Dextranase is one of the hydrolytic enzymes E.C. (3.2.1.11) α1-6 D-glucan-6-glucanohydrolase with occasional α-1,2-, α-1,3-, or α-1,4- branched chains. It hydrolyzes dextran into glucose, isomaltose, maltotriose, and polysaccharides [1,2]. The discovery of dextranase, an enzyme capable of degrading dextran, emerged from pioneering research into microbial enzymes that hydrolyze polysaccharides. During the 1940s and 1950s, scientists investigating the metabolic capabilities of microorganisms identified bacteria and fungi as prolific producers of extracellular enzymes capable of breaking down complex carbohydrates. Among these discoveries, species of Bacillus, Penicillium, and Aspergillus were found to produce dextranase as part of their metabolic machinery when using dextran as a carbon source [3,4]. Microbial enzymes are essential in industrial applications. As a naturally occurring biocatalyst, dextranase offers a sustainable and efficient alternative to chemical additives. Biotechnology and enzyme engineering advances have further enhanced dextranase's production, stability, and activity. These microorganisms produce dextran as an extracellular polysaccharide while processing sugar-based products, such as sugarcane and sugar beet, enabling widespread adoption in food manufacturing [5,6]. The discovery of dextranase emerged from the need to address challenges posed by dextran contamination in industrial processes, particularly in the sugar and food manufacturing sectors. The enzyme’s ability to break down dextran into smaller oligosaccharides or glucose units has since made it an invaluable tool in various applications, from improving food processing efficiency to producing functional food ingredients and food processing applications, such as improving the texture and stability of syrups, confectioneries, and baked goods [7,8,4]. The presence of dextran in food processing systems can cause significant problems, including increased viscosity, inhibition of sugar crystallization, and clogging of filtration systems. These issues are particularly problematic in the sugar industry, where dextran contamination can lead to reduced yields, higher processing costs, and compromised product quality. Dextranase effectively solves these challenges by selectively hydrolyzing dextran into smaller, more manageable oligosaccharides or glucose units. This enzymatic action mitigates the adverse effects of dextran and improves the overall efficiency of food processing operations. For instance, in sugar refining, dextranase ensures optimal crystallization and enhances sucrose's recovery, thereby improving yield and quality [9,10]. The enzyme’s ability to modify food products' viscosity and rheological properties, such as molasses processing, made it a valuable tool for enhancing product quality and consumer appeal. Additionally, the discovery that dextranase could produce dextran-derived oligosaccharides with prebiotic properties opened new avenues for its use in functional foods. These oligosaccharides, which serve as substrates for beneficial gut microbiota, have been shown to promote digestive health and align with the growing demand for health-promoting food ingredients. The discovery of dextranase emerged from the need to address challenges posed by dextran contamination in industrial processes, particularly in the sugar and food manufacturing sectors. The enzyme’s ability to break down dextran into smaller oligosaccharides or glucose units has since made it an invaluable tool in various applications, such as improving the texture and stability of syrups, confectioneries, and baked goods. For example, in producing syrups, confectioneries, and baked goods, dextranase can control viscosity and improve mouthfeel, ensuring a consistent and appealing consumer experience [11,12]. That's why the idea of this study has come about to produce dextranase from a local bacteria isolate and detect the optimal production to achieve the highest efficiency under ideal production conditions and then homogeneous purification of the enzyme to study the characterization of activity and stability regarding pH and temperature molecular weight for determining food applications that align with the enzyme's properties.

This study used a pure isolate of Bacillus amyloliquefaciens MMO registered in NCBI (OR701819.1) obtained from a previous study to produce the dextranase enzyme.

2.1. The Dextranase Production Medium

The submerged culture method was used to produce dextranase according to the method Zohra et al. (2013) included the following components/100 ml of distilled water: 2.0g Dextran, 2.0g yeast extract, 0.2g NaCl, and 2.0g Mg₂PO₄ at pH 7.0 and autoclaved; the medium production was inoculated with 1×10⁸ bacterial suspension cells, and then the conditions were incubated in a shaking at 150 rpm, 37°C for 48 hours. The enzyme was extracted by sedimenting the cells represented by the biomass using centrifugation at a speed of 6000 xg, 4°C for 20 min. Then, the filtrate was considered the crude extract of the enzyme [13] 

2.2. Enzyme Activity Assessment

The reducing sugars released by hydrolysis of the dextran were used to determine the enzyme assay [14,15].  The activity is defined as the amount of dextranase (U/milliliter) released in 1 micromole of glucose/minute under the conditions of the experiment.

