Immobilization and Some Application of Α-Amylase Purified

Turk J Biochem 2019; 44(3): 397–407

Research Article

Umit Uzun and Melike Yildirim Akatin* Immobilization and some application of α-amylase purified from Rhizoctonia solani AG-4 strain ZB-34 Rhizoctonia solani AG-4 strain ZB-34’den Saflaştırılmış α-Amilazın İmmobilizasyonu ve Bazı Uygulamaları https://doi.org/10.1515/tjb-2018-0240 Conclusion: The results showed that immobilized enzymes Received June 11, 2018; accepted November 17, 2018; previously might have potential applications in industry. This is the published online January 8, 2019 first report immobilizing an α-amylase produced from the Abstract fungus R. solani. Keywords: α-Amylase; Desizing; Immobilization; Juice Background: Aim of the study was to immobilize the clarification; Rhizoctonia solani; Wash performance. α-amylase produced earlier from the mesophilic fungus Rhizoctonia solani AG-4 strain ZB-34 by solid-state fermentation and investigate the suitability of immobilized enzymes for some industries. Öz Materials and methods: A novel α-amylase from R. solani AG-4 strain ZB-34 was immobilized in chitosan by cova- Amaç: Bu çalışmanın amacı, katı substrat fermentas- lent binding and Ca-alginate by entrapment. yonu ile daha önceden mezofilik fungus Rhizoctonia Results: The efficiency of chitosan and Ca-alginate immo- solani AG-4 strain ZB-34’den üretilen α-amilazı immobi- bilization was 67.9% and 59.6%, respectively. The immobilize etmek ve immobilize enzimlerin bazı endüstriler için lized enzymes showed the highest activity in the presence uygunluğunu araştırmaktır. of starch. Optimum values for chitosan and Ca-alginate Gereç ve Yöntemler: Rhizoctonia solani AG-4 strain immobilized enzymes were pH 4.50 and 40°C and pH 5.50 ZB-34’den yeni bir α-amilaz, kovalent bağlanma ile kito- and 60°C, respectively. It was found that immobilized sana ve tutuklama yoluyla Ca-aljinata immobilize edildi. enzymes were highly stable in terms of thermal and pH Bulgular: Kitosan ve Ca-aljinat immobilizasyonunun stabilities. When the chitosan immobilized enzyme was verimi sırasıyla %67,9 ve %59,6 olarak bulundu. İmmo- used with detergents, chocolate stains on dirty laundry bilize enzimler en yüksek aktiviteyi nişasta varlığında was better cleaned. Chitosan immobilized R. solani gösterdi. Kitosan ve Ca-aljinat immobilize enzimler için AG-4 strain ZB-34 α-amylase was found to have a higher optimum değerler sırasıyla pH 4,50 ve 40°C ile pH 5,50 desizing effect at 40°C in tap water. As a result of Ca- ve 60°C olarak belirlendi. İmmobilize enzimlerin, termal alginate immobilization, the enzyme clarified apple juice ve pH kararlılığı açısından oldukça kararlı oldukları more than the free enzyme. bulundu. Kitosan immobilize enzim, deterjanlarla kulla- nıldığında çikolata lekeleri içeren kumaş parçaları daha iyi temizlendi. Kitosan immobilize α-amilazın 40°C’deki *Corresponding author: Melike Yildirim Akatin, Department musluk suyunda daha yüksek bir haşıl alma etkisi olduğu of Chemistry and Chemistry Processing Technologies, Macka bulundu. Ca-aljinat immobilizasyonu sonucunda, elma Vocational High School, Karadeniz Technical University, 61750 suyunun serbest enzimden daha fazla berraklaştırıldığı Trabzon, Turkey, Phone: +90 (462)3 777653, Fax: +90 (462) 5123552, e-mail: [email protected]. https://orcid.org/0000-0003-4195-4647 tespit edildi. Umit Uzun: Department of Chemistry, Faculty of Science, Karadeniz Sonuç: Sonuçlar immobilize enzimlerin endüstride potan- Technical University, Trabzon, Turkey, e-mail: [email protected] siyel uygulama alanlarına sahip olabileceğini gösterdi. 398 Umit Uzun and Melike Yildirim Akatin: Immobilization of Rhizoctonia solani AG4 strain ZB-34 α-amylase

Bu çalışma, R. solani fungusundan üretilen bir α-amilazın This is the first report immobilizing an α-amylase pro- immobilize edildiği ilk çalışmadır. duced from a R. solani.

