TR201809310A2 - BIOINTECTIC COMPOSITIONS - Google Patents
BIOINTECTIC COMPOSITIONS Download PDFInfo
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- TR201809310A2 TR201809310A2 TR2018/09310A TR201809310A TR201809310A2 TR 201809310 A2 TR201809310 A2 TR 201809310A2 TR 2018/09310 A TR2018/09310 A TR 2018/09310A TR 201809310 A TR201809310 A TR 201809310A TR 201809310 A2 TR201809310 A2 TR 201809310A2
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- subtilis
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- amylase
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Abstract
Buluş, Bacillus subtilis subtilis, Bacillus thrungiensis, Bacillus cereus bakterilerinin nişasta parçalayan enzim üretiminde kullanımı ve buna bağlı olarak, söz konusu bakteri türlerini içeren biyoinsektisit formulasyonu ile ilgilidir. Şekil 1The invention relates to the use of Bacillus subtilis subtilis, Bacillus thrungiensis, Bacillus cereus bacteria in the production of starch-degrading enzymes, and hence the formulation of bioinsecticides containing said bacterial species. Figure 1
Description
TEKNIK ALAN Bulus, kimya, ziraat, biyoteknoloji gibi farkli alanlarda kullanilmak üzere, nisasta parçalayan enzim üreten mikroorganizma türlerinin saptanmasi ve söz konusu mikroorganizmalarin biyoinsektisit formülasyonu içerisinde kullanimi ile ilgilidir. TECHNICAL FIELD Invention, starch to be used in different fields such as chemistry, agriculture, biotechnology Detection of microorganism species that produce degrading enzymes and It is related to the use of microorganisms in the formulation of bioinsecticide.
Bulus özellikle, Bacillus subtilis subtilis, Bacillus thrungiensis, Bacillus cereus bakterilerinin nisasta parçalayan enzim üretiminde kullanimi ve buna bagli olarak, söz konusu bakteri türlerini içeren biyoinsektisit formulasyonu ile ilgilidir. In particular, the invention includes Bacillus subtilis subtilis, Bacillus thrungiensis, Bacillus cereus. the use of bacteria in the production of starch-degrading enzymes and, accordingly, the It is related to the formulation of bioinsecticide containing bacterial species.
TEKNIGIN BILINEN DURUMU Günümüzde, özellikle tip, eczacilik, tekstil, tarim, deterjan gibi birçok endüstride endüstriyel enzimler kullanilmaktadir. Ihtiyaç duyulan söz konusu enzimleri üretmek üzere yapilan çalismalar son yillarda hiz kazanmis durumdadir. Lipaz, pektinaz, selüloz, amilaz gibi endüstriyel olarak önem tasiyan enzimleri üretmek amaci ile ilgili teknik alanda çesitli mikroorganizmalar kullanilmaktadir. Mikroorganizmalardan enzim üretim süreçlerinde, en efektik üretimi saglayacak mikroorganizmalarin saptanmasi yüksek önem tasimaktadir. Elde edilen enzimler, birçok endüstride kullanilmaktadir. KNOWN STATE OF THE TECHNIQUE Today, it is used in many industries such as medicine, pharmacy, textile, agriculture, detergent. industrial enzymes are used. To produce the enzymes in question Studies on the subject have gained momentum in recent years. lipase, pectinase, related to the production of industrially important enzymes such as cellulose and amylase. Various microorganisms are used in the technical field. Enzyme from microorganisms Determination of microorganisms that will provide the most effective production in production processes is of high importance. The obtained enzymes are used in many industries.
Amilaz da dahil nisasta parçalayan enzimler, nisastayi parçalayarak ve gidalarin kalitesini iyilestirmekte ve totalde amilaz enzimleri, dünya enzim tüketiminin yaklasik % 'unu olusturmaktadir. Bu anlamda, bakteri, maya ve mantarlari kapsayan çesitli mikroorganizmalar sanayide kullanilabilecek enzimleri ve sekonder metabolitleri üretebilme yetenegine sahiptirler. Üretilen enzim ve sekonder metabolitlerin üretim süreçlerinin kalitesinin ve performansin arttirilmasi, ekonomik üretim parametrelerinin belirlenmesi ve optimize edilmesi amaciyla yeni mikroorganizma izolatlarinin saptanmasi bir gereklilik olarak karsimiza çikmaktadir. Starch-degrading enzymes, including amylase, break down starch and make food It improves the quality of the amylase enzymes and in total, approximately % of the world enzyme consumption. constitutes it. In this sense, it covers various types of bacteria, yeast and fungi. microorganisms, enzymes and secondary metabolites that can be used in industry they are capable of producing. Production of enzymes and secondary metabolites produced increasing the quality and performance of the processes, economic production parameters new microorganism isolates in order to identify and optimize detection appears as a necessity.
