Studies on heat tolerance and substrate-recognition mechanism of cellulase

纤维素酶耐热性及底物识别机制研究

• 批准号: 04454076
• 负责人: OHMIYA Kunio
• 金额: $ 3.97万
• 依托单位: Mie University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04454076/
• 关键词: cellulase; xylanase; Clostridium thermocellum; gene; heat tolerance; Clostridium stercorarium; lignocellulose; plant fiber; clostridium thermocellum; clostridium stercorarium; クローニング

The present research aimed at getting some properties of heat tolerance and substrate recognition of cellulases from Clostridium thermocellum and Clostridium stercorarium F-9 by gene analysis and protein characterization. The results obtained here were as follows ; AC.stercorarium gene consisting of 1101 bp, i.c., 367 amino acids with no cysteine was encoded a cellulase XynB, which belongs to cellulase family F.In the amino acid sequence of the protein, 8 conserved regions were found outo by homology analysis. The glutamic acid in the 6th conserved region, ITELD, was estimated to be the active center of the enzyme. Glutamic and aspartic acids in the other conserved regions were evaluated to contribute to the recognition of substrates. Purified XynB revealed cellulase activity in addition to the xylanase activity. This enzyme had maximum activity at pH6.1, 80C and recovered about 60% of initial activity at 60C, even after heating at 100C for 10 min at pH 6.1. This activity recovery was negligible at pH5.8 due to the formation of protein aggregates. Solubilization of the aggregates with 8 M urea showed a partial activity recovery, suggested that activity loss might be caused by the irreversible denaturation in addition to the reversible denaturation. Differential scanning calorimetry (DSC) analysis showed that this protein had a power to rewind the peptide for forming a native protein structure. For studying on the substrate-recognition mechanism, a substrate analog, 1-deoxynogirimicine which binds to active center of the enzyme hydrolyzing glucoside bond, was added to XynB but it was not inhibited. This indicate that the substrate recognition mechanism is different from amylase. The C.thermocellum cellulase(CelC) gene was modified by Sodium nitrate to form random mutation. The mutated gene was encoded the cellulase, in which two amino acids, 107Glu and 209Lys, were replaced to 107Gly and 209 Glu, respectively. This mutation lowered temperature and pH optima. Adding

本研究旨在通过基因分析和蛋白质表征来了解热纤梭菌和粪臭梭菌 F-9 纤维素酶的一些耐热性和底物识别特性。这里获得的结果如下； AC.stercorarium基因全长1101bp，含367个氨基酸，不含半胱氨酸，编码纤维素酶XynB，属于纤维素酶F家族。通过同源性分析，在该蛋白的氨基酸序列中发现8个保守区。第 6 个保守区域 ITELD 中的谷氨酸估计是该酶的活性中心。评估其他保守区域中的谷氨酸和天冬氨酸是否有助于底物的识别。纯化的 XynB 除木聚糖酶活性外还显示纤维素酶活性。该酶在pH6.1、80℃时具有最大活性，并且在60℃时甚至在pH 6.1、100℃加热10分钟后恢复了约60％的初始活性。由于蛋白质聚集体的形成，这种活性恢复在 pH5.8 时可以忽略不计。用8M尿素溶解聚集体显示出部分活性恢复，这表明除了可逆变性之外，不可逆变性也可能导致活性丧失。差示扫描量热法 (DSC) 分析表明，该蛋白质具有使肽倒转以形成天然蛋白质结构的能力。为了研究底物识别机制，将底物类似物 1-deoxynogirimicine 添加到 XynB 中，该底物类似物 1-deoxynogirimicine 与水解葡萄糖苷键的活性中心结合，但它没有受到抑制。这表明底物识别机制与淀粉酶不同。通过硝酸钠修饰C.thermocellum纤维素酶(CelC)基因，形成随机突变。突变基因编码纤维素酶，其中两个氨基酸107Glu和209Lys分别被替换为107Gly和209 Glu。这种突变降低了最适温度和 pH 值。添加

项目成果

S.Karita et al: "Cloning and sequencing of a novel endo-1,4-β-glucanase gene from Ruminococcus albus" J.Ferment.Technol.76. 433-444 (1993)

S.Karita 等人：“来自白色瘤胃球菌的新型内切 1,4-β-葡聚糖酶基因的克隆和测序”J.Ferment.Technol.76 (1993)。

Kazuo Sakka: "Nucleotide sequence of the clostridium stercorarium xyn A gene encoding xylanase A:Identifieation of catalytic and cellulose binding domains." Biosci. Biotech.Biochem.57. 273-277 (1993)

Kazuo Sakka：“编码木聚糖酶 A 的粪梭菌 xyn A 基因的核苷酸序列：催化和纤维素结合域的鉴定。”

Ohmiya, K., K.karita, K.Sakka and K.Shimada: "Genetic engineering of rumen bacteria and their use. I.Properties ofcellulolytic enzymes and their genes." J.Vet.Med.46(8). 687-695 (1993)

Ohmiya, K.、K.karita、K.Sakka 和 K.Shimada：“瘤胃细菌的基因工程及其用途。I.纤维素分解酶及其基因的特性。”

T.Fujino et al: "Nucleotide sequence of cel B gene encoding endo-1,4_1-β-glueanase-2 and ORF1 forming aputative cellulase gene cluster of Clostridiumjosui." J.Ferment.Technol.76. 243-250 (1993)

T.Fujino 等人：“编码内切 1,4_1-β-glueanase-2 和 ORF1 的 cel B 基因的核苷酸序列，形成 Clostridiumjosui 的推定纤维素酶基因簇。”J.Ferment.Technol.76（1993）。

Ohmiya, K., K.Karita, K.Sakka and K.Shimada: "Genetic engineering of rumen bacteria and their use II.Genetic breeding of cellulolytic rumen bacteria." J.BVet.Med.46(9). 767-773 (1993)

Ohmiya, K.、K.Karita、K.Sakka 和 K.Shimada：“瘤胃细菌的基因工程及其用途 II.纤维素分解瘤胃细菌的基因育种。”