Anahtar Kelimeler: α-amilaz; haşıl alma; immobilizasyon; meyve suyu berraklaştırma; Rhizoctonia solani; yıkama performansı. Materials and methods

Production and purification of the enzyme

α-Amylase from R. solani AG-4 strain ZB-34 was produced Introduction previously by solid-state fermentation and purified with starch affinity method [8]. α-Amylase (4-α-D-glucan glucanohydrolase, EC 3.2.1.1) is an enzyme used extensively in a range of industries including food and beverages, textiles and detergents, drugs and Covalent immobilization of the enzyme in pharmaceuticals, brewing and fine chemicals, bioconver- sion of solid waste etc. Due to having so many application chitosan areas it is necessary to produce α-amylases at industrial One gram chitosan was stirred with 100 mL of 0.1 N HCl scale. Amylases have been reported to be produced by solution containing glutaraldehyde (%2.5, v/v) for 2 h plant, animal and microbial sources. Microbial amylase at 25°C. After the addition of 10 mL of 0.1 M NaOH solu- production has been reported to be most effective [1, 2]. tion, the precipitate formed was collected by filtration. One of the main problems for enzymes used in industrial Then, chitosan molecules were washed several times areas is their low stability in these environments. Despite with purified water for removing the non-binding glu- their unique properties, the stability of enzymes needs to taraldehyde from the mixture. Wet chitosan was mixed be improved for industrial applications. For this reason, appropriate volume of purified enzyme and slowly industrial enzymes are frequently immobilized onto solid stirred at 4°C for 1 h. After filtration of the mixture, supports. Immobilization may provide many advantages: immobilized enzyme-chitosan molecules were repeat- efficient recovery, reusability and facile separation of the edly washed with distilled water to remove the non- enzyme from the reaction mixture, increased activity and binding enzyme until the absorbance was lower than improvement of some catalytic features such as stability 0.01 at 280 nm [7, 9]. and specificity [3, 4]. Total protein concentration in the filtrate was deter- Immobilized amylases have generally increased mined and percentage of the amylase binding efficiency stability compared to free enzymes. Entrapment is (E) was defined as follows [10]. one of the most preferable immobilization technique due to it prevents excessive loss of enzyme activity E(= CC10− )/C*1 100 and protects enzyme from microbial contamination.

Physical entrapment of α-amylase in calcium alginate C1: Protein concentration of solution before immobilization beads has shown as a relatively easy, rapid and safe C0: Protein concentration of solution after immobilization technique. Chitosan can be used for immobilization of enzymes by covalent binding. It has many charac- teristics like hydrophobicity, biocompatibility, and Entrapment of the enzyme in Ca-alginate low biodegradability, high permeability toward water, good adhesion and high affinity towards proteins. It Appropriate volume of purified enzyme was added to is an inexpensive, inert, non-toxic, high mechanical 5 mL of sodium alginate solution (2%, w/v). The final strength support [1, 5–7]. mixture was dropped using a syringe into 100 mL solu-

In this study, a novel α-amylase produced from the tion of 1 M CaCl2. The beads were stirred 15–20 min and mesophilic fungus Rhizoctonia solani AG-4 strain ZB-34 left for 24 h at 4°C to get the final hardened form. The final and purified with starch affinity technique previously macro beads were removed and washed several times

[8] was immobilized in chitosan by covalent binding and with deionized water for removal of excess CaCl2. Total Ca-alginate by entrapment. After then, the immobilized protein concentration in the filtrate was determined and enzymes were characterized biochemically and their the percentage of the binding protein was calculated as some industrial applications were investigated. mentioned above [11]. Umit Uzun and Melike Yildirim Akatin: Immobilization of Rhizoctonia solani AG4 strain ZB-34 α-amylase 399