Ayrica, yapilan patent ve literatür arastirmalarinda, biyoinsektisit olarak kullanilma özelliginde çok kisitli sadece birkaç mikroorganizma türünün kesfedilip raporlarndigi tespit edilmistir. In addition, it has been used as a bioinsecticide in the patent and literature researches. that only a few microorganism species with very limited characteristics have been discovered and reported. has been detected.
Sonuç olarak yukarida bahsedilen olumsuzluklardan ve eksikliklerden dolayi, ilgili teknik alanda bir yenilik ve gelistirme yapma ihtiyaci ortaya çikmistir. As a result, due to the above-mentioned negativities and deficiencies, the relevant There is a need for innovation and development in the technical field.
BULUSUN AMACI Mevcut bulus, yukarida bahsedilen gereksinimleri karsilayan, tüm dezavantajlari ortadan kaldiran ve ilave bazi avantajlar getiren, nisasta parçalayan enzim üretilmesini saglayan mikroorganizmalar ve bu mikroorganizmalari içeren biyoinsektisit formülasyon ile ilgilidir. OBJECTIVE OF THE INVENTION The present invention fulfills the above-mentioned requirements, has all the disadvantages production of enzymes that break down starch, which eliminates starch and brings some additional advantages. microorganisms and bioinsecticide containing these microorganisms relates to the formulation.
Bulusun ana amaci, nisasta parçalayan enzim üretmek üzere bakteri türlerinin saptanmasi, bu sayede ekstraselüler olarak endüstriyel enzim üretilmesini saglamaktir. The main object of the invention is to determine bacterial strains to produce starch-degrading enzymes. detection, thereby ensuring the production of industrial enzymes extracellularly.
Bulusun bir diger amaci, biyoinsektisit olarak kullanilabilecek bir formülasyon elde etmektir. Another object of the invention is to obtain a formulation that can be used as a bioinsecticide. is to do.
Bahsedilen amaçlari yerine getirmek üzere, Bacillus subtilis subtilis, Bacillus thrungiensis, Bacillus cereus bakterilerinin nisasta parçalayan enzim üretiminde kullanimi ile ilgilidir. To fulfill the aforementioned purposes, Bacillus subtilis subtilis, Bacillus thrungiensis, in the production of starch-degrading enzymes by Bacillus cereus bacteria relates to its use.
Bahsedilen amaçlari yerine getirmek üzere, bir biyoinstektisik formülasyonu olup, Bacillus subtilis subtilis ve / veya Bacillus thrungiensis ve / veya Bacillus cereus bakterileri ve en az bir yardimci madde içermektedir. It is a bioinsecticidal formulation to fulfill the mentioned purposes, Bacillus subtilis subtilis and/or Bacillus thrungiensis and/or Bacillus cereus bacteria and at least one excipient.
Bulusun yapisal ve karakteristik 'özellikleri ve tüm avantajlari asagida verilen sekiller ve bu sekillere atiflar yapilmak suretiyle yazilan detayli açiklama sayesinde daha net olarak anlasilacaktir ve bu nedenle degerlendirmenin de bu sekiller ve detayli açiklama göz 'Önüne alinarak yapilmasi gerekmektedir. The structural and characteristic features and all advantages of the invention are given in the following figures and It is clearer thanks to the detailed explanation written by making references to these figures. will be understood as such, and therefore the assessment will also include these figures and It should be done with consideration.
BULUSUN ANLASILMASINA YARDIMCI OLACAK SEKILLER Sekil 1'de bakteri izolatlarina ait (izolat no. 1, 2 ve 3) 168 rDNA PCR ürünlerinin elektroforezi bantlari verilmektedir(Tek bant 168 rDNA PCR ürünlerinin elektroforezi. FIGURES TO HELP UNDERSTAND THE INVENTION Figure 1 shows 168 rDNA PCR products of bacterial isolates (isolates 1, 2 and 3). Electrophoresis bands are given (Electrophoresis of single band 168 rDNA PCR products.
M: Marker; B. cereus (A), B. subtilis subtilis (B), B. thuringiensis (C). Ok: 168 rDNA bantlari). M: Marker; B. cereus (A), B. subtilis subtilis (B), B. thuringiensis (C). Arrow: 168 rDNA bands).