2.3. Protein Assays

The protein concentration in the produced enzyme was measured using the Bradford method [16].

2.4. Determine The Optimal Production of Dextranase

To detect the optimum conditions for the production of dextranase, different components and conditions of submerged culture were used, including (carbon sources and concentration and nitrogen sources, pH, temperature, fermentation time, and Inoculum volume); the enzyme efficacy (activity and specific activity) in producing dextranase was assessed after each incubation period.

2.4.1. The Effect of Carbon Source and Concentration

Several types of agricultural and industrial wastes (after cleaning, drying, and ground into powder) were used as media carbon sources for enzyme production, including (wheat bran, rice bran, sunflower meal, soybean meal, wheat straw, and yellow corn meal) and the effect of different concentrations of the optimal carbon source for enzyme production was also studied by adding the selected carbon source to the submerged culture medium at different concentrations: 0.5, 1.0, 1.5, 0.2, 2.5, 3.0, and 3.5%.

2.4.2. The Effect of Nitrogen Sources and Concentrations 

Use multiple and different sources of nitrogen in the production medium to determine the source that gives the highest effectiveness, including (potassium nitrate, ammonium sulfate, ammonium chloride, meat extract, gelatin, yeast extract, peptone) and the effect of different concentrations of the optimal nitrogen source for enzyme production was also studied by adding the selected nitrogen source to the submerged culture medium at different concentrations: 0.5, 1.0, 1.5, 0.2, 2.5, 3.0, and 3.5%.

2.4.3. The Effect of The Initial pH 

The different pH included (5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0) adjustments of the production medium mentioned in paragraph (3.2.) after determining the optimal components and proportions of the carbon and nitrogen sources were prepared to determine the best initial pH to produce dextranase

2.4.4. The Effect of The Temperature of Incubation

The bacterial cell production medium was inoculated at 25, 30, 35, 40, 45, and 50°C for 48 hours. Incubation was established to determine the optimum temperature for the enzyme production.

2.4.5. The Effect of The Fermentation Time

Different periods were used to determine the optimal period for obtaining the highest effectiveness of the enzyme under study. These included: (24, 48, 72, 96, and 120 hours).

2.4.6.The Effect of The Inoculum Volume

The production medium has been modified, following the components of the medium and production conditions according to the results obtained from the previous experiments, where cells were inoculated with the volume of the inoculate (1, 2, 3, 4, 5, 6, 7) × 10⁸ cells/ml to determine the best volume of inoculum to obtain the best enzyme effectiveness.

2.5. The Purification Steps of Dextranase

After determining the optimal conditions for producing dextranase, the crude enzyme was subjected to purification to study its characterization of dextranase. All steps of enzyme purification were performed at 7°C. The purification included concentration by ammonium sulfate, ion exchange with DEAE-cellulose, and gel filtration with Sephadex G-100. After completing each purification step, estimate the volume of enzyme (ml), activity (U/mL), and protein concentration (mg/mL). All data were processed using Microsoft Office, Excel application.

2.5.1. Ammonium Sulfate Precipitation

Crud enzyme was treated with ammonium sulfate for a 20–80% saturation level. Then, it was further purified by removing impurities using dialysis bags with a molecular weight cutoff of 12–14 kDa against 

0.05M potassium phosphate buffer (pH 7.0) for 48 hours.

