白腐真菌Lentinus sp.之新型外切型纖維素水解酵素II之研究

Abstract

白腐真菌是一種已知能有效降解植物木質纖維素的微生物。白腐真菌會外泌許多種類的酵素，可應用於生物能源，生物修復，生物製漿等領域。在先前的研究中，我們從福山植物園中鑑定出一株Lentinus sp.真菌，並在其培養液中發現其具有分泌(半)纖維素水解酶之能力。利用轉錄體分析和PCR技術，一種預測為外切型纖維素水解酶的基因(命名為LsCBHII)，被全長選殖出來。這個論文研究的目的是鑑定此LsCBHII 酵素的生化與催化功能。重組之LsCBHII蛋白藉由Ni-NTA親和性樹脂和Q Sepharose陰離子交換樹脂管柱層析純化。純化之重組LsCBHII蛋白之分子量估計為64.6 kDa，並藉由SDS-PAGE與LC-MS/MS分析驗證具醣基化。酵素動力學研究顯示，重組LsCBHII之最適溫度與pH值分別為50°C和pH 4.5（以Avicel為受質）或60°C和pH 5.0（以RAC為受質）。重組LsCBHII在酸性環境（pH 4.0至5.0）中顯示出較高的穩定性，其t1 / 2在70°C為6.8分鐘，60°C為22.7分鐘，55°C為35.4分鐘，在50°C則為144分鐘。重組LsCBHII對水不溶解之1,4-β-D-葡聚醣（Avicel和RAC）和水溶性之1,3-1,4-β-D-葡聚醣（lichenan）表現出高催化活性，但對修飾過的1,4-β-D-葡聚醣基質（CMC）表現出低催化活性。以Avicel（RAC）為受質之kcat, Km和kcat/Km分別為2.20 s-1 （7.32 s-1）、23.80±1.36 mg / mL（6.83 ± 0.69 mg / mL）和0.092 mL/mg/s（1.072 mL/mg/s）。我們利用來自Phanerochaete chrysosporium的外切型纖維素水解酶II Cel6A（PDBID：5XCY，相似性78.8％）為模板預測LsCBHII可能之3D蛋白質結構。推測LsCBHII具有2個域結構：碳水化合物結合模塊（CBM）與扭曲的β/α桶狀催化區域，兩區域結構由高靈活性的多肽連接。此外，本研究建構且鑑定了LsCBHII的三個突變體（N102A，K167A和T416K），但僅觀測到具醣基化修飾的Asn102上的突變會影響LsCBHII的熱穩定性和對Avicel的親和力。綜合以上所述，這篇研究首次鑑定出一種新型的真菌外切型纖維素水解酶II，它較大多數已發表的真菌CBHII在水解不溶性纖維素時表現出更高的活性，顯示出此酵素極具在工業應用上的潛力。

White-rot fungi are a group of microorganisms known to effectively degrade plant lignocelluloses. White-rot fungi produce a wide-spectrum of enzymes, which can be used in bioenergy, bioremediation, biopulping, among other industrial applications. In our previous study, a fungus Lentinus sp. identified from Fushan botanical garden was detected with (hemi-)cellulose-like activity in its culture broth. We then used transcriptomics analysis and PCR technique to isolate a full-length cellobiohydrolase II gene designated LsCBHII. This thesis study aimed to characterize the biochemical and catalytic function of LsCBHII encoded protein. The full-length LsCBHII gene was subcloned and overexpressed in the Pichia pastoris host cell system. The pure recombinant LsCBHII protein was obtained by Ni-NTA affinity column chromatography and Q Sepharose anion exchange column chromatography. The recombinant LsCBHII protein was shown glycosylated, with an estimated molecular mass 64.6 kDa, verified by SDS-PAGE and LC-MS/MS analyses. Recombinant LsCBHII exhibited optimal temperature and optimal pH at 50°C and pH 4.5 with Avicel, and 60°C and pH 5.0 with RAC as the substrate. Recombinant LsCBHII displayed higher stability in the acidic environment (pH 4.0 to 5.0), and its t1/2 was determined at 70°C for 6.8 min, at 60°C for 22.7 min, at 55°C for 35.4 min, and at 50°C for 144 min. Recombinant LsCBHII displayed high catalytic activity on the insoluble form of 1,4-β-D-glucan (Avicel and RAC) and 1,3-1,4-β-D-glucan (lichenan) but low activity on modified 1,4-β-D-glucan (CMC). The kcat, Km and kcat/Km were 2.20 ± 0.04 (7.32 ± 0.45) s-1, 23.80 ± 1.36 (6.83 ± 0.69) mg/mL and 0.092 (1.072) mL/mg/s with Avicel (RAC) as substrate. The hypothetical 3D structure of LsCBHII was predicted using a protein template of cellobiohydrolase Cel6A from Phanerochaete chrysosporium (PDBID: 5XCY, sequence identity 78.8%). LsCBHII is predicted to contain 2 domains in its protein structure, i.e., a carbohydrate binding module (CBM) and a distorted β/α barrel catalytic domain connected by a flexible linker. Three mutants (N102A, K167A, and T416K) of LsCBHII were constructed and characterized. Only mutation on the predicted glycosylated residue Asn102 affected thermostability and cellulose (Avicel) binding affinity of LsCBHII. Taken together, this study is the first to identify a novel fungal cellobiohydrolase II exhibited higher hydrolytic activity towards insoluble cellulose than most of the published fungal CBHII, which shows a great potential in industrial application.