Sekil 2'de, B. subtilis subtilis, B. thuringiensis ve B. cereus'un hareketsiz ve serbest kültür kosullarindaki amilaz üretim sonuçlari verilmektedir(B. subtilis subtilis (A), B. thuringiensis (B) ve B. cereus (C) ile immobilize (I) ve serbest (A) kosullarda amilaz üretimi.) Sekil 3'de, Bacillus izolatlarinin ürettigi toplam protein seviyelerini gösteren grafikler verilmektedir( B. subtilis subtilis (A), B. thuringiensis (B) ve B. cereus (C) ile immobilize (I) ve serbest (A) kosullarda toplam protein üretimi). Figure 2 shows the dormant and free strains of B. subtilis subtilis, B. thuringiensis, and B. cereus. amylase production results in culture conditions are given (B. subtilis subtilis (A), B. amylase in immobilized (I) and free (A) conditions with thuringiensis (B) and B. cereus (C) production.) Figure 3 graphs showing the total protein levels produced by Bacillus isolates. (Immobilized with B. subtilis subtilis (A), B. thuringiensis (B) and B. cereus (C) Total protein production in (I) and free (A) conditions).
Sekil 4'te, biyoreaktörde 9 operasyon günü boyunca amilaz aktivitesi ve toplam protein seviyeler verilmektedir( Bir pilot-ölçekli biyoreaktörde B. subtilis subtilis kullanilarak (C) amilaz (A) ve toplam protein üretimi (B).). Figure 4 shows amylase activity and total protein over 9 operating days in the bioreactor. levels are given (using B. subtilis subtilis in a pilot-scale bioreactor (C) amylase (A) and total protein production (B).
Sekil 5'de B. subtilis subtilis tarafindan bir karbon kaynagi olarak melaz varliginda amilaz ve toplam protein üretimi verilmektedir(B. subtilis subtilis tarafindan bir karbon kaynagi olarak melaz varliginda Amilaz (A) ve toplam protein (B) üretimi.) BULUSUN DETAYLI AÇIKLAMASI Bu detayli açiklamada, bulusun tercih edilen yapilanmalari, sadece konunun daha iyi anlasilmasina yönelik olarak ve hiçbir sinirlayici etki olusturmayacak sekilde açiklanmaktadir. Figure 5 in the presence of melase as a carbon source by B. subtilis subtilis amylase and total protein production are given (by B. subtilis subtilis one carbon Production of Amylase (A) and total protein (B) in the presence of melase as a source.) DETAILED DESCRIPTION OF THE INVENTION In this detailed description, preferred embodiments of the invention are merely better suited to the subject. for understanding and without any limiting effect is explained.
Bulus, Bacillus subtilis subtilis, Bacillus thuringiensis ve Bacillus cereus mikroorganizmalarinin nisasta parçalayici enzim (tercihen amilaz) üretmek üzere kullaniminin konu almaktadir. Söz konusu mikroorganizmalardan, nisasta parçalayici enzim üretilmesi, bu mikroorganizmalarin endüstriyel amaçli biyoinsektisit formulasyonu eldesinde kullanimina da imkan vermektedir. Nisasta parçalayan enzim üretiminde ve biyoinsektisit formulasyonunda kullanilan söz konusu mikroorganizmalar (Bacillus subtilis subtilis, Bacillus thuringiensis ve Bacillus cereus) genetigi degistirilmemis dogal izolatlardir. Söz konusu mikroorganizma türleri, dogal toprak kaynagindan izole edilerek ve moleküler teknikler kullanilarak tanimlanmistir. Invention, Bacillus subtilis subtilis, Bacillus thuringiensis and Bacillus cereus microorganisms to produce starch-degrading enzyme (preferably amylase) subject of use. Starch-degrading micro-organisms production of enzymes, the use of these microorganisms as an industrial-purpose bioinsecticide It also allows its use in obtaining the formulation. enzyme that breaks down starch These microorganisms used in the production and formulation of bioinsecticide (Bacillus subtilis subtilis, Bacillus thuringiensis and Bacillus cereus) genetics are unmodified natural isolates. Microorganism species in question, natural soil was isolated from its source and identified using molecular techniques.
Tanimlanan türlerim ekstraselüler enzim (amilaz) üretimleri spektrofotometrik olarak ölçülerek karsilastirilmistir. Bacillus subtilis subtilis izolatinin daha yüksek amilaz aktivitesi oldugu saptanmistir. The extracellular enzyme (amylase) production of the described species was determined spectrophotometrically. measured and compared. Higher amylase level of Bacillus subtilis subtilis isolate activity was detected.
