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أمثلة إنتاج إنزيم غلوكوز إيزوميراز Glucose Isomerase من عزالت محلية

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أمثلة إنتاج إنزيم غلوكوز إيزوميراز Glucose Isomerase من عزالت محلية وزارة التعليم العالي جامعة دمشق وزارة التعليم العالي كلية الزراعة الهيئة العامة للتقانة الحيوية قسم علوم اﻷغذية أمثلة إنتاج إنزيم غلوكوز إيزوميراز Glucose isomerase من عزﻻت محلية من بكتريا.Streptomyces spp وتحسين إنتاجه باستخدام بعض الطرائق الوراثية Optimization of Glucose isomerase production from local isolates of Streptomyces spp. and improvement of its production using some genetic methods رسالة أُ ِع دت لنيل درجة الدكتوراه في علوم اﻷغذية إعــــــــداد م . سهى عيسى حبيب إشـــــــــــراف أ. د. صباح يازجي د. لينه اﻷمير أستاذ في قسم علوم اﻷغذية أستاذ مساعد في التقانة الحيوية Ministry of Higher Education Ministry of Higher Education National Commision Damascus University for Biotechnology College of Agriculture Department of Food Science Optimization of Glucose isomerase production from local isolates of Streptomyces spp. and improvement its production using some genetic methods A THESIS Submitted in partial fulfillment of the requirements for the Ph. D. degree Of Food Science Section Prepared by Suha Issa Habeeb (National Commission for Biotechnology) Supervision Supervisor Co-Supervisor Dr. Sabah Yazajy Dr. Lina Al-Amir Prof. Of Food Science Assistant Researcher Faculty of Agriculture National Commission for Damascus University Biotechnology المحتويات الموضوع رقم الصفحة 1- المقدمة ..........................................................................1 1-1- أهداف البحث .................................................................2 2 - الدراسة المرجعية .................................................................4 2 -1- كيمياء اﻹنزيمات ...............................................................4 2-2- لمحة تاريخية عن اﻹنزيم ........................................................7 2-3- تسمية اﻹنزيمات ................................................................9 2-4- المماكبة الكيميائية واﻻنزيمية ...................................................11 2-5- طبيعة إنزيم GI وتركيبه .......................................................12 2-6- آلية عمل إنزيم GI .............................................................14 2-7- مصادر إنزيم GI ...............................................................16 2-8- إنتاج GI باستخدام الجنس Streptomyces .....................................19 2-8-1- مجموعة اﻷكتينومايسيتات .....................................................19 2-8-1-1- جنس Streptomyces.....................................................20 2-8-1-1-1- موطن Streptomyces.................................................21 2-8-1-1-2- دورة حياة Streptomyces..............................................22 2-9- العزل ...........................................................................23 2-10- الغربلة ..........................................................................24 2-10-1- الغربلة اﻷولية ...............................................................25 2- 10-2- الغربلة الثانوية .............................................................25 2-11- التشخيص ......................................................................25 2-12- استخﻻص اﻹنزيم ...............................................................26 2-13- تقدير الفعالية اﻹنزيمية 28........................................................... 2-13-1- طريقة (Dische and Borenfreund (1951 ..............................28 2-13-2- طريقة (Kulka (1956 .....................................................29 2-13-3- طريقة الفحص المناعي اﻹنزيمي )ELISA( .................................29 2-14- العوامل المؤثرة في إنتاج إنزيم GI ..............................................30 2-14-1- تأثير درجة الحرارة ...........................................................30 2-14-1-1- الحرارة المثلى لنشاط اﻹنزيم..............................................30 2-14-1-2- درجة حرارة حفظ اﻹنزيم..................................................30 2-14-1-3- درجة حرارة إنتاج اﻹنزيم..................................................31 2-14-2- تأثير الرقم الهيدروجيني اﻻبتدائي .............................................31 2-14-3- تأثير زمن التخمير)مدة التحضين( ............................................32 2-14-4- تأثير نسبة الزيلوز ............. ..……………….…………..