2.5.2. Ion Exchange DEAE-Cellulose

The DEAE-cellulose was prepared according to the method described.suspending 20 grams (Whatman Co., England) in 500 ml of distilled water and allowing it to settle. The upper liquid was poured off, and the exchanger was washed several times with distilled water until the overlying liquid became clear. It was filtered through a Buchner funnel under vacuum and suspended in a 0.25 M sodium hydroxide 0.25 M sodium chloride solution. The exchanger was washed several times with distilled water after filtration. Then washed with a 0.25M hydrochloric acid and distilled water solution, then equilibrated with a 0.1M sodium acetate buffer solution of pH 6.0 containing 0.3M sodium chloride and 0.02% sodium azide and filled to give a column of dimensions 2.5×17cm at a rate of 40 ml/h in 3 ml tubes with 0.05M potassium phosphate buffer (pH 7.0). Then, it was eluted by a NaCl gradient from 0.1 to 0.5M solution at pH 7.0. The absorbance of the recovered fractions was read at 280 nm. The fractions that showed enzyme activity were subjected to the next step.

2.5.3. Sephadex G-100

They Prepared according to the instructions of the company (Pharmacia Co., Sweden) by suspending 40 grams in a liter of distilled water and heating the suspension in a water bath at a temperature of 85–90°C for 3 hours with continuous stirring, then left at a temperature of 7°C until the next day. After removing the air, it was filled in a column to give dimensions of 60 x1.5cm at a rate of 45 ml/h in 3 ml tubes. They were collected in 5 ml tubes. Then, it was equilibrated with a 0.1 M sodium acetate buffer solution of pH 6.0 containing 0.3 M sodium chloride and 0.02% sodium azide. The absorbance of the recovered fractions was read at 280 nm. The active fractions were collected and dialyzed for concentration by sodium acetate solution buffer (0.01 M) at pH 7.0.

2.6. Study The Characterization of Dextranase

2.6.1. Molecular weight

Gel filtration Sephadex G-100 estimated the molecular weight (MW) with the same conditions mentioned for the purification enzyme. The blue dextran concentrically (3 mg/ml) was used to determine the void value (Vo) on (600 OD) and then the elution value (Ve) on (280 OD) using standard proteins, which included Trypsin (23 kDa), Ovalbumin (45 kDa), Bovine serum albumin (67 kDa), and Alkaline phosphatase (140 kDa), with a concentration of 5 mg/ml. The molecular weight value was calculated using the straight-line equation of the relationship between the logarithm of the MW of the standard proteins and the (Ve/Vo).

2.6.2. Determination of The Optimum pH for Dextranase Activity

To determine the optimal pH for the enzyme's activity, 1 ml of 1% dextran dissolved in potassium phosphate buffer at 0.05 M with pH values ranging from 4.0–8.0 was added to 1 ml of the purified enzyme, and then the enzyme activity was determined.

2.6.3. Determine The Optimum pH for Dextranase Stability

To determine the optimum pH for enzyme stability, a known volume of purified enzyme solution was mixed with an equal volume of solution buffer in pH values ranging from 2.0-8.0 and incubated at 35°C for 30min and then transferred to an ice bath and used to estimate relative of the enzyme activity.

2.6.4. The Effect of  Temperature on Dextranase Activity

The enzyme activity was estimated over a temperature range of (30-75)°C.

2.6.5. The Effect of  Temperature on Dextranase Stability

The pure enzyme was dissolved in acetate-phosphate buffer solution 0.05M with a pH of 5.5 and treated at a temperature ranging from (30-70)°C for 30min, then transferred to an ice bath, and the relative of the enzyme activity was estimated after the temperature treatments.

3. Results and Discussion

3.1. The Optimal Production of Dextranase

3.1.1. The Effect of Carbon Source and Concentration on Produce Dextranase

The results shown in Figure (3-1) showed that the best carbon source for enzyme production from the bacterial isolate Bacillus amyloliquefaciens is yellow cornmeal when incubated for 48 hours at 37°C based on the enzyme activity of 10.19 U/ml and the specific activity of 93.1 U/mg, followed by dextran with an enzyme activity of 9.28 U/ml and a specific activity of 84.8 U/mg. This is evidence of the possibility of using agricultural waste to produce dextranase, thus reducing production costs [17]. In a study in which the effect of five carbon sources on the production of dextranase enzyme from Bacillus subtilis NRC-B233 bacteria was studied, namely corn flour, wheat flour, wheat bran, commercial starch, and milled rice, the best carbon source was corn flour with an enzyme activity of 75.276 U/ml using a shaking incubator and an enzyme activity of 61.323 using a static incubator at a temperature of 30°C for 24 hours [18].