Determination of α-amylase activity and optimum reaction conditions. The percentage residual protein concentration enzyme activity was calculated by comparison with non- incubated enzyme. One-hundred microliter of 1% soluble starch, 700 μL of To determine the thermal stability, the enzyme solu- phosphate buffer (50 mM, pH 7.00) and 0.1 g immobilized tions were separately incubated at 4, 28, 40, 50 and 60°C. enzyme was mixed in an Eppendorf tube for determina- Aliquots were withdrawn at 15, 30, 60, 90 and 120 min, tion of the enzyme activity. The mixture was incubated at and α-amylase activity was determined at optimum con- 50°C for 15 min. The immobilized enzyme was separated ditions. The activity of non-incubated enzyme was used to from the mixture by quick-centrifugation. Eight-hundred determine the 100% activity value. microliter of dinitrosalicylic acid (DNS) reagent was added to supernatant and the resulting mixture was boiled in a water bath for 10 min. After cooling to room temperature, Effect of some metal ions and detergent on the absorbance was recorded at 540 nm and the liber- the enzyme activity ated reducing sugar was calculated from a standard curve using glucose. One unit of enzyme activity was defined as The effect of Li+, Na+, Mn2+, Cu2+, Zn2+, Ni2+, Ca2+, Co2+, Mg2+ the amount of enzyme producing 1 μmol reducing sugar and EDTA on the enzyme activity was investigated by per minute under the standard assay conditions [12]. adding them separately to the standard reaction mixture Protein concentration was determined by Bradford at a final concentration of 1 mM. The residual enzyme method, using BSA as a standard [13]. activity (%) was calculated in comparison to that without any additives. To study the effect of Tween 20, SDS, Triton X-100 and Substrate specificity Triton X-114, they were directly added to the standard reaction mixture at the final concentration of 1%. The percent- Soluble starch, maltose, glycogen, β-cyclodextrin, amy- age residual activities were expressed by comparison with lopectin and maltotriose were used to determine the sub- standard assay mixture including no detergent. strate specificity of the immobilized enzymes. All assays were performed in the standard reaction conditions [11]. In all subsequent studies, soluble starch was used as Determination of salt tolerance substrate. To determine salt tolerance of immobilized enzymes, enzyme activities were assayed in the presence of NaCl Optimum pH and temperature with different concentration (0.5–5 M). The enzyme activity in the absence of NaCl was taken as 100% [14]. Enzyme activities were determined in glycine-HCl (pH 3.0), sodium acetate (pH 4.00, 4.50, 5.00, 5.50) and phosphate (pH 6.00, 7.00, 8.00) buffers. The highest enzyme Reusability of the immobilized enzymes activity was defined as 100%, and others were calculated as relative activities. The effect of repeated usage on the activity of enzyme was The optimum temperature of the each enzyme was determined under optimum conditions. At the end of reac- determined at optimum pH value by measuring the activ- tion, the immobilized enzyme beads were separated from ity at 10–80°C. The activity was expressed as percent the assay mixture by quick centrifugation and activity relative activity in relation to the temperature optimum, assay was done in supernatant. The beads were washed which was considered as 100%. three times in distilled water and then a new reaction was repeated under identical conditions [15]. pH and thermal stability Evaluation of the chitosan immobilized α-Amylase activity was determined by incubating the enzyme for use in detergent formulations immobilized enzyme with acetate (pH 5.5) and phosphate (pH 7.0–8.0) buffer solutions at 4°C for 24 h. At the end Usability of chitosan immobilized enzyme as a detergent of the incubation, enzyme activity was assayed under additive was examined by using some commercial laundry 400 Umit Uzun and Melike Yildirim Akatin: Immobilization of Rhizoctonia solani AG4 strain ZB-34 α-amylase and dish washing detergents available at a local market. After the cotton fabrics were washed using tap water, dried at the solutions were prepared from solid detergents as 7 mg/mL 105°C to a constant weight and weighed. The percent (%) and liquid detergents as 10% (v/v) to simulate washing con- removal of starch was calculated by applying the follow- ditions, they were pre-heated at 100°C for 60 min to destroy ing formula [18]. the endogenous enzyme activity. Enzyme activity was determined in the presence of 1 mg/mL and 1% final concentra- Desizing (%) = {Weight of starch removed by enzyme (g)/ tion of solid and liquid detergents, respectively, at 50°C. The Total starch present on the fabric (g)} × 100 activity of the immobilized enzyme assayed in the absence of detergents was taken as 100% [16]. To test the stability of chitosan immobilized enzyme The use of Ca-alginate immobilized enzyme in the presence of protease, 0.1 g immobilized enzyme was mixed with 100 μL of a commercial protease preparation in apple juice clarification (proteinase K, 0.1 mg/mL) and incubated for up to 150 min Apples (Malus domestica cv. Golden Delicious) were cut at room temperature. Thereafter, the residual activity was into cubes and mashed in a mixer grinder and manually determined against the control (amylase without protease pressed using double layer cheesecloth. After calcium treatment) [8]. chloride was added to the raw apple juice at a final concentration of 10 mM, aliquots were pasteurized (5 min at 90°C) and immediately cooled to 50°C. A mixture Wash performance analysis of the chitosan containing 5 mL apple juice and 1 g Ca-alginate immo- immobilized enzyme bilized enzyme was incubated at 60°C for 1 and 3 h. After centrifugation at 17136 × g for 10 min, absorbance Wash performance analysis of the chitosan immobilized (440 nm) of the supernatant was determined. Also, the enzyme was evaluated by determining its chocolate stain total reducing sugar content was determined by the DNS releasing capacity from cotton fabrics. After the choco- method [5]. late was liquefied at 70°C, cotton fabrics (5 cm × 5 cm) were stained with 300 μL of the liquefied chocolate and then dried overnight under in a hot air oven. Each piece of stained cloth was dipped in one of the following flasks Results containing: (a) 25 mL of tap water (control), (b) 25 mL of tap water and 1 g immobilized enzyme, (c) 20 mL of tap α-Amylase from R. solani AG-4 strain ZB-34 produced pre- water and 5 mL of commercial detergent (Persil®, 1%), and viously by solid state fermentation (SSF) and purified with (d) 20 mL of tap water and 5 mL of commercial detergent starch affinity method was immobilized in chitosan and (Persil®, 1%) containing 1 g immobilized enzyme. Flasks were stirred at 200 rpm, 40°C for 60 min. Stain removal capabilities of the chitosan immobilized enzyme Table 1: Substrate specificity of the immobilized α-amylases. was examined visually by looking at the pieces of dried cloth. The chocolate stained cloth piece washed with tap Substrate Relative activity (%) water was considered as a control [17]. Chitosan Ca-alginate immobilized enyzme immobilized enyzme

