Crystallization, structural characterization of a recombinant B-mannanase from Klebsiella oxytoca KUB-CW2-3 and its application for prebiotic preparation

Name: Nawapan Pongsapipatana

LCSH: Kasetsart University -- Dissertations. Ph.D. (Biotechnology) 2021

Classification :.LCCS: QR82.E6

LCSH: Kasetsart University. -- Department of Biotechnology -- Dissertations

LCSH: Klebsiella

LCSH: Escherichia coli

LCSH: Polysaccharides

LCSH: Prebiotics

Abstract: β-Mannanase (EC 3.2.1.78) is an enzyme that cleaves within the backbone of mannanbased polysaccharides at β-1,4-linked-D-mannan resulting in manno oligosaccharides (MOS), a potential prebiotic. To understand the digestion mechanism of β-mannanase KMAN-3, its crystal structure was successfully obtained, and the structure was analyzed. This is the first crystal structure of β-mannanase from Klebsiella species reported. The crystal structure of βmannanase KMAN-3 at the resolution of 2.57 Å revealed an open and narrow cleft shaped active site with the typical structure of glycosyl hydrolase clan A (GH-A) enzyme, (β/α)8-barrel or TIM (triosephosphate isomerase) barrel architecture. The putative catalytic residues were predicted as E183 and E282 located on the loop connected to β-strand 4 and at the end of βstrand 7, respectively. The β-mannanase KMAN-3 exhibited preferential to long linear substrates which no smaller than DP4 and was unable to digest branched substrates. The interaction between β-mannanase KMAN-3 and substrates required at least 4 binding subsites to efficiently bind and hydrolyze substrates to the end-products of mainly mannobiose (M2) and mannotriose (M3). The protruding walls formed by loop 2 and 6 affected the binding of substrates leading to difference in hydrolysis patterns compared to other β-mannanase sources. The application on copra meal hydrolysate (H-DCM) production and its effect on fecal and cecal gut microbiota have also been observed. H-DCM at the concentration of 2.13-2.38 g/L was sufficient to enhance the growth of Lactobacillus johnsonii KUNN19-2 and Limosilactobacillus reuteri KUB-AC5 with the specific growth rate of 0.32-0.33 h-1 . It significantly enhanced the growth of Clostridium coccoides-Eubacterium rectale at 24 h with high acetic acid concentration of 87.43 mM in human GI tract simulation. While the growth enhancing effect of Lactobacillus spp. and growth suppression of Enterobacteriaceae were observed in chicken GI tract simulation with acetic acid concentration of 22.94 mM suggesting a strong beneficial effect on cecal fermentation.

Kasetsart University. Office of the University Library

Address: Bangkok

Email: tdckulib@ku.ac.th

Name: Suttipun Keawsompong

Role: Thesis Advisor

Name: Sunee Nitisinprasert

Role: Thesis CO-Advisor

Created: 2021

Modified: 2025-07-12

Issued: 2025-07-12

วิทยานิพนธ์/Thesis

application/pdf

URL: https://www.lib.ku.ac.th/KUthesis/2564/nawapan-pon-all.pdf

CallNumber: QR82.E6 .N39

eng

DegreeName: Doctor of Philosophy

Level: Doctoral Degraee

Descipline: Biotechnology

Grantor: Kasetsart University

©copyrights Kasetsart University

ลำดับที่.	ชื่อแฟ้มข้อมูล	ขนาดแฟ้มข้อมูล	จำนวนเข้าถึง	วัน-เวลาเข้าถึงล่าสุด
1	nawapan-pon-all.pdf	4.9 MB

ใช้เวลา

0.022401 วินาที