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Effects of Carbohydrate Binding Modules on Characteristics of Xylanases from Thermophilic Bacteria

Thesis Info

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Author

Shahid, Saher

Program

PhD

Institute

University of the Punjab

City

Lahore

Province

Punjab

Country

Pakistan

Thesis Completing Year

2019

Thesis Completion Status

Completed

Subject

Biological Sciences

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/11541/1/Saher%20shahid%20biological%20sci%20year%202019.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726017330

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Xylanases degrade the hemicellulosic component of plant biomass and find potential applications in poultry, paper, textile and biofuel industries. In this study, a novel, family GH10 enzyme, Xyn10B.CB3B2 from Acidothermus cellulolyticus 11B was characterized. This enzyme was found to be a trifunctional enzyme having endo xylanase, arabinofuranosidase and acetyl xylan esterase activities. Native xylanase, Xyn10B.CB3B2 had carbohydrate binding modules (CBM), CBM3 and CBM2 in tandem at the C-terminus. CBMs are protein domains that bind carbohydrate ligands and are found in carbohydrate active enzymes. Truncation of CBM2 was done to create Xyn10B.CB3 while CBM3 was fused to N-terminus of catalytic domain to form Xyn10B.B3C. Fusion of CBM2 at the C- and N-termini of the catalytic domain resulted in Xyn10B.CB2 and Xyn10B.B2C, respectively. In addition, only the catalytic domain (Xyn10B.C) was also characterized in this study. All of the enzyme variants were successfully expressed in soluble fraction of Escherichia coli cells and purified through binding with regenerated amorphous cellulose except Xyn10B.C that was obtained as inclusion bodies and purified by refolding. Activities of Xyn10B.CB3B2, Xyn10B.CB3, Xyn10B.B3C, Xyn10B.CB2, Xyn10B.B2C and Xyn10B.C on beechwood xylan were 118,305, 68,325, 65,825, 49,261, 44,518 and 40,368 U/μmol, respectively. Activities of Xyn10B.CB3B2, Xyn10B.CB3, Xyn10B.B3C, Xyn10B.CB2, Xyn10B.B2C and Xyn10B.C towards p nitorphenylarabinofuranoside were 9,042, 4,532, 4,026, 5,672, 5,137 and 4,340 U/μmol, respectively. Activities of Xyn10B.CB3B2, Xyn10B.CB3, Xyn10B.B3C, Xyn10B.CB2, Xyn10B.B2C and Xyn10B.C towards p-nitrophenylacetate were 15,545, 10,485, 8,856, 7,820, 7,571 and 7,342 U/μmol, respectively. All of the enzyme variants had optimum temperature 70 °C and optimum pH 6.0, under the vii assay conditions used. However, Xyn10B.C had optimum temperature and pH of 60 °C and 5.0-6.0, respectively. Binding assays revealed that all of the variants bound to insoluble oat spelt xylan and Avicel expect Xyn10B.C that did not bind to Avicel. Incubation of all enzyme variants with Mn2+ had negative impact on the activity of enzymes while other metal ions had no effect on the activity. Xyn10B.CB3B2 was stable up to 70 °C while Xyn10B.CB3, Xyn10B.B3C, Xyn10B.CB2 and Xyn10B.B2C were stable up to 60 °C. Xyn10B.C was stable only up to 50 °C as thermal unfolding was observed beyond these temperatures during CD spectroscopy analysis. All of the enzyme variants were highly active producing xylobiose and xylose as end products, as well as debranching the substrates by removing arabinose and acetyl side chains as observed by HPLC analysis of the lysates and arabinose/acetate assays. This study successfully elucidated the characteristcs of a novel trifunctional xylanase, Xyn10B. Due to its specific characteristics, Xyn10B.CB3B2 and its variants seem to be of importance for industrial applications. In another study, XynI from Caldicellulosiruptor saccharolyticus DSM 8903 was expressed in E. coli as 35.8 kDa protein in soluble form, but the expression level was rather low. MFOLD analysis of the sequence between the ribosomal binding site and the 5¢-end codons of the gene showed that the start codon AUG was trapped in the mRNA secondary structure. Cloning the gene using pET28a(+) increased expression to a level of 35% as compared to about 4% when expressed using pET22b(+). pET28a(+), having His-tag before the start codon, would prevent strong secondary structure formation thus allowing higher expression level. Activity of XynI was found to be 10, 5 and 6 U/mg on beechwood, birchwood and oat spelt xylan, respectively. Further studies are required to elucidate the reasons behind low activity through molecular modelling and docking analyses.
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ہوئے سجن بے پرواہ

ہوئے سجن بے پرواہ

ساتھوں ہوندا نہیں نبھاہ
اپنے جد بیگانے ہوئے

ہُن دس لگیے کیہڑے راہ
منگے ہوئے نیں ساہ قدیمی

اس دا نہیں کوئی ہے وساہ
دکھاں درداں عشقے اندر

کیتا میرا حال تباہ
ادھیں راتیں اٹھ اٹھ رو

بھر بھر دکھی ٹھنڈے ساہ

ANALISIS KESULITAN MEMBACA PADA SISWA KELAS V SDI WAIROTANG

Tujuan dari penelitian ini adalah untuk mendeskripsikan kesulitan membaca yang di alami siswa kelas V SDI Wairotang dan untuk mengetahui faktor-faktor yang menghambat siswa dalam  kesulitan membaca. Penelitian ini menggunakan pendekatan kualitatif dengan jenis metode penelitian deskriptif kualitatif. Sumber data dalam penelitian ini adalah guru dan siswa SDI Wairotang yang berkesulitan membaca. Teknik pengumpulan data menggunakan teknik wawancara dan dokumentasi. Teknik analisis data menggunakan Miles and Huberman yaitu mereduksi data, menyajikan data dan penarikan kesimpulan. Hasil penelitian yang menunjukkan kesulitan yang dialami siswa adalah membaca belum lancar, mengeja, dan penggunaan  tanda baca kurang tepat dan faktor-faktor yang menghambat siswa dalam membaca yakni kurangnya minat belajar membaca serta kurangnya dukungan keluarga dalam belajar membaca. Berdasarkan hasil penelitian yang dilakukan maka dapat disimpulan bahwa kesulitan-kesulitan siswa dalam membaca dan faktor penghambat dalam membaca di kelas V SDI Wairotang yaitu: belum  lancar  membaca, mengeja dan penggunaan tanda baca yang kurang tepat serta kurangnya minat belajar membaca dan kurangnya dukungan keluarga dalam belajar membaca.  

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