It was noted from the results shown in Figure (3-2) that the enzyme activity and specific activity at the concentration of 0.5% were low, reaching 1.28 U/ml and 11.7 U/mg. The enzyme and specific activity gradually increased until it reached the highest activity at the concentration of 2.0%, where the enzyme activity reached 10.19 U/ml and the specific activity 93.1 U/mg. It gradually decreased until it reached 1.11 U/ml and 10.1 U/mg at a concentration of 3.5%. Using low concentrations of carbon to produce, the enzyme gives better results regarding enzyme activity in quantity and quality [19].

3.1.2. The Effect of Nitrogen Sources and Concentrations 

The results shown in Figure (3-3) showed that peptone is the best at producing dextranase compared to other sources, as the enzyme activity reached 15.66 U/ml and the specific activity reached 143.1 U/mg. This result is consistent with what the researcher [20] reached, where he stated that the best nitrogen source for the production of dextranase enzyme from Bacillus licheniformis bacteria is peptone, as the researcher used yeast extract, tryptone, and peptone, each separately, where the effectiveness reached approximately 90 U/ml. While the results shown in Figure (3-4) show that the best concentration of yeast extract is 3.0% because it achieved the highest enzyme productivity in terms of enzyme activity and specific activity of 20.48 U/ml and 187.1 U/mg, respectively, this concentration was adopted as the best concentration of yeast extract. It was added to the production medium in subsequent experiments of this study. A nitrogen source in the enzyme production medium is essential in building several organic compounds in the cell, including proteins and amino acids. Also, the benefit of microorganisms from these sources is affected by the difference in the source and the degree of their readiness for the cell [21].

3.1.3. The Effect of The Initial pH

A decrease in the production of dextranase enzyme was observed at low pH numbers and an increase in production with increasing pH of the medium as a function of enzyme activity and specific activity, reaching a maximum of 20.48 U/ml and 187.1 U/mg, respectively, at pH 7.0, as shown in Figure (3-5). It was found that the pH affects the production of the enzyme through its effect on the properties of the culture medium, the solubility of the nutrients, their transfer and ionization, and the concentration of bicarbonates formed from the dissolution of carbon dioxide, which affects the buffer capacity of the medium, which affects the growth of the organism and the productivity of the enzyme [22].

3.1.4. The Effect of The Temperature of Incubation

Figure (3-6) shows the low enzyme production at 25°C through the enzyme activity and specific activity, which reached 8.21 U/ml and 75.0 U/mg, respectively. The activity increased with increasing temperature until it reached its maximum at 45°C, giving an enzyme activity of 27.72 U/ml and a specific activity of 253.2 U/mg. After that, the activity began to decrease gradually with the gradual increase in temperature. The optimum temperature for enzyme production from microorganisms varies depending on the organisms, as the optimum temperature may be correct when under specific environmental conditions. Therefore, all studies on enzyme production from microorganisms, whether by solid-state fermentation, have given this aspect special attention to determine the temperature for production to obtain the enzyme effectively and with high production [23].

3.1.5. The Effect of The Fermentation Time

The results showed that the highest enzyme production was after 48 hours of incubation in the shaking incubator; the enzyme activity was 27.72 U/ml, and the specific activity was 253.2 U/mg, as shown in Figure (3-7). The decrease in activity with increasing incubation period can be attributed to the fact that a significant portion of the enzyme produced by the bacteria may have been exposed to deterioration either due to changes that occurred in the production medium that caused a change in the culture conditions due to the products of metabolic processes with the continued growth of the bacteria or due to the secretion of other enzymes from the bacteria themselves into the production medium, including proteolytic enzymes such as proteases, which may have degraded part of the enzyme. The dextranase enzyme molecules present in the medium or the depletion of the medium components during the incubation period negatively affects production [24].

