Isolation and Identification of Bacillus atrophaeus from Root Soil of the Işgın: Obtaining and Characterization of α-Amylase

Abstract

Amylase; enzymes that break down starch produced by plants, animals and microorganisms. Amylases produced by microorganisms; pharmaceutical, food, detergent, textile, paper and pulp industry. However, there are still limitations in the isolation of amylase-producing microorganisms. Işgin plant is an important plant used in many fields, especially in medicine The aim of this study was to isolate the bacteria from the useful plant ışgın and to obtain amylase which is of biotechnological importance. Bacterial isolation was performed using dilution technique. Soil in the root part of the plant was removed and dilution was performed. Morphological, physiological and biochemical tests were performed after isolation.16 S rRNA analysis was performed for species identification of microorganism. Bacterial sequence analysis result detected Bacillus atrophaeus with 864 base pairs. As a result of biochemical tests which were effective in identification, the bacteria were gram positive (+), bacillus and immobile. Catalase, hemolysis and glucose positive (+) results; Biochemical tests such as oxidase, H2S and indole were found to be negative (-).Firstly, bacterial growth was optimized. Important parameters that directly affect bacterial growth such as time, temperature and pH were studied. Optimum bacterial production was determined at 72 hours, 30 oC and pH 6.0, respectively. In order to determine strong amylase production, bacteria were produced from starch solid media. After incubation, it was determined that microorganism produced amylase by using lugol solution. The optimal production conditions of the bacteria determined to be amylase synthesized were determined. The maximum production conditions of α-amylase from Bacillus atrophaeus were 36 hours, 35 oC and pH 6.0. Enzyme characterization was performed using the supernatant portion of the bacteria produced under optimum conditions. Enzyme temperature and pH were found to have maximum activity at 40 oC and pH 6.0, respectively. As a result of these determinations, the desired bacteria were obtained. Bacteria identified and found to produce amylase were determined to be used in different biotechnological areas.

Keywords

• α- Amylase
• Bacillus atrophaeus
• Biotechnology
• Identification

Işgının Kök Toprağından Bacillus atrophaeus’un İzolasyonu ve Tanımlanması: α-Amilaz’ın Elde Edilmesi ve Karakterizasyonu

Öz

Amilazlar; bitkiler, hayvanlar ve mikroorganizmalar tarafından üretilen nişastayı parçalayan enzimlerdir. Mikroorganizmalar tarafından üretilen amilazlar; eczacılık, gıda, deterjan, tekstil, kağıt ve kağıt hamuru endüstrisi gibi çok çeşitli endüstriyel uygulamalarda kullanılabilme özelliğine sahiptir. Bununla birlikte, amilaz üreten mikroorganizmaların izolasyonunda hala kısıtlamalar vardır. Işgın bitkisi tıp başta olmak üzere birçok alanda kullanılan önemli bir bitkidir. Bu çalışmanın amacı yararlı ışgın bitkisinden bakterinin izolasyonu gerçekleştirerek biyoteknolojik öneme sahip olan amilazın elde edilmesidir. Bakteri izolasyonu dilüsyon tekniği kullanılarak yapıldı. Bitki kök kısmında bulunan toprak alınarak seyreltme işlemi gerçekleştirildi. İzolasyon sonrası morfolojik, fizyolojik ve biyokimyasal testler yapıldı. Mikroorganizmanın tür teşhisi için 16 S rRNA analizi gerçekleştirildi. Bakteri sekans analiz sonucunda 864 baz çiftine sahip Bacillus atrophaeus olduğu tespit edildi. Tanımlamada etkili olan biyokimyasal testler sonucunda bakterinin gram pozitif (+), basil ve hareketsiz olduğu belirlendi. Katalaz, hemoliz ve glikoz un pozitif (+) sonuç verdiği; oksidaz, H2S ve indol gibi biyokimyasal testlerin negatif (-) olduğu tespit edildi. Öncelikle bakteri üremesinin optimizasyonu sağlandı. Zaman, sıcaklık ve pH gibi bakteri üremesine doğrudan etki eden önemli parametreler çalışıldı. Optimum bakteri üretimi sırasıyla 72.saat, 30 oC ve pH 6.0 belirlendi. Güçlü amilaz üretimini belirlemek amacıyla nişastalı katı besi yerinde bakteriler üretildi. İnkübasyon sonrası lügol çözeltisi kullanılarak mikroorganizmanın amilaz ürettiği tespit edildi. Amilaz sentezlendiği belirlenen bakterinin optimal üretim koşulları tespit edildi. Bacillus atrophaeus’tan α-amilaz’ın maksimum üretim koşulları 36. saat, 35 oC ve pH 6.0 olduğu görüldü. Optimum şartlarda üretilen bakterinin süpernatant kısmı kullanılarak enzim karakterizasyonu gerçekleştirildi. Enzim sıcaklık ve pH’sı sırasıyla 40 oC ve pH 6.0 da maksimum aktivite gösterdiği tespit edildi. Bu tespitler sonucunda istenilen bakteri elde edildi. Tanımlanan ve amilaz ürettiği tespit edilen bakteri, farklı biyoteknolojik alanlarda kullanılma özelliğine sahip olduğu belirlendi.

Anahtar Kelimeler

• α-Amilaz
• Bacillus atrophaeus
• Biyoteknoloji
• Tanımlama

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