Proteolytic degradation of salivary proteins by oral Streptococcus and selection for attenuated mutants

Other Title(s)

التفكك الإحيائي لبروتينات اللعاب بواسطة المسبحيات الفموية-و دراسة الطوافر المضعفة

Dissertant

al-Sadi, Ali Jabbar Rishk

Thesis advisor

al-Jumayli, Isam Fadil A.
Hamzah, Subhi Jawad

Comitee Members

al-Azzawi, Hasan Fayyd Samir
al-Mahdi, Jabbar F.
Salman, Ihab Dawud

University

University of Baghdad

Faculty

College of Science

Department

Biotechnology Department

University Country

Iraq

Degree

Master

Degree Date

2012

English Abstract

Two hundred and seven samples random collected from human oral cavity by sterile swabs, and these samples include 104, 50 and 53 male fe-male and children respectively.

The samples were primary screening by us-ing MS-agar (Mitis-Salivirius agar) medium according to these screening we obtained eighty eight isolates which include Streptococcus sanguis , Streptococcus mutans , Streptococcus oralis , Streptococcus salivaruis , Enterococcus faecalis, and Enterococcus casseliflavus, depending on mor-phology and biochemical identification system.

The isolates were tested for the production of protease enzyme by measuring their clearance zone ratios on the surface of skim milk agar medium.

All isolates were able to produce this enzyme.

S.oralis N 49 was chosen as the best for production.

Optimization of cultural conditions for protease production from S.oralis N49 revealed that the optimum conditions are, Inoculum size 8×106 cell/ml, Efficient medium Tood-Hewitt broth medium, temperature 37 ᴼC, incubation period 20 hours, and pH 7.0.

Separation and purification of S.

oralis N49 proteolytic enzyme was done by precipitated with 0-50 % saturated ammonium sulfate, then ion-exchange chromatography on DEAE-cellulose column .

Partial purified protease gave an activity of 19.8 U/ml, protein concentration of 0.037 mg/ml, specific activity of 535.1 U/mg with purification fold of 2.9 and a yield of 46 %.

Determination of S.

oralis N49 partial purified protease molecular weight was elucidated by the use of gel filtration on Sepharose-6B column with the presence of standard proteins, which determined as 129 KDa.

Summary II Separation of human salivary mucin was done using 4 M guanidinium chloride containing 10 mM EDTA, then mixed and centrifuged at 4400 rpm for 10 min.

at 4 ᴼC.

The supernatant was separated and dialyzed for concentration, and then purification of crud salivary mucin was done using gel filtration on Sepharose-4B column.

The presence of mucin was detected using acetic acid assay and depending on high molecular weight of mucin.

Protein concentration for purified mucin by gel filtration was estimated as 0.11 mg/ml.

Separation of human salivary agglutinin was done using centrifugation and the pellet was dissolving in 1/10 volume of phosphate buffer solution supplemented with EDTA, and then purification of crud salivary agglutinin was done using gel filtration on Sepharose-4B column.

The presence of ag-glutinin was detected depending on high molecular weight of agglutinin.

Protein concentration for purified agglutinin by gel filtration was estimated as 0.03 mg/ml.

Determination of purified mucin and agglutinin molecular weight was elucidated by the use of gel filtration on Sepharose-6B column with the presence of standard proteins, which determined as 437 KDa and 309 KDa, respectively.

Partial purified protease was tested for cleavage of mucin and aggluti-nin proteins by gel filtration chromatography on Sepharose-6B column.

Partial purified protease was able to cleave mucin protein substrate into three fractions, while it's was cleave agglutinin protein into two fractions.

Nitrosoguanidine used as mutagenic factor for S.oralis bacteria.

The ef-fects mutagenic nitrosoguanidine was after one hour of treatment where killing percent was 90 %, and obtaining 9 mutant colonies.

Results were showed that 4 mutant colonies were low-protease productive, where was III clearance zone ratios between 12-18 millimeter, while one colony was non-protease productive.

Remaining living colonies were high-protease produc-tive, as compared with wild type.

Main Subjects

Biology

No. of Pages

147

Table of Contents

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Language

English

Data Type

Arab Theses

Record ID

BIM-600824