3.1.6. The Effect of The Inoculum Volume

Figure (3-8) shows a gradual increase in enzyme production with increasing inoculum volume in terms of enzyme activity and specific activity, reaching a maximum of 28.98 U/ml and 264.7 U/mg by adding an inoculum volume containing 2×10⁸ cells/ml. We also found that inoculating the medium with small inoculum volumes gave low enzyme productivity. This decrease in activity may be due to the small number of cells and thus not utilizing the components of the medium efficiently, and increasing the cells beyond the optimal limits leads to their competition for nutrients and an increase in toxic metabolic materials, which results in a decrease in enzyme production. This result that we obtained showed that a high inoculum volume does not give a high production of the enzyme, but rather may result from adding a high inoculum volume to the depletion of nutrients present in the medium early and the depletion of the quantity of Oxygenation due to rapid growth of the culture and cell clumping, which leads to decreased enzyme production [25]

Figure (3-1): The effect of carbonic source on dextranase production

Figure (3-2): The effect of concentration carbonic source on dextranase production

Figure (3-3): The effect of nitrogenic source on dextranase production

Figure (3-4): The effect of concentration nitrogenic source on dextranase production

Figure (3-5): The effect of pH on dextranase production

Figure (3-6): The effect of temperature on dextranase production

Figure (3-7): The effect of incubation time on dextranase production

Figure (3-8): The effect of Inoculum Volume ×10⁸ on dextranase production

3.1.7. Estimating the amount of increase resulting from estimating the optimal production

From the results table (3-1) obtained from determining the optimal conditions for production, an increase in productivity was estimated based on enzyme activity and specific activity effectiveness According to the following equation:

Increase productivity

Table (3-1): Development of dextranase Production by Bacillus amyloliquefaciens after the six stages of determining the optimum conditions

3.2. The Purification Steps of Dextranase

Table (3-2) shows that the number of purification folds and the enzyme recovery obtained after the enzyme precipitation step with ammonium sulfate between the 20–80% addition ratios were 1.68 and 45.50%, respectively. Then, the number of purifications folds to 3.43, and recovery decreases to 34.88% after the ion exchange chromatography step. It was noted that the parts not bound to the exchanger (due to washing) were utterly free of activity, which confirms the association of the enzyme with the negative ion exchanger and that the net charge carried on the enzyme under the conditions used is negative (Figure 3-9). The dextranase recovery from the ion exchanger was not achieved until the concentration of sodium chloride reached 0.5 M. As for the last step of enzyme purification by gel filtration (Figure 3-10), it was possible to obtain purification folds of 6.57, where the enzyme recovery reached 23.95%.

Figure (3-9): Ion exchange dextranase purification step

Figure (3-10): Gel filtration dextranase purification step

Studies have shown different results for purifying dextranase enzymes from microorganisms characterized by their high diversity.  Ren et al., 2018, were able to use multiple steps to purify the dextranase enzyme from Catenovulum sp. The method of ultrafiltration, alcohol precipitation, ammonium sulfate precipitation, and then ion exchange was used, which gave an enzyme activity of 1069.5 U/ml, a specific activity of 2309 U/mg, several purification times of 29.6 times, and an enzyme yield of 16.9%. The researcher used the following steps in purifying the enzyme: precipitation with ammonium sulfate, a PEG 6000 supernatant step, dialysis, ultrafiltration, and then an ion exchange step using a DEAE-Sepharose column, which gave an enzyme activity of 33700 units/ml, a specific activity of 78500 units/mg, and an enzyme yield of 19%. This purified enzyme was produced from Bacillus sp. [26]. It was able to purify the dextranase enzyme produced from Paecilomyces lilacinus using a DEAE-Cellulose column, which gave an enzyme activity of 108.76 units/ml, a specific activity of 42.32 units/mg, and a purification number of 6.37 times. [27] We purified the enzyme using ultrafiltration, which gave an enzyme activity and specific activity of 29866 units/ml and 388.6 units/mg, respectively, and a purification number of 1.4 times and an enzyme yield of 94.8%.

Table (3-2): Purification Steps of dextranase Produced from Bacillus amyloliquefaciens by submerged Fermentation culture.