Detayli açiklamanin bundan sonraki kisminda, söz konusu mikroorganizmalarin nasil saptandigi, yapilan testler, söz konusu test çiktilari ve biyoinsektisit formülasyonu açiklanmaktadir. Söz konusu açiklamalar ile herhangi bir kisitlamaya gidilmeksizin, bulusun anlasilir olmasi amaçlanmaktadir. Detayli açiklama boyunca, Bacillus subtilis subtilis; sus no 1 olarak, Bacillus thuringiensis; sus no 2 olarak, Bacillus cereus ise sus no 3 olarak isimlendirilmektedir. In the next part of the detailed explanation, how the microorganisms in question are determined. determined, the tests performed, the test outputs in question and the bioinsecticide formulation is explained. Without any restrictions with the said explanations, The invention is intended to be understandable. Throughout the detailed description, Bacillus subtilis subtilis; as sus no 1, Bacillus thuringiensis; sus no. 2, Bacillus cereus as sus It's called number 3.
Söz konusu mikroorganizama türlerinin saptanmasi için öncellikle Bacillus subtilis subtilis (Sus no. 1), Bacillus thrungiensis (Sus. N0.2), Bacillus cereus (Sus No. 3) bakterilerinin izolasyonu ve Luria Bertani (LB) besiyeri ortaminin hazirligi yapilmaktadir. Kültür malzemelerinin de hazirlanmasinin ardindan, inokulasyon islemi ve söz konusu kültürün, tek karbon kaynagi olarak kullanilan melaz içeren kosullarda büyütülmesi (inkübasyon) islemleri uygulanmaktadir. Kültürün santrifüje tabii tutulmasinin ardindan, ekstraselüler olarak amilaz üretimi saptanmaktadir. Söz konusu yöntem detayli olarak asagida açiklanmaktadir. In order to determine the microorganism species in question, first of all, Bacillus subtilis subtilis (Sus no. 1), Bacillus thrungiensis (Sus. N0.2), Bacillus cereus (Sus No. 3) isolation of bacteria and preparation of Luria Bertani (LB) medium is being done. After the preparation of the culture materials, the inoculation process and the culture in question in conditions containing melase used as the sole carbon source. growth (incubation) procedures are applied. Centrifugation of culture After retention, extracellular amylase production is detected. Aforementioned The method is described in detail below.
Sus no 1, Sus no 2 ve Sus no 3 izolatlarini elde etmek üzere çesitli toprak örnekleri temin edilmektedir. Bulusta, sus no 1 ve sus no 2 içeren toprak örnegi; Istanbul Altunizade parkindan, sus no 3 içeren toprak örnegi ise Istanbul Bostanci sahilinden alinmistir. Söz konusu toprak örnekleri, içerisinde 200 uL steril ultra saf su bulunan mikrosantrifüj tüplerine ilave edilip, homojen bir süspansiyon olusturmak için vortekslenmistir. Söz konusu süspansiyon, 10 dakika boyunca 80 ° C'de ayarlanmis bir isi blogu içinde inkübe edilmistir. Various soil samples were used to obtain Sus no 1, Sus no 2 and Sus no 3 isolates. is provided. In the invention, soil sample containing sus no 1 and sus no 2; Istanbul The soil sample containing sus no 3 is from the Altunizade park, and the soil sample is from the Bostanci coast of Istanbul. has been taken. The soil samples in question were contained in 200 µL sterile ultrapure water. to be added to microcentrifuge tubes to form a homogeneous suspension. vortexed. Said suspension was prepared at 80 °C for 10 min. incubated in the heat block.
