03. Rini.cdr Vol.15, No.1, March 2021, p 15-20 DOI: 10.5454/mi.15.1.3 Thermostable Alkaline Protease Activity from DUCC- K225 and Aspergillus flavus Its Compatibility to Local Detergents ALMALINA NABILA SULISTYO RINI SWORO UKMI RI UJIYANTO, I R *, S PAND Department of Biology, Faculty of Science and Mathematics, Universitas Diponegoro Jl. Prof Soedharto, SH, Tembalang, Semarang 50275, Indonesia. Protease important enzyme in many industries, including detergent. DUCC-K225 is a Aspergillus flavus thermotolerant indigenous molds isolated from Madura island which is potential in producing thermostable alkaline protease enzymes. The enzyme produced by submerged culture on modified Czapeks Dox liquid medium containing glucose as carbon source and 1% of casein. The aims of this study were to determine the activity and stability of thermostable alkaline protease produced by DUCC-K225 at various temperatures, also the A. flavus compatibility to 5 local detergents. Research were using , conducted Completely Randomized Design in triplicate with temperature variation for protease activity as treatment. The results showed that the activity of highest thermostable alkaline proteases was 214.503 U/mL, with retained activities up to 78% in 60 minutes at 55°C. Th is enzyme compatible with 5 local detergents tested, with the retained activity varied 59.48%-99.48% at 29 C and o 62.83%-98.05% at 55 C. The compatibility to detergent confirmed by blood o all tested were the increament of solubility. alkaline protease, DUCC-K225, compatibility, detergent, thermostableKey words: A. flavus Protease merupakan enzim yang penting untuk industri, termasuk industri deterjen. Aspergillus flavus DUCC-K225 kapang termotoleran indigenous dari Pulau Madura, yang berpotensi menghasilkan merupakan enzim protease alkalis termostabil. Produksi enzim dilakukan dalam kultur terendam pada medium Czapexs Dox broth yang mengandung glukosa sebagai sumber karbon, dan kasein 1%. Tujuan dari penelitian ini untuk men aktivitas dan stabilitas ensim protease alkalis yang dihasilkan oleh DUCC-K225 pada getahui A. flavus berbagai suhu, serta kompatibilitasnya terhadap 5 jenis deterjen lokal. Hasil yang diperoleh menunjukkan bahwa ensim tetap stabil pada suhu yang menunjukkan bahwa ensim ini termostabil, A tertinggi ensim ini 55 C ktivitas o adalah 9 48 9,48 dan 62.83214.503 U/mL, dengan aktivitas yang dipertahankan sebesar 5 . %-9 % pada suhu 29 C %-o 98,05 Ensim ini kompatibel terhadap semua deterjen yang diujikan, karena dapat % pada suhu 55 C. o meningkatkan kelarutan noda darah pada kain. Kata kunci: DUCC-K225 deterjen, kompatibilitas, protease alkalis, termostabil A. flavus , MICROBIOLOGY INDONESIA Available online at http://jurnal.permi.or.id/index.php/mionline ISSN 1978-3477, eISSN 2087-8575 *Corresponding author: Phone ; ;: +62-811288428 Fax: +62- E-mail: mgiswororukmi@lecturer.undip.ac.id nature, including in extreme environment (Coral et al. 2003). Some microorganisms have the potential to produce thermostable alkaline proteases, among others are fungi that can produce many kinds of extracellular enzymes . The enzyme production from fungi has several advantages, the because enzymes are more easily obtained in fermentation eassubmerged , ily separated from the medium, and can be grown on a cheap medium (Souza 2015; Soares 010). et al. et al. 2 According to Souza (2015 the genus et al. ) the mold of Aspergillus known as a largest producer of widely alkaline proteases such as , which Aspergillus flavus has been known as a species that generally has high a ability in producing extracellular protease. The production of microbial enzymes by were influenced the composition, while the enzyme activitymedium affected by the incubation temperature (Packianathan et al. 2008). The aims of this study were to examined the production of thermostable alkaline protease from Protease (EC 3.4) is an enzyme that able to hydrolyze the peptide bonds between amino acids in proteins (Nallusami, Remya & Al-Bahri 009). The 2 availability of proteases is important for several industries, especially the proteases which are stable at high temperature and pH According to Gupta . et al (2002) Chimbekujwoa ome and . (2020) set al industries need thermostable alkaline proteases which is active at pH 8-12 and temperatures of 50°C-70°C The . presence of alkaline proteases is one of the 3 major groups of enzymes needed in industrial processes, which have a contribution of 60% in the sale of enzymes worldwide (Sahib 2009; Lanka, Anjali, & Pydipalli 2 , . 