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العنوان
Studies on the Production of
Bacterial Cellulases /
المؤلف
Morgan, Mery Sameh Waheeb Gabrah.
هيئة الاعداد
باحث / ميرى سامح وهيب جبره مرجان
مشرف / محمود عبد المجيد يس
مشرف / نادية عبد الحليم حسونة
مشرف / وليد فيصل الخطيب
تاريخ النشر
2021.
عدد الصفحات
201 P. :
اللغة
الإنجليزية
الدرجة
ماجستير
التخصص
العلوم الصيدلية
تاريخ الإجازة
1/1/2021
مكان الإجازة
جامعة عين شمس - كلية الصيدلة - قسم الميكروبيولوجيا والمناعة
الفهرس
Only 14 pages are availabe for public view

from 201

from 201

Abstract

Forty-three soil samples were collected from different Egyptian governorates from which one hundred and sixty-seven isolates, with the characteristic morphology of Genus Streptomyces, were recovered. One hundred and sixty-five isolates of them were mesophilic (28°C), while the other two isolates were thermophilic (49°C). Congo red method was used to confirm the isolates’ ability to produce cellulase. According to the clear zones diameter formed around the isolates, they were classified into five groups; very strong (17 isolates), strong (99 isolates), moderate (39 isolates), weak (6 isolates), and very weak (6 isolates) producers. The 17 isolates that showed very high cellulase production were submitted to a further quantitative test utilizing dinitrosalicylic acid assay. The highest two producers (S11-6 and TS4-2) were identified by 16S rRNA gene sequencing as Streptomyces coelicoflavus and Streptomyces thermodiastaticus, respectively. Because of the high demand for thermotolerant cellulase and its advantages especially on the industrial scale, the thermophilic strain, Streptomyces thermodiastaticus, was selected to complete the current study.
Optimization of cellulase production by S. thermodiastaticus was carried out using One-Factor-at-a-time (OFAT) method followed by Response Surface Methodology (RSM). OFAT included optimization of several factors affecting cellulase production (incubation temperature, agitation speed, initial pH of fermentation medium, incubation period, inoculum size, different nitrogen sources, CMC concentration, and different salts) and determination of their optimum ranges. The optimum incubation temperature (45°C) and agitation speed (150 rpm) were determined first and kept during the rest of the OFAT and RSM experiments. Using RSM, the initial pH of the medium, CMC concentration, tryptone, and NaCl were optimized using the statistical software package. The model showed that the optimum conditions for cellulase production were pH 6, CMC concentration of 2%, tryptone concentration of 0.03%, and NaCl concentration of 0.12 %. This optimization resulted in 3.25 fold increase in cellulase productivity (2034 U/L) compared to productivity under basal conditions (625 U/L).
Moreover, S. thermodiastaticus was subjected to gamma irradiation to also improve cellulase production. Counting the survivors revealed that a dose of 4 Kilo Gray (KGy) resulted in a 99.99 percent kill rate. Upon screening, mutant coded M1 showed a 1.5 fold increase in cellulase production as compared to the wild-type strain. Finally, optimization of cellulase production by the selected strain, S. thermodiastaticus, using gamma radiation and model-based optimization of nutritional and environmental factors increased the cellulase productivity by 5.1 fold as compared to that obtained by the wild type strain under the basal conditions.
Purification of cellulase was carried out using ammonium sulfate precipitation followed by dialysis. The purified enzyme had a specific activity of 4.21U/mg and 1.74-fold purification. Cellulase had a molecular mass of 63 kDa as indicated by SDS-PAGE and confirmed by zymogram analysis. The optimum temperature and pH for enzyme activity were determined as 60°C and 5.0 and it retained 91.2 and 95% of activity even at 90°C, and pH 10.0 respectively. The enzyme showed outstanding thermal stability as it could retain its full activity after incubation at 90°C for 12 h.
Finally, enzyme immobilization via cross-linked cellulase aggregates (XCA) formation was carried out to improve the efficiency of cellulase by S. thermodiastaticus. First, the enzyme was precipitated to form physical aggregates followed by crosslinking using glutaraldehyde. The most suitable precipitating agent was 100% acetonitrile. The optimum conditions for XCA preparation were glutaraldehyde concentration of 20 mM, the cross-linking temperature of 50°C, agitation speed of 50 rpm, initial pH of crude enzyme 4, and cross-linking time of 90 min. These conditions could increase the recovered activity of the immobilizes crude cellulase from S. thermodiastaticus by approximately 1.9 times as compared to that obtained under basal conditions. XCA could retain approximately 50% of its activity on the second run and only 15% on the third run.