Soluble starch 100 100 Desizing of cotton fabrics with the chitosan Glycogen 61.69 ± 2.5 42.52 ± 0.8 immobilized α-amylase Amylopectin 90.94 ± 1.2 37.10 ± 1.5 Maltose 64.51 ± 4.1 75.17 ± 3.0 Cotton fabrics (5 cm × 5 cm) were weighed and treated Maltotriose 38.90 ± 3.7 55.47 ± 2.5 β-cyclodextrin 0 0 with 25 mL of soluble starch solution (1% w/v) at room temperature for 15 min. After then, they were dried and The enzyme activity was performed in the standard reaction weighed again. The cotton fabrics were desized in 25 mL conditions by using soluble starch, maltose, glycogen, β-cyclodextrin, amylopectin and maltotriose as substrates. acetate buffer (50 mM, pH 4.5) containing 1 g immobi- The highest enzyme activity (0.065 U/mg protein and ° lized enzyme at 40 and 50 C for 1 h by stirring at 200 rpm. 0.118 U/mg protein for chitosan and Ca-alginate immobilized The same procedure was applied by using tap water enzyme, respectively) was defined as 100%, and other enzyme instead of buffer. Upon the completion of the reaction, activities were calculated as relative activities. Umit Uzun and Melike Yildirim Akatin: Immobilization of Rhizoctonia solani AG4 strain ZB-34 α-amylase 401

Ca-alginate. The binding efficiency of the enzyme to chi- hydrolytic activity was observed for β-cyclodextrin, the tosan and Ca-alginate was 67.9% and 59.6%, respectively. enzymes catalyzed hydrolysis of other substrates used at different ratios.

Substrate specificity Optimum pH and temperature Soluble starch, maltose, glycogen, β-cyclodextrin, amylopectin and maltotriose were used to determine the sub- Optimum pH and temperature values were determined strate specificity of the immobilized enzymes. All assays as pH 4.50, 40°C and pH 5.50, 60°C for chitosan and Ca- were performed in the standard reaction conditions. Both alginate immobilized enzyme, respectively (Figure 1A, B). of the immobilized enzymes showed the highest activity in the presence of soluble starch (Table 1). Although no

pH and thermal stability A Chitosan immobilized enzyme 120 Ca-alginate immobilized enzyme As can be seen from Figure 2A, the chitosan immobilized enzyme retained all of its original activity at all tested pH 100 values after 1 day. At the end of 5 days, approximately 80 70–80% of activity was still present. These results suggest

40 A Relative activity (%) 120 pH 5,50 20 100 pH 7,00 pH 8,00 0 80 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 pH 60

B Chitosan immobilized enzyme 40

120 Residual activity (% ) Ca-alginate immobilized enzyme 20 100 0 80 012345678 60 Time (day) pH 5,50 40 B

Relative activity (%) 120 pH 7,00 20 100 pH 8,00 0 0102030405060708090 80 Temperature (°C) 60 Figure 1: (A) Effect of pH on the activity of immobilized enzymes. Enzyme activity was determined in glycine-HCl (pH 3.0), sodium 40 acetate (pH 4.00, 4.50, 5.00, 5.50) and phosphate (pH 6.00, 7.00, Residual activity (%) 20 8.00) buffers. The highest enzyme activity (0.035 U/mg protein and 0.096 U/mg protein for chitosan and Ca-alginate immobilized 0 enzyme, respectively) was defined as 100%, and other enzyme 012345678 activities were calculated as relative activities. (B) Effect of Time (day) temperature on the activity of immobilized enzymes. The optimum temperature of the enzyme was determined at optimum pH value by Figure 2: pH stability of chitosan immobilized enzyme (A) and of measuring the activity at 10–80°C. Ca-alginate immobilized enzyme (B). The activity was expressed as percent relative activity in relation Enzyme activity was determined by incubating the immobilized to the temperature optimum, which was considered as 100% enzyme with acetate (pH 5.5) and phosphate (pH 7.0–8.0) buffer (0.058 U/mg protein and 0.132 U/mg protein for chitosan and solutions at 4°C for 24 h. At the end of the incubation, enzyme Ca-alginate immobilized enzyme, respectively). activity was assayed under standard reaction conditions. 402 Umit Uzun and Melike Yildirim Akatin: Immobilization of Rhizoctonia solani AG4 strain ZB-34 α-amylase