Tüm bakteri örnekleri, Luria Bertani (LB) agar 'üzerinde büyütülmüs ve 37 °C'de inkübe edilmistir. Söz konusu bakteri örnekleri içerisinden morfolojik olarak farkli koloniler seçilip, gram boyama teknigi uygulanarak gram pozitif ve gram negatif bakterilerin ayirt edilmesi amaçlanmistir. Gram pozitif çubuk seklindeki endospor olusturan 3 sus ayrilarak her biri taze bir LB agar besiyerine inoküle edilmistir. Her bir sus için tek bir koloni seçilmis ve tekrar gram boyama uygulanmistir. Çizgi olusturma ve gram boyama prosedürü 5 kez tekrarlanarak, her sustan saf bir kültür izolesi saglanmistir. Gram boyama ile saf koloni morfolojisine ulasildiktan sonra, bu saf koloni, M9 (% 0.5 nisasta eklenmis) olarak isimlendirilen agar besiyerine ekilmis ve 37 cC'de 2 gün süre ile inkübe edilmistir. 2 günlük bu inkübasyon isleminden sonra, iyot çözeltisi petri yüzeyine dökülmüstür. Bahsedilen petri yüzeyinde, nisastanin amilaz tarafindan bozundugu ve testte pozitif oldugu alanlar saptanmistir. Amilaz-pozitif suslar 60 mL LB besiyeri içeren 100 mL kültür siselerine inoküle edilmis ve optik yogunluk, toplam protein ve amilaz etkinligi için 37 cC'de çalkalamali inkübat'orde inkübe edilmistir. Bakteri izolatlarinin gliserol stoklari sonraki testler için ertesi gün hazirlanmistir. All bacterial samples were grown on Luria Bertani (LB) agar and incubated at 37 °C. has been made. Morphologically different colonies among the aforementioned bacterial samples Distinguish between gram positive and gram negative bacteria by applying gram staining technique. is intended to be. 3 strains forming Gram-positive rod-shaped endospores separated and each inoculated on a fresh LB agar medium. One for each sound Colonies were selected and Gram staining was applied again. Line creation and gram staining By repeating the procedure 5 times, a pure culture isolate was obtained from each strain. Gram After obtaining pure colony morphology by staining, this pure colony was stained with M9 (0.5% starch). inoculated on agar medium called (added) and incubated for 2 days at 37 °C. has been incubated. After this 2-day incubation process, the iodine solution is poured onto the petri surface. spilled. On the said petri dish, starch is degraded by amylase and positive areas were detected in the test. Amylase-positive strains containing 60 mL of LB medium It was inoculated into 100 mL culture flasks and measured for optical density, total protein and amylase. For its effectiveness, it was incubated at 37 °C in a shaken incubator. Bacterial isolates Stocks of glycerol were prepared the next day for further testing.
DNA Izolasyonlari. Bacillus sp., LB besiyerinde büyütülmüs ve 3 farkli kültür izolatina ait DNA izolasyonlari yapilmistir. DNA izolasyonu için 1 mL numune (n = 2) alinmistir. DNA Isolations. Bacillus sp. was grown in LB medium and isolates from 3 different cultures. DNA isolations were made. 1 mL sample (n = 2) was taken for DNA isolation.
DNA, ticari bir DNA izolasyon kiti (Zymo Research Company, Mantar/ Bakteriyel DNA miniprep TM, Katalog No: D kullanilarak ekstre edilmistir. DNA was obtained from a commercial DNA isolation kit (Zymo Research Company, Fungal/Bacterial DNA Extracted using miniprep TM, Catalog No: D.
Izole edilen DNA numuneleri -20 °C'de saklanmistir ve bu izole numunelerin Polimeraz Zincir Reaksiyonu (PCR) analizi ve DNA dizi analizleri yapilmistir. PCR, her bir primer ve 1 uL dNTP, ve 5 uL DNA'nin bir kalip olarak 20 uL toplam PCR hacmi içinde bulundugu 1 pL karisimdan olusmaktadir. Reaksiyonlar için bir termal döngüleyici (BIO-RAD T100, Singapur) kullanilmistir. Bu islem için, Medsantek firmasindan (Istanbul, Türkiye) temin edilen ileri CGG WGT GTA CAA GGC-39) kullanilmistir. PCR ürünleri -20 °C'de saklanmis olup, PCR ürünleri, Genetic Analyzer kullanilarak dizilenmis ve sonuçlar, Chromas Software (sürüm 2.6.2) kullanilarak degerlendirilmistir. The isolated DNA samples were stored at -20 °C and the Polymerase Chain Reaction (PCR) analysis and DNA sequence analysis were performed. PCR, each primer and 1 µL of dNTP, and 5 µL From 1 pL of mixture with DNA as a template in 20 µL total PCR volume is formed. A thermal cycler for the reactions (BIO-RAD T100, Singapore) used. For this process, advanced technology obtained from Medsantek (Istanbul, Turkey) CGG WGT GTA CAA GGC-39) is used. PCR products were stored at -20 °C, PCR products using Genetic Analyzer sequenced and results evaluated using Chromas Software (version 2.6.2).
