Characterization and molecular study of alkaline phosphatase produced from local isolates of bacillus genus and immobilize the enzyme with different methods

العناوين الأخرى

دراسة توصيفية و جزيئية لإنزيم الفوسفاتيز القاعدي المنتج من عزلات محلية لجنس Bacillus وتقييد الإنزيم بطرائق مختلفة

مقدم أطروحة جامعية

al-Hilli, Hasan Majid Rashid

مشرف أطروحة جامعية

Hassan, Shadha Salman

الجامعة

جامعة بغداد

الكلية

كلية العلوم

القسم الأكاديمي

قسم علوم الحياة

دولة الجامعة

العراق

الدرجة العلمية

دكتوراه

تاريخ الدرجة العلمية

2014

الملخص الإنجليزي

Twenty one bacterial isolates were obtained from food (9 isolates), soil (9 isolates) and water (3 isolates) , they were identified by morphological and biochemical tests , all of them were belong to the genus Bacillus.

Bacillus FH4 isolate(from potato) was determined as the highest producer for alkaline phosphatase (1.433 U/ml).

Bacillus FH4 isolate was identified by 16s rRNA , the phylogenetic tree was done by MEGA5 program.

The isolate was closet to Bacillus lichenifrmis, the bacterial strain was submitted to NCBI and it dependent as Bacillus licheniformis FH4.IRQ, with accession number KF531930.

Effect of cobalt chloride was studied, no effect was determined on the productivity.

The optimum conditions for alkaline phosphatase production on PB ALP medium were determined as : incubation temperature 45-50 C, pH 9, inoculum age and size 24 hr and 4% respectively, arabinose 3%, maltose 2% and 3% , cellobiose 1% and 2% , gave high rate of alkaline phosphatase productivity , they used as carbon source.

Aspartic acid , serine and histidine showed complete inhibition on Bacillus licheniformis FH4,IRQ growth .

FeCl3 and ZnSO4 activate alkaline phosphatase production while Cd , FeSO4 , Hg , Mn , EDTA , and CuCl2 inhibited the alkaline phosphatase production .

Alkaline phosphatase was produced after 18 hr of inoculation and it increased during the beginning of stationary phase .

Alkaline phosphatase was precipitated by ammonium sulphate (60%-95% saturation ) and purified using DEAE Sepharose CL6B ion exchange and Sephacryl S 300 gel filtration , 34.85% of total enzyme was recovered with specific activity 7.066 U/mg and 71.59 folds of purification .

Alkaline phosphatase was characterized , Mwt was determined by PAGE and gel filtration , it ranged between 117400 to 118500 Dalton and it composed of two identical subunits (58.748 Dalton each) .

PI was 4.85±0.1 , maximum activity was at pH 10 and temperature 70 C .

The enzyme was activated by Ca(0.1 mM ) and Co(1 mM) and inhibited by Hg , Cd , Mn and EDTA(0.1 , 1 , 5 and 10)mM .

Km of alkaline phosphatase was 0.5426 mM for pNPP and Vmax was 0.1075μmol/min .

Mass spectrometry result indicated that the enzyme was more closet to alkaline phosphatase PhoB of Bacillus licheniformis 9945A , the peptide segments covered 47% of the original peptide chains of alkaline phosphatase PhoB of Bacillus licheniformis 9945A .

Gene sequencing of alkaline phosphatase gene was achieved and submitted to NCBI that depended it now as alkaline phosphatase gene sequence of Bacillus licheniformis FH4.IRQ with accession number KF531931.

Alkaline phosphatase was immobilized by entrapment method in gelatin gel , PVC and calcium alginate , the enzyme immobilized by Ca-alginate was more efficient , it can be reused for 11 times with 57% of remaining activity .

The properties of immobilized ALP were determined , the maximum activity was obtained at pH 10 and temperature 70-80 C , it was more stable when treated with Mn and Cd.

Km of immobilized alkaline phosphatase decreased to 0.138 mM for pNPP and Vmax was 0.0465 μmol/min .

التخصصات الرئيسية

الأحياء

الموضوعات

• الأنزيمات
• البروتينات

عدد الصفحات

181

قائمة المحتويات

• APA
• MLA
• AMA

لغة النص

الإنجليزية

نوع البيانات

رسائل جامعية

رقم السجل

BIM-599004