A that the chitosan immobilized enzyme was highly stable 120 at 4°C for tested pH values. About 90% of the activity of the Ca-alginate immo- 100 4 °C ity (%) 80 bilized enzyme was maintained at pH 5.5 after 7 days 28 °C (Figure 2B). At pH 7.00 and 8.00, activity was conserved at activ 60 40 °C the rate of 65% and 50%, respectively, after 7 days. 50 °C 40

The activity of chitosan and Ca-alginate immobilized Residual 60 °C α-amylases was conserved completely at storage tempera- 20 ture (4°C) and the fungus growth temperature (28°C) for 0 120 min. They also conserved approximately 60–70% of 020406080 100 120 140 Time (min) their original activity at 40°C after 120 min (Figure 3A, B). B Ca-alginate immobilized enzyme seems to maintain its 120 activity much more than chitosan immobilized enzyme at ) 100 4 °C 4°C and 50°C. The enzyme had more than about 10% of its 80 ity (% 28 °C activity even after 60 min at 60°C. 40 °C

activ 60 50 °C 40 60 °C

Residual 20 Effect of some metal ions and detergent on 0 020406080 100 120 140 the enzyme activity Time (min)

When the activity of α-amylase was examined in the pres- Figure 3: Thermal stability of chitosan immobilized ence of metal ions, it was noted that in the case of some enzyme (A) and Ca-alginate immobilized enzyme (B). metal ions there were differences between immobilized The enzyme solutions were separately incubated at 4, 28, 40, ° enzymes (Figure 4). The activation rate was higher in chi- 50 and 60 C. Aliquots were withdrawn at different times and α-amylase activity was determined at optimum conditions. 18.8 U/ tosan immobilized enzyme then Ca-alginate immobilized mg protein and 112.51 U/mg protein for chitosan and Ca-alginate 2+ 2+ 2+ enzyme in the presence of Li and Mn . Cu activated the immobilized enzyme, respectively, was considered as 100% chitosan immobilized enzyme but inhibited Ca-alginate residual activity.

160 Chitosan immobilized enzyme 140 Ca-alginate immobilized enzyme

120

100

Residual activity (% ) 40

SDS LiCI2 NaCI NiCI2 Control MnCI2 CuCI2 ZnCI2 CaCI2 CoCI2 MgCI2 EDTA Tween 20 Triton X-100Triton X-114

Figure 4: Effect of some metal ions, EDTA and detergents on the activity of immobilized enzymes. The effect of some chemicals was investigated by adding them separately to the standard reaction mixture at a final concentration of 1 mM or 1%. The percentage residual activities were expressed by comparison with standard assay mixture with no chemical added. 17.1 U/mg protein and 102.1 U/mg protein for chitosan and Ca-alginate immobilized enzyme, respectively, was considered as 100% residual activity. Umit Uzun and Melike Yildirim Akatin: Immobilization of Rhizoctonia solani AG4 strain ZB-34 α-amylase 403

α-amylase approximately 10%. In contrast, while Ca2+ to be inhibited in a considerable amount at all salt con- inhibited the chitosan immobilized enzyme about 10%, centration more than 1.0 M (Figure 5). activated the Ca-alginate α-amylase about 30%.

Reusability of the immobilized enzymes Determination of salt tolerance Reusability of immobilized enzymes was determined by performing seven repetitions. For both of the immobilized Whereas no significant change was found in the activity enzymes, only about 20% of the activity was lost at the of chitosan immobilized enzyme at salt concentrations end of seven measurements (Figure 6). of 0.5, 1.0 and 2.0 M, the enzyme was inhibited approximately 20–25% in the presence of 4.0 and 5.0 M NaCl, respectively. The alginate immobilized enzyme was found Evaluation of the chitosan immobilized enzyme for use in detergent formulations Chitosan immobilized enzyme 120 Ca-alginate immobilized enzyme Chitosan immobilized enzyme was tested for its potential to be used as a commercial detergent additive. The highest 100

80 Table 2: Testing the compatibility of chitosan immobilized enzyme 60 with various commercial detergents. 40 Detergents Residual activity (%) Residual activity (% ) 20 Omo® (Solid laundry) 76.9 ± 0.5 0 Ariel® (Solid laundry) 74.1 ± 1.2 Control 0.5 12345 Persil® (Solid laundry) 81.1 ± 1.5 [NaCl] Alo® (Solid laundry) 73.4 ± 0.9 Etimatik® (Solid laundry) 51.1 ± 2.5 Figure 5: Salt tolerance of immobilized enzymes. Enzyme activities Perwol® (liquid laundry) 66.2 ± 3.0 were assayed in the presence of NaCl with different concentration. Fairy® (liquid dishwasher) 65.1 ± 0.7 The activity in the absence of NaCl (17.1 U/mg protein and 102.1 U/mg protein for chitosan and Ca-alginate immobilized Enzyme activity was determined in the presence of 1 mg/mL and enzyme, respectively) was taken as 100%. 1% final concentration of solid and liquid detergents, respectively, at 50°C. The activity of the immobilized enzyme assayed in the absence of detergents (19.8 U/mg protein) was taken as 100%. 120