* The results are represented after dialysis with 0.05M potassium phosphate at pH 7.0 for 48 h. 

3.3. Study The Characterization of Dextranase

3.3.1. Molecular weight

The molecular weight value was calculated using the straight-line equation of the relationship between the logarithm of the MW of the standard proteins and the (Ve/Vo), as shown in Figure 3-11. The results showed that the logarithm of the molecular weight of the enzyme under study reached 1.76; thus, its molecular weight reached approximately 58 kilodaltons.

The previous study found the molecular weights of dextranase ranging from 29 to 78 kDa across fungal, bacterial, and yeast sources. Most studies used gel filtration chromatography, SDS-PAGE, and HPLC for molecular weight determination [28].

Figure (3-11): Gel filtration method to determine the molecular weight of dextranase 

3.3.2. The Optimum pH for Dextranase Activity

The results shown in Figure 3-12 show that the optimum pH for enzyme activity was 7.0 and that the values decreased at pH higher than 8.0 and lower than 7.0. These results indicate that the enzyme has neutral properties. Studies have shown that the optimal pH for dextranase activity varies among microbial sources. For Talaromyces sp., the optimum pH was 7 [29], while Microbacterium sp. XD05 exhibited optimal activity at pH 7.5 [10]. Penicillium roquefortii TISTR 3511 dextranase showed maximum activity at pH 6 [30], and Arthrobacter oxydans G6-4B dextranase had an optimum pH of 7.5 [31] 

3.3.3. The Optimum pH for Dextranase Stability
As shown in Figure 3-13, it has stability towards the pH at neutral and acidic values (6.0-7.0), as the enzyme retains its effectiveness but loses 50% at pH 3.0. It is noted that the enzyme's effectiveness decreases when the pH rises above 7.5. The reason for the decrease in effectiveness, when the enzyme is incubated at extreme pH numbers is due to the effect of the charges in the active sites, and thus, the secondary and tertiary structure of the enzyme changes, which in turn leads to a change in the shape of the active site and a decrease in its effectiveness [32].

3.3.4. The Effect of  Temperature on Dextranase Activity
As shown in Figure 3-14, the enzyme activity increases with the increase in temperature up to 50°C until it reaches its maximum, then decreases with the increase in temperature above 50°C. It was observed that the rate of the enzymatic reaction increases with the increase in temperature and within a specific range due to the increase in the kinetic energy of the molecules and the increase in collisions between the enzyme molecules and the substrate. As a result of the increase in the kinetic energy of the molecules due to the increase in temperature, the rise in temperature above certain limits leads to the denaturation of the enzyme and the damage of its tertiary structure and then a decrease in its effectiveness [33,34]found that 50°C is the optimum temperature for the dextranase activity produced by Pochonia chlamydosporia VC4.

3.3.5. The Effect of  Temperature on Dextranase Stability

This experiment showed that the enzyme maintained full activity when incubated at 30-45 °C for 30 minutes. Then, the enzyme activity began to decrease with the increase in temperature. The enzyme lost a high percentage of its activity, as shown in Figure 3-15. The decrease in enzyme activity at high temperatures is attributed to the change in the natural state of the enzyme and its denaturation, as the rapid change in the enzyme causes the destruction of weak hydrogen bonds, which leads to the complete loss of enzyme activity [35].

Figure (3-12): The effect of pH on dextranase activity

Figure (3-13): The effect of pH on dextranase stability

Figure (3-14): The effect of temperature on dextranase activity

Figure (3-15): The effect of temperature on dextranase stability

The conclusions of the present study determined the optimal production from components and conditions to increase dextranase from Bacillus amyloliquefaciens to enhance its activity, which reached 184%. As for purification, although the enzyme yield may seem low, it is acceptable, especially if the goal is to obtain a highly purified enzyme. Additionally, from the dextranase characteristics, the molecular weight is 58 kDa, which means that the enzyme is medium-sized and consists of about 527 amino acids. Additionally, activity increases with temperature increase. 

The authors declare that they have no conflict of interest

No funding sources

The study was approved by the University of Kirkuk/College of Agriculture.

