student's t-test. Every single impact t-test assesses the probability that nding the observed effect was pure chance. If this is highly unlikely, then the affect is thought to be caused by the variable when it is below the accepted level, such as 5%. The con dence level re ects a percentage of the P-value. The activities were assessed using the Microsoft Excel solver add-in program. Each response was simultaneously visualized in threedimensional graphics created using STATISTICA 10.0 software for the four largest independent factors.

Model veri cation
Experimentally, optimal conditions were veri ed from the optimization experiments. The predictions were examined and compared to the basic conditions, near-optimal conditions, and conditions different to the optimum levels of the independent variables.
Puri cation of amylase enzyme Amylase was puri ed from Streptomyces rochei HMM 13 using various steps, including precipitation of ammonium sulfate, dialysis, and Sephadex G-50 (Mohammed et al 2017

Characterization of amylase enzyme
The pH (3-10) of puri ed Streptomyces rochei HMM 13 amylases were tested under standard assay conditions. The temperature effect was established on decontaminated amylase by hatching the improved amylase at a temperature extending from 30°C to 80°C utilizing phosphate buffer for 4h and measuring activity as portrayed already (Nithya et al 2017). Amylase thermal stability was determined when an enzyme was incubated at 55°C, 65°C, and 70°C for 4h.

Results
The marine actinomycetes strains were initially qualitatively assessed for extracellular enzyme development. Initial screening showed that amylases could be produced from isolated marine actinomycetes on culture plates. Among the isolates of actinomycetes, the Streptomyces rochei strain HMM 13 has been very active on starch agar (24 mm) when compared to other isolates (Table 4 & Fig. 1). The Streptomyces rochei HMM 13 strain selected was Gram-positive, mycelic coenocytic, and branched. It was catalase and protease positive, reduced nitrate, hydrolyzed starch, Voges Proskauer, indole, and H 2 S production negative.
Following ooding with Gram's Iodine on starch agar plates, the Streptomyces rochei HMM 13 strain showed a 24 mm zone. Based on 16S rRNA analysis, this organism was identi ed as Streptomyces rochei; the sequence was accessed by KR108310 Optimization of amylase production from actinomycetes isolates The Plackett-Burman design has been used to assess the critical impact of utilizing starch casein agar medium components for the generation of amylase enzyme using Streptomyces griseorubens strain (MMH 9), Streptomyces rochei strain (HMM 13), Streptomyces Parvus strain (8), and Streptomyces sp. strain (M12). The most signi cant effect of the four isolates has been found for Streptomyces rochei HMM 13. The t-test showed that the most critical independent variable in uencing amylase enzyme development was starch, k 2 HPO 4 , pH, and temperature. Consequently, speci c activities were detected (Table 5). The primary effects of the examined factors on the speci c activity results were estimated and are represented in (Fig 2).  Table 5 bolsters this perception. This approach has shown that the implemented design is correct. A veri cation experiment was used to evaluate the basic versus optimized medium.

Veri cation experiment
Veri cation was achieved by comparing the expected ideal levels of autonomous factors and basic conditions.
Cultivation of S. griseorubens MMH9, S. rochei HMM13, S. parvus (8), and Streptomyces sp. M12 in the veri ed medium adjusted to a pH of six for seven days resulted in a 1.7, 9.8, 7.7, and 3.12 -fold increment in the speci c activity measurement compared to the basal conditions (Table 6 and Fig. 3). Among the BBD design, The signi cant independent variables (Starch X 1 , K 2 HPO 4 X 2 , pH X 3 , and Temperature X 4 ) suggested by the Plackett-Burman design were used to investigate the optimum response region for amylase production by Streptomyces rochei HMM 13 at three levels (-, 0, +) in the BBD (Table 7).    in Starch (X 1 ), K 2 HPO 4 (X 2 ), pH (X 3 ), and Temperature (X 4 ) were effective within the examined concentration ranges and under the present experimental conditions. These gures suggest that increasing the starch concentration to (19 g/l) with a high level of temperature will promote the production of amylase enzyme. However, a higher level of enzyme production was accomplished with diminished K 2 HPO 4 concentration.