2020017 Chimbekujwoa ). Protease enzymes et al can be obtained from various sources such as plants, animals and microorganisms that are widespread in indigenous thermotolerant fungi Aspergillus flavus DUCC-K225 isolated from lime soil in Madura , Island East Java and examined using glucose as carbon source the effect of temperature on the enzyme activity, as well as the compatibility to detergents. enzyme local MATERIALS AND METHODS C u l t u r e M a i n t e n a n c e a n d I n o c u l u m Preparation. The mold DUCC-K225 was A. flavus maintained on PDA Agar slant, and sub cultured before used in the study. The spore inoculum was prepared by adding sterile distilled water containing 1% of Tween 80 into 5 days old mold culture on PDA (Coral et al. 2003). Enzyme Production. The fungal alkaline protease from DUCC-K225 produced A. flavus according to Coral 2003 Packianathan . 2008) by et al. et al ( ) and ( submerged fermentation in the modified Czapeks Dox medium composed of 30 g/L glucose; 0.5 g/ L KCl; 0.01 g /L FeSO ; 0.5 g/L MgSO ; 1 g/L K HPO ; 2 g/L 4 4 2 4 NaNO , and 1 % casein. The media pH was arranged at 3 9. One percent fungal spore suspension of s A. flavus DUCC-K225 (10 /ml) inoculated into the medium, the 8 flasks were then incubated on rotary shaker with 120 rpm for 7 days at room temperature. The fungal cultures filtered using Whatman paper. No. 1, the supernatant obtained centrifuged at 4000 rpm for 20 minutes and use as crude enzymes. Protease Assay. The protease activity was measured by modified method of Keay . (et al 1970 in Coral 003; Rani & Prasad 013 Chimbekujwoaet al. 2 2 ; 2020) using casein as substrate. One ml of culture supernatant was mixed thoroughly with 1ml of 2% of casein solution, incubated at 37°C for 10min and the reaction was stopped by adding 2ml of 0.4M trichloroacetic acid and incubated for 20min at 37°C. The solution filtered using Whatman No. 1 paper. One ml of filtrate then mixed thoroughly with 5ml of 0.4M Na CO and 1ml of 0.5N Folin phenol reagent, 2 3 incubated at 37°C for 20min. The absorbance of the final solution was measured at 660nm. One unit of protease activity was defined as the amount of enzyme required to liberate 1 µmol of tyrosine in 20min at 37°C. T h e r m o s t a b i l i t y A s s a y. T h e e n z y m e thermostability examined according to Kamoun et al. (2008) by measuring the enzyme activity for 1 hour at various temperatures with interval of 15 minutes at pH 9.8. Thermal stability of enzyme was determined by incubating 2 mL of culture supernatant mixed with 2% casein solution for 60 minutes at 29 C, 40 C, 45 C, o o o 50 C, 55 C, and 60 C. The heated enzyme was used o o o un as control. The Compatibility to Commercial Detergents. alkaline protease of DUCC-K225 was tested A. flavus for its compatibility to 5 commercial detergents i.e. B, D, S, R, and J . A , according to Choudhary (2012) detergent solution of 0.7g/100mL were heated at 100 C o for 1 hour, to destroy indigenous protease that might be present. The detergent solution mixed with the culture supernatant in a ratio of 1:1 (v/v) incubated at 40ºC for 20 min. The residual protease activity examined using standard assay procedure as mentioned previously. A mixture of culture supernatant and tap water (1:1) used as control 100%. The relative enzyme activity was expressed as percentage activity considering the activity of control. The enzyme activities were examined in 3 treatments, i.e. 0.3mL of culture supernatant mixed with 5.7mL of detergent solution (0.7g/100mL); 0.3mL of detergent solution (0.7g/100mL) mixed with 5.7mL of distilled water; and 0.3 mL of culture supernatant mixed with 5.7mL of distilled water. The enzyme assays conducted at room temperature (29 C) and the optimum temperature of o protease activity obtainedwhich previously (55 C). o Cleansing Power as Additive in Local Detergents. The