Bahsedilen test süreçlerinde, amilaz etkinligi ve mikrobiyal büyüme degerlendirmesi su sekilde yapilmistir. Adapazari Seker Fabrikasindan (Adapazari, Türkiye) temin edilen melaz temin edilerek, söz konusu melaz içeren (6.7 g/L ve 13.4 g/L) modifiye edilen besiyeri kullanilmistir. Besiyeri ortami MgSO4.7H20 (1.0 g/L), NaCI (10 g/L), CaClz (2 g/L) ve KH2PO4 (2 g/L) bulundurmaktadir ve karbon kaynagi olarak sadece melaz içermektedir. In the mentioned test processes, the evaluation of amylase activity and microbial growth was carried out. has been made in this way. Procured from Adapazari Sugar Factory (Adapazari, Turkey) by supplying the molasses, modified with said molasses (6.7 g/L and 13.4 g/L) medium was used. Medium medium MgSO4.7H2O (1.0 g/L), NaCl (10 g/L), CaClz (2 g/L) and KH2PO4 (2 g/L) and only molasses as a carbon source. contains.
Mikroorganizma izolatlarin immobilizasyonu için söz konusu mikroorganizmalara sodyum aljinat immobilizasyonu uygulanmistir. % 3 sodyum aljinat otoklav (Nuve, OT90L, Türkiye) kullanilarak 121 ° C'de 15 dakika süreyle sterilize edilmis ve daha önce LB ortaminda yetistirilen üç izolat aljinat çözeltisi ile karistirilmistir. izolatlarin kapsüllenmesi için 250 mM steril CaClz çözeltisi kullanilmistir. Aljinat boncuklar bir çalkalama sisesindeki sterilize edilmis suya aseptik kosullar altinda aktarilmis ve 150 rpm'de oda sicakliginda çalkalanmistir. Tüm deneyler, bir Laminar hava akisli kabinde (Thermo Fisher Scientific, ABD) aseptik kosullar altinda yürütülmüstür. For the immobilization of microorganism isolates, the microorganisms in question sodium alginate immobilization was applied. 3% sodium alginate autoclave (Nuve, OT90L, Turkey) for 15 minutes at 121 °C and then First, three isolates grown in LB medium were mixed with alginate solution. your isolates 250 mM sterile CaClz solution was used for encapsulation. alginate beads a It was transferred to sterilized water in the rinsing bottle under aseptic conditions and 150 shaken at room temperature at rpm. All experiments were carried out in a Laminar air flow cabinet. (Thermo Fisher Scientific, USA) was conducted under aseptic conditions.
Amilaz üreten izolatlar (izolat no. 1, 2 ve 3), LB besiyerine inoküle edilmis ve 150 rpm'de çalkalayici inkübatör (Daihan inkübatör, G. Kore) içinde 37 cC'de büyütülmüstür. Kültür ortaminin optik yogunluklari ölçülmüs ve 1.5 mL besiyeri örnekleri, 10.000 rpm'de 5 dakika boyunca santrifüje tabi tutulmustur. Biyokimyasal analizler için UV-görünür bir spektrofotometre (Shimadzu, UV-vis 2600, Japonya) kullanilmistir. Toplam protein seviyeleri standart olarak sigir serum albümini kullanilarak ölçülmüstür. Amilaz aktivitesi spektrofotometrik olarak ölçülmüstür. Amilaz etkinligi (U/ml) asagidaki formül kullanilarak hesaplanmistir. Buna göre; denklemi olusturulmustur. Burada bahsedilen A620 kontrolü, enzim eklenmeden nisastadan elde edilen absorbansdir. A620 numunesi ise, enzim ile sindirilmis nisasta absorbansini tanimlamaktadir; A620 / mg nisasta, standart egriden türetilen 1 mg nisasta için absorbanstir, 5 dakika bekleme süresini ve 0.1 mL deneyde kullanilan örnek hacmini ifade etmektedir. Amylase-producing isolates (isolates no. 1, 2, and 3) were inoculated into LB medium and 150 rpm at 37 cC in a shaker incubator (Daihan incubator, S. Korea) is enlarged. Optical densities of the culture medium were measured and 1.5 mL of medium was added. The samples were centrifuged at 10,000 rpm for 5 minutes. Biochemical a UV-visible spectrophotometer for analyzes (Shimadzu, UV-vis 2600, Japan) used. Total protein levels were standardized with bovine serum albumin. measured using. Amylase activity was measured spectrophotometrically. Amylase efficacy (U/ml) was calculated using the formula below. According to this; equation is created. The A620 control mentioned here is without the addition of enzyme. is the absorbance obtained from starch. The A620 sample is enzyme digested starch. defines its absorbance; A620 / mg starch, 1 mg derived from standard curve is the absorbance for starch, the 5 min dwell time and the 0.1 mL used in the experiment. represents the sample size.