………33 2-14-5- تأثير اﻷمﻻح والعناصر المعدنية .............................................35 2-15- تحسين إنتاجية اﻹنزيمات باستخدام التطفير للكائنات الدقيقة .......................36 2-16- فصل وتنقية اﻷنزيم .............................................................38 2-17- الصفات الحركية لعمل إنزيم GI.................................................42 2-17-1- تأثير درجة الحرارة في فعالية اﻹنزيم..........................................42 2-17-2- تأثير درجة الحرارة في ثباتية اﻹنزيم............................................42 2-17-3- تأثير الرقم الهيدروجيني في فعالية اﻹنزيم........................................44 2-17-4- تأثير الرقم الهيدروجيني في ثباتية اﻹنزيم........................................44 2-18- اﻷهمية التطبيقية ﻹنزيم GI.....................................................45 3 - مواد البحث وطرائقه ...............................................................49 3-1- اﻷجهزة والمواد المستخدمة في الدراسة ............................................49 3-1-1- اﻷجهزة.........................................................................49 3-1-2- المواد الكيميائية المستخدمة ....................................................50 3-1- 3- اﻷوساط الزرعية .............................................................50 3-1-3-1- وسط العزل (Gauza) ......................................................50 3-1-3-2 وسط اﻷساس الصلب )وسط إنتاج اﻹنزيم(...................................51 3-1-3-3- وسط اﻹنتاج السائل........................................................52 3-1-4- أقراص المضادات الحيوية.......................................................52 3-1-5- البروتينات القياسية.............................................................53 3-2- طرائق العمل......................................................................53 3-2-1 جمع العينات....................................................................53 3-2-2- عزل بكتريا Streptomyces...................................................54 3-2-3- تصنيف بكتريا Streptomyces..............................................54 3-2-3-1 دراسة بعض الخواص المزرعية والبيوكيميائية للمستعمرات الناتجة...............54 3-2-3-2 اختبار حساسية البكتريا للمضادات الحيوية...................................55 3-2-4- انتخاب السﻻﻻت المنتجة ﻷنزيم غلوكوز إيزوميراز...............................55 3-2-4-1- الغربلة اﻷولية.............................................................55 3-2-4-2- الغربلة الثانوية.............................................................56 3-2-5- تحديد الطريقة المثلى لتحطيم الخﻻيا واستخﻻص إنزيم GI....................56 3-2-6- تقدير الفعالية اﻹنزيمية لـ GI.................................................57 3-2-6-1- المنحنى القياسي لمحلول الفركتوز..........................................58 3-2-7- التطفير باﻷشعة فوق البنفسجية..................................................59 3-2-8- أمثلة ظروف إنتاج أنزيم GI....................................................60 3-2-9- دراسة بعض الصفات الحركية ﻷنزيم GI الخام المفرز من بكتريا 62................................................ Streptomyces roseiscleroticus 3-2-9-1- تأثير درجة الحرارة ...........................................................63 3-2-9-2- اﻻستقرارية الحرارية............................................................63 3-2-9-3- تأثير الرقم الهيدروجيني........................................................63 3-2-9-4- اﻻستقرارية الحمضية...........................................................65 3-2-9-5- تأثير تركيز الركيزة على فعالية اﻹنزيم..........................................65 3-2-9-6- تأثير بعض الشوارد المعدنية...................................................66 3-2-10- تنقية إنزيم غلوكوز إيزوميراز...................................................66 3-2-10-1- الحصول على المستخلص الخام لﻷنزيم.......................................66 3-2-10-2- مراحل تنقية GI ...........................................................67 3-2-10-2-1- الترسيب بأمﻻح كبريتات اﻷمونيوم.....................................67 3-2-10-2-2- التنقية باستخدام أعمدة الكروماتوغرافيا...................................68 3-2-10-2-2-1- عمود الـ Sephadex G-100........................................68 3-2-10-2-2-2- عمود الـ Sephacryl S-200 SF................................69 3-2-11 - تقدير البروتين..................................................................70 3-2-11-1- المواد والمحاليل المستخدمة................................................70 3-2-11-2- تحضير المنحنى القياسي لتقدير تركيز البروتين..........................71 3-2-11-3- تقدير تركيز البروتين في المستخلص اﻹنزيمي............................71 3-2-12- طريقة الرحﻻن الكهربائي للبروتين باستخدام طريقة SDS-PAGE 72.......................................(Poly Acrylamide Gel Electrophoresis) 3-2-12-1- المواد والمحاليل المستخدمة...............................................73 3-2-12-2- طريقة الرحﻻن الكهربائي ﻹنزيم GI باستخدام SDS-PAGE..............74 3-2-13- التطبيقات العملية لـ GI.....................................................77 3-2-13-1- طريقة كروماتوكرافيا الطبقة الرقيقة TLC .................................77 3-2-14- التحليل اﻹحصائي...........................................................78 4- النتائج و المناقشة .................................................................79 4-1- العزل ............................................................................79 4-2- تصنيف بكتريا ستربتوميسيس ......................................................79 4-2-1- الوصف المورفولوجي لبكتريا ستربتوميسيس .....................................79 4-2-2- مقاومة العزﻻت للمضادات الحيوية .............................................82 4-2-3- قابلية
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