cleansing power of the enzyme carried out by based on Niyonzima & More (2015) soaking a piece of 4x4cm white cloth which square have been stained with blood in 4 solutions, containing (1) 50mL of distilled water 1mL of culture supernatant , and 1mL of detergent solution (0.7g /100 mL) which has been heated before at 100 C for 1 hour; (2) 51mL of o distilled water and 1 mL culture supernatant; (3) 51mL of distilled water and 1mL of detergent solution (0.7g /100mL) heated at 100 C; (4) 52mL of distilled o and water as control. The bloodstained clothes were soaked in the solution at room temperature (29 C) and 55 C for o o 10 minutes. The clothes were then dried and visually observed for the stain removal. The blood solubility in the soaking solution were also observed by measuring the absorbance at λ . 200 RESULTS Effect of Temperature on Protease Activity. The protease activity examination at various temperatures carried out to obtain the optimum temperature of . A flavus DUCC-K225 protease. Figure 1 showed that the , activity of alkaline protease increased along with the temperature increment. The highest protease activity reached at 55°C with the value of 214,50U/mL, 16 RINI ET AL . Microbiol Indones Volume 15, 2021 Microbiol Indones 17 although it is statistically at par with 50 C and 60 C. o o Thermostability of Alkaline Protease. The enzyme thermostability have been examined at the optimum temperature of 55 C, which obtained from o previous examination of temperature effect on protease activity he result showed that the enzyme was still . T active for 60 minutes with retained activity value of 77.8 – 78.8% (Fig 2). Compatibility with Detergents. The detergent compatibility test was carried out at room temperature ( and to determine the ability of . 29°C) 55°C A flavus DUCC-K225 thermostable alkaline protease as additive for commercial detergents. The enzyme compatibility to detergents determined by the retained activity value of enzyme in the detergent solutions. Table 2 t retained showed hat the activity of the enzyme in the detergent solution were varied. in three commercial detergents D, J and R were , which is in slightly higher 55°C 29°C while B at than at , for and S detergent at 29 C 55 Cwere slightly higher than at o o (Table 2) , but not different significantly. Cleansing Potential as Detergent Additive. The effect of alkaline protease produce by . DUCC-A flavus K225 on detergent's cleansing power was carried out by soaking the blood stained fabrics in the detergent solution mixed with enzyme at two different temperature. The visually examination found that the enzyme increased the detergent's removing power of the blood stain from the fabrics (Table 3). The measurement of soaking solution's absorbance at λ 200 were to ensure that the blood dissolved to carried out the water. The obtained of absorbance values were higher 55°C than 29°C indicated that more at at dissolved blood occurred at higher temperature (Fig 3). The highest absorbance showed on the enzyme and S detergent mixed solution, this result in line with the Detergents* Protease activity (U/mL) Retained activity (%) 29°C 55°C 29°C 55°C Enzyme 66.76 ± 1.46 111.77 ± 2.70 100.00 100.00 B 61.55 ± 2.83 102.23 ± 1.33 92.19 91.47 D 57.01 ± 1.29 99.42 ± 2.19 85.39 88.96 J 48.11 ± 1.78 85.43 ± 2.83 72.07 76.44 R 39.71 ± 2.70 70.22 ± 3.10 59.48 62.83 S 66.41 ± 2.66 109.58 ± 1.66 99.48 98.05 Table 2 The of DUCC-K225 alkaline proteas various detergentscompatibility e to A. flavus Table 3 The of DUCC-K225 alkaline proteas various detergentscompatibility e to A. flavus Control (water) Water + Detergent 29oC 55oC Water + Enzyme + Detergent 29oC 55oC No detergent No detergent S S D D B B R R J J 18 RINI ET AL . Microbiol Indones value of detergent's compatibility (Table 2). DISCUSSION Aspergillus flavus DUCC K225 is an indigenous mold isolated from lime soil of Madura Island. The temperature of the sampling site was 38 C with the pH 0 of 8.3. This fungus can grow up to 45 C at the pH 8 on o PDA medium and showed proteolytic activity with the index of 1.38. Based on the data obtained, study in the production of alkaline thermotolerant protease from this isolate were conducted, to examine