1. Lambré et al.; "Safety Evaluation of the Food Enzyme Dextranase from the Collariella Gracilis Strain AE-DX" 20.5 (2022) Pp e07279, doi: https://doi.org/10.2903/j.efsa.2022.7279.

2. Hu et al.; "Research Progress on Structure and Catalytic Mechanism of Dextranase" 4.1 (2023) Pp e60, doi: https://doi.org/10.1002/efd2.60.

3. Khalikova et al.; "Microbial Dextran-Hydrolyzing Enzymes: Fundamentals and Applications" 69.2 (2005) Pp 306-325, doi: https://doi.org/10.1128/MMBR.69.2.306-325.2005.

4. Barzkar et al.; "Marine Bacterial Dextranases: Fundamentals and Applications" 27.17 (2022) Pp 5533, doi: https://doi.org/10.3390/molecules27175533.

5. Okpara; "Microbial Enzymes and Their Applications in Food Industry: A Mini-Review" 10.1 (2022) Pp 23-47, doi: https://doi.org/10.4236/aer.2022.101003.

6. Kumar et al.; "Microbial Enzymes and Major Applications in the Food Industry: A Concise Review" 6.1 (2024) Pp 85, doi: https://doi.org/10.1007/s43014-024-00185-7.

7. Jiménez; "Dextranase in Sugar Industry: A Review" 11 (2009) Pp 124-134, doi: https://doi.org/10.1007/s12355-009-0021-7.

8. Ahmed et al.; "Pattern of Contraceptive Use Among Women Attending Family Planning Clinic in Kirkuk Governmental Hospitals, Kirkuk-Iraq" 14.3 (2019) Pp 68-78.

9. Bukhari et al.; "Investigations of the Influence of Dextran on Sugar Cane Quality and Sugar Cane Processing in Kenana Sugar Factory" 7.4 (2015) Pp 381-392.

10. Boualis et al.; "Dextranase Production Using Marine Microbacterium sp. XD05 and Its Application" 21.10 (2023) Pp 528, doi: https://doi.org/10.3390/md21100528.

11. Juvonen et al.; "The Impact of Fermentation with Exopolysaccharide Producing Lactic Acid Bacteria on Rheological, Chemical and Sensory Properties of Pureed Carrots (Daucus carota L.)" 207 (2015) Pp 109-118, doi: https://doi.org/10.1016/j.ijfoodmicro.2015.04.027. Here is your updated reference list in the requested format:

12. Khatun et al.; "Transformation of Sugarcane Molasses into Fructooligosaccharides with Enhanced Prebiotic Activity Using Whole-Cell Biocatalysts from Aureobasidium pullulans FRR 5284 and an Invertase-Deficient Saccharomyces cerevisiae 1403-7A" 8 (2021) Pp 1-12, doi: https://doi.org/10.1186/s40643-021-00373-5.

13. Omar & Awda; "Cloning and Expression of Levansucrase (sacB) Gene from Bacillus licheniformis MJ8 in Escherichia coli and Enzymatic Synthesis of Levan" 55.5 (2024) Pp 1801-1812.

14. Lin et al.; "A Gene Encoding for α-Amylase from Thermophilic Bacillus sp. Strain Ts-23 and Its Expression in Escherichia coli" 82 (1997) Pp 325-334, doi: https://doi.org/10.1046/j.1365-2672.1997.00351.x.

15. Omar; "Isolation and Identification of Bacillus sp. Producing of Amylase from Different Sources in Kirkuk, Iraq" 20.1 (2020) Pp 1867-1872.

16. Bradford; "A Rapid and Sensitive Method for the Quantitation of Microorganism Quantities of Protein Using the Principles of Protein–Dye Binding" 72 (1976) Pp 248-254, doi: https://doi.org/10.1016/0003-2697(76)90527-3.

17. Mouafi et al.; "Production of Dextranase from Agro-Industrial Wastes by Aspergillus awamori F-234 under Solid-State Fermentation" 7.6 (2016) Pp 1451-1459.