Tablo 1'de izole edilen dogal bakterilerin moleküler tanimlama sonuçlari gösterilmistir. Molecular identification results of isolated natural bacteria are shown in Table 1.
Söz konusu tabloda, sekans benzerlik oranlari ve erisim numaralari yer almaktadir. The table in question contains sequence similarity rates and access numbers.
Sekans benzerlik oranlari dikkate alindiginda, söz konusu mikroorganizmalarin en yüksek benzerlik yüzdesine sahip olduklari görülmektedir. Hagström ve arkadaslarina ait makalede (2000), 168 rDNA sekansi benzerliginin, bir izolatin filogenetik iliskisini belirttigi ve benzerlik degeri % 97'den yüksek olanlarin ayni tür olabilecegi ifade edilmektedir. Buna göre, Tablo 1rdeki sonuçlar, saptanan izolatlarin (izolat no. 1, 2 ve 3) amilaz enzimini ürettigini göstermistir. Considering the sequence similarity ratios, it is seen that the microorganisms in question are the most It is seen that they have a high similarity percentage. To Hagström and friends (2000), 168 rDNA sequence similarity, an isolate phylogenetic relationship It is stated that the ones with a similarity value higher than 97% may be the same species. is being done. Accordingly, the results in Table 1 show that the detected isolates (isolates no. 1, 2 and 3) showed that it produced the enzyme amylase.
Sus No. Tür Sekans Benzerlik (%) GenBank Erisim No. Hush No. Species Sequence Similarity (%) GenBank Access No.
Tablo 1. Izole edilen dogal bakterilerin moleküler tanimlama sonuçlari. Table 1. Molecular identification results of isolated natural bacteria.
Sekil 1'de bakteri izolatlarina ait (izolat no. 1, 2 ve 3) 168 rDNA PCR ürünlerinin elektroforezi bantlari verilmektedir. Söz konusu sekilde; Marker M ile, B. cereus A ile, B. subtilis subtilis B ile, B. thuringiensis C ile gösterilmektedir. Sagdan sola gösterimli ok ise 168 rDNA bantlarini göstermektedir. Figure 1 shows 168 rDNA PCR products of bacterial isolates (isolates 1, 2 and 3). electrophoresis bands are given. In the said section; with Marker M, with B. cereus A, B. subtilis is indicated by subtilis B, B. thuringiensis by C. Right-to-left display arrow indicates 168 rDNA bands.
Sekil 2, B. subtilis subtilis, B. thuringiensis ve B. cereus'un hareketsiz ve serbest kültür kosullarindaki amilaz üretim sonuçlarini göstermektedir. Söz konusu bakteri izolatlari tarafindan üretilen amilaz üretimi günlük olarak ölçülmüstür. Söz konusu sekile göre, her üç Bacillus izolati amilaz enzimini üretmistir. Her üç bakteri izolatinin kullanildigi tüm deneylerde enzim üretim seviyelerinin benzer oldugu ve yaklasik 10 U / mL amilaz enziminin basariyla üretildigi saptanmistir. Bakteri izolatlarinin immobilizasyonun ise, enzim üretiminde artirici bir etkisinin olmadigi saptanmistir. Figure 2, quiescent and free culture of B. subtilis subtilis, B. thuringiensis and B. cereus shows the results of amylase production under the conditions. The bacterial isolates The production of amylase produced by was measured daily. According to the said figure, All three Bacillus isolates produced the enzyme amylase. All three bacterial isolates were used enzyme production levels were similar in all experiments and approximately 10 U/mL of amylase It was determined that the enzyme was produced successfully. The immobilization of bacterial isolates, It was determined that it did not have an increasing effect on enzyme production.
Sekil 3ide, Bacillus izolatlarinin ürettigi toplam protein seviyelerini gösteren grafikler verilmektedir. Söz konusu grafiklerde, maksimum protein üretimine, B. subtilis subtilis'in kültürünün 3. gününde ulasildigi görülmektedir. B. thuringiensis'in protein üretimi, hareketsiz kosullarda 18 ± 3 mg / L'ye ulasirken, serbest kültür ortaminda ise üretim; mg/L seviyesinde bulunmustur. Bu sonuçlar, üç Bacillus sp. izolatlarinin incelenen kosullar altinda protein ürettigini göstermektedir. In Figure 3, graphs showing the total protein levels produced by Bacillus isolates are given. In the aforementioned graphs, the maximum protein production, B. subtilis subtilis It is seen that the culture is reached on the 3rd day. Protein production of B. thuringiensis, production reached 18 ± 3 mg / L in sedentary conditions, while in free culture medium; It was found at the level of mg/L. These results indicate that three Bacillus sp. examined isolates shows that it produces protein under these conditions.