100 100 (%) 80 80 ity

activ 60 Chitosan immobilized enzyme 60

40 Ca-alginate immobilized enzyme 40 Residual

20 Residual activity (% ) 20

0 0 012345678 020406080 100 120 140 160 180 200 Number of cycle Time (min)

Figure 6: Reusability of the immobilized enzymes. The immobilized Figure 7: Stability of the chitosan immobilized enzyme in the presence enzyme beads were separated from the assay mixture by quick of protease. 0.1 g immobilized enzyme was mixed with 100 μL of a centrifugation at the end of reaction and activity assay was done in commercial protease preparation (proteinase K, 0.1 mg/mL) and supernatant. The beads were washed three times in distilled water incubated for up to 150 min at room temperature. Thereafter, the and then a new reaction was repeated under identical conditions. residual activity was determined against the control (amylase without Activity assays were done at optimum pH and temperature values for protease treatment, 19.3 U/mg protein and 108.6 U/mg protein for each of the immobilized enzyme. chitosan and Ca-alginate immobilized enzyme, respectively). 404 Umit Uzun and Melike Yildirim Akatin: Immobilization of Rhizoctonia solani AG4 strain ZB-34 α-amylase

A B C D

Figure 8: Wash performance analysis tests. After the chocolate was liquefied at 70°C, cotton fabrics (5 cm × 5 cm) were stained with 300 µL of the liquefied chocolate and then dried overnight. The cloth pieces washed separately with (A) control: tap water, (B) detergent, (C) chitosan immobilized enzyme and (D) chitosan immobilized enzyme + detergent at 40°C for 60 min and stain removal capabilities of the immobilized enzyme was examined visually.

Table 3: Desizing capacity of chitosan immobilized enzyme. The use of Ca-alginate immobilized enzyme

Temperature (°C) Desizing (%) in acetate Desizing (%) in the apple juice clarification buffer (pH 4.5) in tap water The alteration effect of Ca-alginate immobilized 40 31.0 23.6 enzyme on some parameters of apple juice was inves- 50 20.9 16.7 tigated (Figure 9 and Table 4). It has been observed that activity of the enzyme was detected in the presence of Persil brand detergent (Table 2). When the stability of the chitosan immobilized enzyme in the presence of protease was examined, it appeared that there was a loss in enzyme activity about 15% after 150 min (Figure 7).

Wash performance analysis of the chitosan immobilized enzyme

The utility of the chitosan immobilized enzyme as a detergent additive was visually tested. For this purpose, the fabric pieces on which the chocolate stain was formed A B C were individually washed in Erlenmeyer flask contain- Figure 9: Effect of calcium alginate immobilized enzyme on fruit ing tap water, immobilized enzyme, detergent (Persil®), juice clarity. detergent and immobilized enzyme together. At the end of After CaCl2 was added to the raw apple juice at a final concentration the washing process, it was observed that the fabric piece of 10 mM, aliquots were pasteurized (5 min at 90°C) and immediately washed with detergent and immobilized enzyme together cooled to 50°C. A mixture containing 5 mL apple juice and 1 g was cleaner than the fabric piece cleaned by only deter- Ca-alginate immobilized enzyme was incubated at 60°C for 1 and 3 h. (A) Unclarified juice, (B) after 1 h incubation, (C) after 3 h incubation. gent (Figure 8).

Table 4: Some properties of unclarified and clarified apple juice.

Properties Unclarified Clarified Clarified Desizing of cotton fabrics with chitosan juice juice after 1 h juice after 3 h immobilized α-amylase incubation incubation pH 4.58 4.38 4.32 Chitosan immobilized R. solani AG-4 strain ZB-34 Absorbance (440 nm) 1.537 0.542 0.343 α-amylase was found to have a higher desizing effect at Reducing sugar 27.74 28.57 29.12 (μg/mL) 40°C and in acetate buffer (pH 4.5) (Table 3). Umit Uzun and Melike Yildirim Akatin: Immobilization of Rhizoctonia solani AG4 strain ZB-34 α-amylase 405