18. Mansour et al.; "Production of a Novel Halophilic Dextranase from a Honey Isolate, Bacillus subtilis NRC-B233 under Solid-State Fermentation" 46 (2011) Pp 55-78.

19. Bednik et al.; "Enzyme Activity and Dissolved Organic Carbon Content in Soils Amended with Different Types of Biochar and Exogenous Organic Matter" 15.21 (2023) Pp 15396, doi: https://doi.org/10.3390/su152115396.

20. Zohra et al.; "Dextranase: Hyper Production of Dextran Degrading Enzyme from Newly Isolated Strain of Bacillus licheniformis" 92.2 (2013) Pp 2149-2153, doi: https://doi.org/10.1016/j.carbpol.2012.11.080.

21. Walsh & Wright; Nitrogen Metabolism and Excretion. CRC Press, 1995.

22. Chen et al.; "Effect of Environmental and Cultural Conditions on Medium pH and Explant Growth Performance of Douglas-Fir (Pseudotsuga menziesii) Shoot Cultures" 3 (2015) Pp 298, doi: https://doi.org/10.12688/f1000research.5926.1.

23. Nigam; "Microbial Enzymes with Special Characteristics for Biotechnological Applications" 3.3 (2013) Pp 597-611, doi: https://doi.org/10.3390/biom3030597.

24. Zhang et al.; "Biodegradation of Polyethylene and Polystyrene: From Microbial Deterioration to Enzyme Discovery" 60 (2022) Pp 107991, doi: https://doi.org/10.1016/j.biotechadv.2022.107991.

25. Li et al.; "Cell Culture Processes for Monoclonal Antibody Production" 2.5 (2010) Pp 466-479, doi: https://doi.org/10.4161/mabs.2.5.12720.

26. Bhatia et al.; "Kinetic and Thermodynamic Properties of Partially Purified Dextranase from Paecilomyces lilacinus and Its Application in Dextran Removal from Cane Juice" 18.2 (2016) Pp 204-213, doi: https://doi.org/10.1007/s12355-015-0394-5. Here are the new references in your requested format:

27. Virgen-Ortíz et al.; "Kinetics and Thermodynamics of the Purified Dextranase from Chaetomium erraticum" 122 (2015) Pp 80-86, doi: https://doi.org/10.1016/j.molcatb.2015.09.008.

28. Huang et al.; "Purification, Characterization and Degradation Performance of a Novel Dextranase from Penicillium cyclopium CICC-4022" 20.6 (2019) Pp 1360, doi: https://doi.org/10.3390/ijms20061360.

29. Ebaya et al.; "Purification, Characterization, and Biocatalytic and Antibiofilm Activity of a Novel Dextranase from Talaromyces sp." 2020 (2020) Pp 9198048, doi: https://doi.org/10.1155/2020/9198048.

30. Juntarachot et al.; "Optimization of Fungal Dextranase Production and Its Antibiofilm Activity, Encapsulation and Stability in Toothpaste" 25.20 (2020) Pp 4784, doi: https://doi.org/10.3390/molecules25204784.

31. Liu et al.; "Purification, Characterization, and Hydrolysate Analysis of Dextranase from Arthrobacter oxydans G6-4B" 9 (2022) Pp 813079, doi: https://doi.org/10.3389/fbioe.2021.813079.

32. Robinson; "Enzymes: Principles and Biotechnological Applications" 59 (2015) Pp 1-41, doi: https://doi.org/10.1042/bse0590001.

33. Arcus & Mulholland; "Temperature, Dynamics, and Enzyme-Catalyzed Reaction Rates" 49.1 (2020) Pp 163-180, doi: https://doi.org/10.1146/annurev-biophys-121219-081543.

34. Sufiate et al.; "Statistical Tools Application on Dextranase Production from Pochonia chlamydosporia (VC4) and Its Application on Dextran Removal from Sugarcane Juice" 90.1 (2018) Pp 461-470, doi: https://doi.org/10.1590/0001-3765201820170212.

35. Markin et al.; "Revealing Enzyme Functional Architecture via High-Throughput Microfluidic Enzyme Kinetics" 373.6553 (2021) eabf8761, doi: https://doi.org/10.1126/science.abf8761