Bacillus subtilis subtilis izolati, pilot ölçekli bioreaktör kullanilarak nisasta parçalayan enzim (amilaz) üretimi için kullanilmistir. Biyoreaktöre, önceden çalkalama sisesi içinde yetistirilen B. subtilis subtilis inoküle edilmistir. Sekil 4'te, biyoreaktörde 9 operasyon günü boyunca amilaz aktivitesi ve toplam protein seviyeler verilmektedir. Incelenen kosullar altinda, sadece 13.4 9 / L melazin tek karbon kaynagi olarak kullanildigi ortamda B. subtilis subtilis tarafindan yaklasik 8 U / mL amilaz enzimi, 20 mg / L toplam protein, üretilmistir. Bacillus subtilis subtilis isolate, starch degrading using a pilot scale bioreactor used for enzyme (amylase) production. Into the bioreactor in a pre-shake bottle grown B. subtilis subtilis was inoculated. In Figure 4, 9 operations in the bioreactor Amylase activity and total protein levels are given throughout the day. examined conditions, only 13.4 9/L melassin was used as the sole carbon source. approximately 8 U/mL of amylase enzyme, 20 mg/L total by B. subtilis subtilis in medium protein has been produced.
Sekil 5'de B. subtilis subtilis tarafindan bir karbon kaynagi olarak melaz varliginda amilaz ve toplam protein üretimi verilmektedir. Figure 5 in the presence of melase as a carbon source by B. subtilis subtilis amylase and total protein production are given.
Biyoinsektisit formülasyonu: Bulus ayni zamanda, söz konusu mikroorganizmalari içeriginde bulunduran biyoinsektisit formülasyonlarini konu almaktadir. Dogadan toplanan ve izole edilen söz konusu mikroorganizmalar, kültüre edildikren sonra yardimci maddeler ile birlikte formülize edilerek uzun raf ömürlü biyoinsektisit eldesinde kullanilabilmektedir. Ayrica, izole edilip büyütülen bu mikroorganizmalarin izole edilip büyütülerek, IiyofiIizasyon sonrasi toz formunda zirai amaçli olarak kullanimlari da mümkündür.Söz konusu biyoinsektisit formülasyonunda, kütlece esit miktarlarda kültür besiyerinde yetistirilen mikroorganizmalari bulunmaktadir. Söz konusu mikroorganizmalara ek olarak, yardimci madde olarak stabilizatör de biyoinsektisit form'ulasyonu içeriginde yer almaktadir. Gelistirilen bu formülasyon, özellikle içerigindeki mikroorganizmalar tarafindan üretilen enzimler vasitasiyla toprakta polimerik karbonhidratlarin parçalanmasini, monomerik substratlarin olusmasini tesvik ederek, toprak florasinda yarali mikroorganizmalarin zenginlesmesini ve ayrica içerigi ile tarim zararlilarinin etkisizlestirilmesini saglamaktadir. Yardimci madde olarak kullanilan stabilizör; 1 litre biyoinsektisit formülasyonu içerisine, 10 g borik asit, 30 g bugday nisastasi, 10 mL bitkisel gliserin, 10 g benzoik asitten olusmaktadir.Bioinsecticide formulation: The invention also contains the microorganisms in question. deals with bioinsecticide formulations. The word collected and isolated from nature The microorganisms in question, after being cultured, together with the auxiliary substances It can be formulated and used to obtain long shelf-life bioinsecticide. Moreover, By isolating and growing these microorganisms isolated and grown, lyophilization It is also possible to use them for agricultural purposes in powder form after in bioinsecticide formulation, grown in culture medium in equal amounts by mass have microorganisms. In addition to these microorganisms, As an auxiliary substance, the stabilizer is also included in the bioinsecticide formulation. takes. This formulation developed, especially the microorganisms in its content, polymeric carbohydrates in the soil via enzymes produced by in the soil flora, promoting the degradation of monomeric substrates and the formation of monomeric substrates. enrichment of injured microorganisms and also the content of agricultural pests ensures its deactivation. The stabilizer used as an auxiliary substance; 1 liter into the bioinsecticide formulation, 10 g boric acid, 30 g wheat starch, 10 mL vegetable glycerin consists of 10 g of benzoic acid.
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