Ca-alginate immobilized enzyme affected the change of immobilized enzymes maintain their activity at 60°C relevant parameters positively. after 1 h [19]. In another study, α-amylase from B. subtilis (ITBCCB148) bacterium lost almost all its activity when it was kept at 60°C for 1 h. In the thermal stability of the enzyme immobilized on alginate, an increase of about Discussion 10.5 times occurred compared to the free enzyme [22]. Commercial Bacillus sp. α-amylase (Sigma, St Louis, Both of the immobilized enzymes had no hydrolytic activ- MO, USA) was immobilized to N-isopropylacrylamide ity in the presence of β-cyclodextrin as in the case of free (NIPAAm) polymer matrix. Free and immobilized enzyme enzyme [8]. This situation can be attributed existence of conserved their activity about 33% and 46%, respectively, the cyclic α-(1,4)-glycosidic bounds in β-cyclodextrin. for 35 min at 70°C [23]. It was previously reported that the free α-amylase It has been reported that α-amylase from Aspergil- from R. solani AG-4 strain ZB-34 had a pH optima at 5.50 lus sclerotiorum was immobilized on Ca-alginate beads [8]. When the literature data are examined, it can be and the immobilized enzyme had a half-life of 164 min at seen that immobilization can change optimum pH of an 60°C [24]. Commercial Aspergillus oryzae α-amylase from enzyme. pH is one of the most important parameters with D. Fine-Chem Ltd. (Mumbai, India) was immobilized in the ability to change the enzyme activity in the reaction Ca-agar microcapsules and the immobilized enzyme was mixture. Immobilization may cause optimal pH to shift found to have a half-life of 17 min at 75°C [25]. as it causes conformational changes on the enzyme. This It can be said that immobilized α-amylases from R. shift occurs when the ionization of the acidic and basic solani AG-4 strain ZB-34 may be either advantageous amino acid side groups in the microenvironment around or disadvantageous over other α-amylases in terms of the active site of the enzyme changes. It was reported that thermal stability. However, the most important point here optimum pH of the immobilized α-amylases decreased is that the industrial application area of the enzyme to be from pH 6.0 to pH 5.75 and from 6.0 to 5.25 in DEAE-cellu- used and therefore the evaluation must be done accord- lose and chitin, respectively [19]. ingly. As a result of immobilization, there was a significant Optimum temperature of the free α-amylase from increase in the stability of the R. solani AG-4 strain ZB-34 R. solani AG-4 strain ZB-34 was 50°C [8]. Shifting of the α-amylase, especially at low temperatures, up to 50°C. It optimum temperature from 50°C to 40°C by chitosan is clear that this increase will contribute to the availability immobilization leads to the need to investigate the suit- of immobilized enzymes in industrial processes especially ability of the immobilized enzyme for industrial processes at low temperatures. or applications that occur at low temperatures. For Ca- Immobilization is one of the most preferred ways to alginate immobilized enzyme, the optimum temperature increase the stability of enzymes under extreme condi- increased from 50°C to 60°C. In a study, commercial pig tions. If the immobilization process is carried out prop- pancreatic α-amylase was covalently immobilized on glass erly, the properties of the enzyme will recover in many beads and the optimum temperature of the enzyme was ways, especially stability, activity, specificity and reduced reported to increase from 30°C to 50°C [20]. When the par- inhibition [26]. Immobilization prevents the opening tially purified α-amylase enzyme from Bacillus subtilis was of protein folds leading to decrease in enzyme activity. immobilized on the chitin surface, the optimum tempera- Thus, the stability and other properties of the enzymes ture value increased to 65°C with an increase of 20°C [19]. are favorable to develop [22]. The fact that immobilized R. Furthermore, when the pH stability of free and immo- solani AG-4 strain ZB-34 α-amylases have higher thermal bilized enzymes was compared, it can easily be seen that stability than the free enzyme overlaps these facts. the stability increases with immobilization [8]. This will In the presence of EDTA, immobilized enzymes were make a positive contribution to the availability of the found to have higher activity losses than free enzymes [8]. enzyme in industrial areas. It was reported that commer- α-Amylase obtained from Bacillus isolates and immobi- cial Bacillus sp. α-amylase was immobilized on acrylic lized on polyglycidyl methacrylate beads was found to be solid support materials and it was stable for 1 h at pH 3.5– inhibited by Mg2+, Mn2+ and Cu2+ ions at different rates [27]. 8.0 [21]. The pig pancreatic α-amylase covalently immobi- It has also been reported that α-amylase from Aspergillus lized to glass beads was determined to be stable at pH 6.9 niger immobilized to glutaraldehyde modified polyaniline and 4°C for 5 days [20]. was inhibited by Ca2+, Zn2+ and Cu2+ ions [28]. It has been reported that α-amylase from B. subti- The activity of chitosan immobilized enzyme lis I was immobilized to DEAE-cellulose and chitin and increased about 8% in the presence of Tween 20. 406 Umit Uzun and Melike Yildirim Akatin: Immobilization of Rhizoctonia solani AG4 strain ZB-34 α-amylase