Veri cation experiment
The optimum conditions obtained from the optimization experiment was experimentally veri ed and compared with measured data from the model. The positive relationship between anticipated and test values beneath ideal conditions demonstrates the precision and legitimacy of the model. Thus, we predicted that to produce the highest production of amylase enzyme by isolate Streptomyces rochei HMM 13, the medium formula should be kept at 30.6°C for seven days and formulated as follows (g/L): starch 19 g, KNO 3 3 g, casein 0.5 g, MgSO 4 0.75 g, FeSO 4 0.001 g, K 2 HPO 4 0.2 g, CaCO 3 0.1 g, agar 18 g, and 50/50 DW. An investigative study was conducted to compare the anticipated optimal levels of autonomous factors and fundamental conditions. Development of Streptomyces rochei HMM13 within the con rmed medium balanced to pH 8 for seven days resulted in a 12.22-fold increment within the speci c activity estimation compared to the basal conditions (Table 7). Amylase enzyme puri cation and characterization Amylase was purified from Streptomyces rochei HMM 13 using ammonium sulfate, dialysis, and Sephadex G-50. The crude amylase showed 12.22-fold purification (Fig 5.), and the specific activity of amylase was 7.37 U/mg with activity of 64.895 U/ml. At 90%, ammonium sulfate was ideal for the fractionation of amylase from S. rochei HMM 13, and the others showed no important amylase activity. The precipitated sample was dialyzed against buffer and water overnight, and the activity of the enzyme was inspected. After purification utilizing Sephadex G-50 chromatography, the specific activity of the enzyme was 10.5 U/mg with an activity of 103.73 U/ml. Dynamic amylase fractions were stacked onto the SDS-PAGE, and the atomic mass of decontaminated amylase was 45, 43, and 53 kDa at three peaks within the gel ltration chromatography (Fig. 6).
The digestion rate of starch with a puri ed enzyme was evaluated at different pH-values to assess the impact of varying pH on amylase activity. Enzyme characterization studies revealed stability in the wide pH range (3-10) of puri ed amylase with maximum strength and activity at a pH of 6 ( Fig 7A).
Amylase was found to be profoundly dynamic at 65 °C (Fig. 7B). Decontaminated S. rochei amylase with optimum stability at 70°C was astoundingly steady inside the temperature extend 55-70°C S. rochei amylase was thermostable up to 70°C and held 52.8% initial activity after 100 min incubation at 70°C. The enzyme maintained its unique action for 40 min at 55°C, 65°C, and 70 °C (Fig 7C). It was uncovered that the amylase from S. rochei is thermostable. Moreover, the puri ed enzyme was characterized by the kinetical characteristics that illustrate a consistent Michaelis-Menten (Km) and Vmax of 6.58 mg/ml and 21.93 mg/ml/min, respectively (Fig. 7D). The purified enzyme seems to withstand saltiness up to 0.8 M NaCl, with the most elevated movement at 22.71 U/ml (Fig. 7E). The impact of different metal particles at concentrations of 0.05 M on puri ed S. rochei HMM 13 amylase activity is shown in Fig. 7F. The addition of Cu+2, Zn+2, and Fe+2 (152.21%, 207.24%, and 111.89%) upgraded enzyme activity. However, Mn+2 and Mg+2 had a limited impact on enzyme activity (91.47% and 90.96%).

Discussion
The diversity of marine actinomycetes is important in many areas of science and medicine (Magarvey et al 2004). They are a rich source of bioactive, chemically diverse substances (Bernan et al 1997). Gopinath has recently been using agar plates to determine the amylase activity of Penicillium sp. and Aspergillus versicolor (Gopinath et  , which is different from our ndings. Amylase was found to be profoundly dynamic at 65°C (Fig. 7B). Comparable outcomes were found in another study (Al-Dhabi et al 2020a), in which most extreme amylase activity was notable at 60°C.It was uncovered that the amylase from S. rochei is thermostable. Numerous past studies have detailed that amylase from Streptomyces sp. MSC702 and Streptomyces avermitilis 5981 were steady at 60°C and 50°C, respectively ( . The puri ed enzyme seems to withstand saltiness up to 0.8 M NaCl, with the most elevated movement at 22.71 U/ml (Fig. 7E). Numerous past studies have described that a-amylase from Streptomyces sp. SNAJSM6 and Nocardiopsis dassonvillei KaS11 were steady at 3% and 9% concentrations of NaCl, respectively (Anand ety al 2019 and Rathore et al 2020). Finally, Cu + 2, Zn + 2, and Fe + 2 expanded amylase activity as reported by (Du et al 2018) and (Asgher et al 2007). The results of our study indicate the possible testing of the factors that have a positive impact on enzyme production using the Plackett-Burman and Box-Behnken designs. The amylase from Streptomyces rochei KR108310 strain HMM13 is steady at a wide range of temperatures and an acidic pH. Due to their importance, further study should concentrate on improving methods for using the enzyme in industrial processes. The ndings obtained were consistent with other worldwide studies that suggest that the Streptomyces genus from marine sediments is suitable for the production of industrial enzymes.