Protein engineering of the cellulase catalyzing transglycosylation and condensation of lactose unit.

在里氏木霉的粗酶制备中，我们发现该酶不仅具有将乳糖（Lac）和n -乙酰乳胺（LacNAc）双糖转移到各种糖基（R-OH）羟基上的活性，而且还具有催化糖基与糖基之间缩合反应的活性。我们一直认为缩合酶是特定的内切- b -1,4-葡聚糖酶，但它们尚未被确定。在本研究中，我们尝试用序列柱层析法纯化乳糖的缩聚活性，并通过定点诱变等蛋白质工程技术提高缩聚活性。粗酶经70%硫酸铵饱和沉淀，采用两种阴离子交换层析（HiTrap DEAE FF和UNO-Q1）纯化。纯化后的酶在SDS-PAGE上均相，具有Lac凝聚和双糖释放活性。该酶的Mr值为54000，pi值为4.8 ~ 5.1，与EGI接近。在Mono P色谱柱上聚焦后，观察到多糖有明显的缩聚和释放活性。该酶具有较强的CMC酶活性，但不具有Avicel酶活性，这是内切- b -1,4-葡聚糖酶的特征。在不同宿主细胞中表达的EGI和EGII重组酶，由长冈工业大学的okada博士赠送，也进行了不同活性的测试。在a.m oryzae中表达的rEGI具有凝聚Lac和释放Lac和LacNAc的活性，而在s.m obbe中表达的rEGI则不具有这些活性。结果表明，其中一种双糖浓缩酶为EGI。利用RT-PCR技术制备了T reesei EGI的cDNA，用于EGI蛋白工程。hiroaki University的dr . yoshida等人也在高表达内型纤维素酶Ce1B的突变株Aspergillus oryzae TB1的培养液中发现了Lac凝聚活性。T reesei的EGI与A. oryzae的Ce1B氨基酸序列同源性约为50%，均为糖苷水解酶家族7中的内切- b -1,4-葡聚糖酶。我们还试图比较细胞b与T - reesei、EGI和rEGI的双糖释放、转糖基化和凝聚活性。少

In a crude enzyme preparation of Trichoderma reesei, we found activities not only to transfer lactose (Lac) and N-acetyllactosamine (LacNAc) disaccharides to hydroxyl groups of various aglycons (R-OH) but also to catalyze the condensation reaction between those disaccharides and the aglycons. We have been suggesting the condensation enzyme are particular endo-B-1,4-glucanases, however, they have not been identified yet. In the study, we tried to purify the lactose condensation activity by serial column chromatographic methods to improve the condensation activity by protein engineering such as the site-directed mutagenesis.The crude enzyme was purified by two kinds of anion exchange chromatography (HiTrap DEAE FF and UNO-Q1) after 70% ammonium sulfate saturated precipitation. The purified enzyme was homogeneous in SDS-PAGE and showed Lac condensing and disaccharide-releasing activities. The enzyme had Mr 54,000 and pI4.8-5.1, which are close to those of EGI. Several Lac condensing and d … More isaccharide-releasing activity were observed after chromato-focusing in Mono P column. The enzyme showed strong CMC'ase but no Avicel'ase activity, which is characteristic of endo-B-1,4-glucanase. Recombinant enzymes of EGI and EGII expressed in different host cells, gifted from Dr.Okada in Nagaoka University of Technology, were also tested for various activities. The rEGI expressed in A. oryzae showed both activity condensing Lac and releasing Lac and LacNAc while rEGII expressed in S. pombe showed none of them. In result, we concluded one of the disaccharides-condensing enzymes from T resei as EGI. The cDNA of EGI from T reesei was prepared by RT-PCR for protein engineering of EGI.Dr.Yoshida et al. in Hirosaki University also found Lac condensation activity in culture broth of Aspergillus oryzae TB1 which is a mutant strain with highly expression of Ce1B, an endo-type cellulase. The homology of amino acid sequence is approximately 50% between EGI of T reesei and Ce1B of A. oryzae, both of which are endo-B-1,4-glucanases in Glycoside Hydrolase Family 7. We also tried to compare the dissacharides-releasing, transglycosylating, and condensing activities of CelB to these of T reesei EGI and rEGI. Less