Ca-alginate immobilized enzyme activated 5% in the pres- enzyme in the presence of protease was higher than the ence of Triton X-114. Activity of the Ca-alginate immobi- free enzyme [8]. lized enzyme could not be determined in the presence In one study, it was reported that α-amylase (terma- of SDS because the Ca-alginate beads disintegrated. It mylaze) obtained from Novozymes Co. (Tehran, Iran) was can be easily stated that immobilization of the R. solani immobilized on silica nanoparticles and used in detergent AG-4 strain ZB-34 α-amylase contributes positively to the formulations to aid the removal of starch stains. Removal availability of the enzyme in some detergents. of starch stains was been found to be better with the use of The enzyme purified from Marinobacter sp. EMB8 was detergent containing immobilized enzyme [36]. reported to maintain its activity at 75% as a result of 1 h treat- In this study α-amylase from R. solani AG-4 strain ment with SDS and Triton X-100 detergents at 0.1% concen- ZB-34 was immobilized covalently on chitosan. In this tration [29]. The effects of detergents such as Triton X-100 case immobilized enzyme can interact with the stains on and Tween-20 and surfactants such as SDS on the activity of dirty laundry. Because of the enzyme was immobilized the α-amylase produced by Bacillus licheniformis AI20 were in Ca-alginate by entrapment, substrates of the enzyme studied and found that detergents tested at 0.25% concen- within laundry stains cannot enter Ca-alginate beads. For tration did not alter the enzyme activity. Anionic surfactant this reason Ca-alginate immobilized enzyme cannot be SDS at the concentration of 1% was found to cause a 35% used as detergent additive. reduction in enzyme activity [30]. It has been reported that When the effect of Ca-alginate immobilized enzyme B. licheniformis NH1 α-amylase was treated with Tween-20 on some parameters of apple juice was investigated, it was and Triton X-100 at 1% concentration at 40°C for 1 h and found that Ca-alginate immobilized enzyme affected the there was no loss of activity at this time [31]. change of relevant parameters positively. This effect was The increase of amylase activity in the presence of better than free enzyme [8]. some detergents suggests that these detergents may have In this work, an α-amylase from R. solani AG-4 strain positive effects on the enzyme conformation and on the ZB-34 was immobilized for the first time. The immobilized hydrophobic interactions involved in stabilizing the enzymes were characterized biochemically. The suitability buccal structure of the protein molecule [32]. The decrease of the chitosan immobilized enzyme for detergent indus- in enzyme activity in the presence of certain detergents try as an additive and Ca-alginate immobilized enzyme for can also be correlated with a possible deterioration of juice clarification were investigated. Also, chitosan immo- protein conformation. bilized enzyme was used for desizing of the cotton fabrics. It has been reported that having high salt tolerance is a It can be seen from results that some properties of the useful feature for amylases used in starch sugar processing enzyme changed after immobilization. For example, while and in different applications in the industry [33]. Salt toler- the optimum pH and temperature values of the enzyme ance testing has been reported to be important in the treat- decreased upon chitosan immobilization, they were ment of pollution control mechanisms, cellulose, starch and increased with Ca-alginate immobilization. Hence, it is waste water containing high salt [34, 35]. In our work, the necessary to look at where the enzymes are used to decide high salt tolerance of chitosan immobilized enzyme sup- which method is more advantageous. But in conclusion it ports that it might be used in these industrial applications. can be said that immobilized R. solani AG-4 strain ZB-34 An α-amylase immobilized to N-isopropylacrylamide α-amylases might have potential application in different (NIPAAM) polymer was reported to maintain 54% activity industrial areas. in 12 consecutive measurements [23]. It was reported that α-amylase immobilized on Ca-agar microcapsules lost its Acknowledgements: This work was financed by TUBITAK activity by 22% after six measuring [26]. α-Amylase pro- (Project number is 115Z109). duced by SSF from A. sclerotiorum was immobilized on Ca-alginate beads. Beads prepared under optimal immo- Conflict of interest: The authors declare no conflicts of bilization conditions could be used for up to 7 times, interest of any kind. losing only 35% of the initial activities [24]. The utility of the chitosan immobilized enzyme as a detergent additive was visually tested. At the end of the washing process, it was observed that the fabric piece References washed with detergent and immobilized enzyme together 1. Pandey A, Nigam P, Soccol CR, Soccol VT, Singh D, Mohan R. was cleaner than the fabric piece cleaned by only deter- Advances in microbial amylases. Biotechnol Appl Biochem gent. Also, the stability of the chitosan immobilized 2000;31:135–52. Umit Uzun and Melike Yildirim Akatin: Immobilization of Rhizoctonia solani AG4 strain ZB-34 α-amylase 407

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