on the thermotolerant characteristic and the compatibility to comercial detergent. The result of protease activity from DUCC K225 examination showed that A. flavus the enzyme is a thermostable one. The enzyme activity at 55 C-60 C higher than at lower temperatures o o (Fig.1.), this indicates that the enzyme structure remains stable up to 60 C. According to Yeoman o et al., (2010) the thermostable enzymes can theirmaintain Fig 1 The effect of temperature on the alkaline protease activity of . DUCC-K225.A flavus Remarks: the same superscripts show no significant difference (p<0.05ly ). Fig 2 The stability of alkaline protease enzyme produced by . DUCC-K225 at 55°C.A flavus Fig 3 The blood stain solubility in the soaking water. W- water; E – enzyme; B,D,J,R,S - detergents. Volume 15, 2021 Microbiol Indones 19 structural integrity at above 55 C Coral . 2003 o . ( ) et al reported the thermostable alkaline protease produced by an strain stable at 40 C, Aspergillus niger which is o while alkaline protease from AS2 A. flavus studied by Rani & Prasad 2013 stable at 55 C. ( ) found Based on o previous data obtained in this study, the temperature of 55 C was stated as optimum temperature of this 0 protease. The observation at showed that this 55 C o enzyme still active for 60 minutes with 78% retained activity (Fig. 2.). This result supported the previous indication that the alkaline protease produced by . A flavus DUCC-K225 was thermostable. The similar result also found in previous study on the alkaline proteinases produced by which Aspergillus fumigatus Fresenius TKU003 and which Aspergillus terreus is thermostable at 50°C and 60°C (Nirmal 2011). . et al The enzyme retained activity value of the alkaline protease produced by . DUCC-K225A flavus in five commercial indicated that detergents (Fig 3), this enzyme compatible with all detergents tested. Choudhary 2012 Niyonzima & More 201( ), ( 5) and Devi 2008 the alkaline et al ( ) have been reported that protease from several species Aspergillus were compatible to detergents. The stability of enzyme was strong related to the retained activity in detergent solutions. s The difference in enzyme stability of A. flavus DUCC K225 in the five detergent solution used, may corelated with detergent . Surfactant is ingredient one of the main ingredient in commercial detergents, which have the ability to interact with proteases in increasing or inhibit protease activity (Zhang & ing Zhang 2015). The detergent S, B, and D contain ed Sodium Alkyl Benzene Sulfonate as surfactant with the concentrations of 22%, 20% and 15% respectively, while J and R detergents 16% and 19%contained surfactant respectively, but the types of surfactant were not clearly defined Samanta & Mitra . According to ( ) t2004 here are 4 groups of surfactants, i.e. anionic surfactants, non-ionic surfactants, cationic surfactants, and amphoteric surfactants An anionic surfactants . such as Sodium Alkyl Benzene Sulfonate also known as Linear Alkyl Benzene Sulfonate (LAS) have the greatest on protease activity, compared to other effect anionic surfactants such as Sodium Duodesyl Sulfate (SDS) and Sodium Lauryl Sulfate (SLS) (Zhang & Zhang 2015). The protease activity will be increased or persists constantly when the concentration of anionic surfactants increases (Barberis 2013). The low et al concentration of LAS in water solution (<20%) caused low protease activity, whereas in the high concentration caused enzyme denaturation. (>45%) The highest compatibility of the alkaline protease from A flavus. DUCC-K225 shown in S detergent which is contained , 20% LAS as surfactant with the activity retained 9 . % and 9 . % at 29 C and 55 C 9 48 8 05 o o respectively The ability of alkaline protease of . . A flavus DUCC-K225 in removing blood stains on cloth indicates that this enzyme has the potential to be used in detergent formulas (Tambekar & Tambekar 2013). Shahid & Ahmed 2016 roteases capable ( ) stated that p in removing proteinic stains such as blood, often used in various detergent industries. The alkaline protease of A flavus. DUCC-K225 showed highest compatibility to S detergent, hence the A flavus. 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