CAJ | 학술논문

以宇佐美曲霉(Aspergillus usamii)YL-01-78的5家族β-甘露聚糖酶(AuMan5A)为研究对象，对其底物亲和力的定向改造进行理性设计及定点突变以获取米氏常数 Km 值较低的突变酶AuMan5AY111F .首先采用同源建模和分子对接模拟等方法预测AuMan5A与甘露二糖对接复合物的空间结构；在此结构上使用PyMol软件统计到距甘露二糖8?以内的38个氨基酸位点.其次对不同来源的、与AuMan5A一级结构序列全同率大于50％的β-甘露聚糖酶进行多序列比对；排除21个保守氨基酸位点，依据17个非保守位点上的氨基酸性质及其所处空间位置，选择AuMan5A中的Tyr111、Phe206和Tyr243作为拟突变氨基酸，将它们分别替换为性质相似的或在其他β-甘露聚糖酶序列中出现频率高的氨基酸，形成一系列拟突变酶.最后采用MM-PBSA法计算 AuMan5A及其拟突变酶与甘露二糖的结合自由能(ΔGbind )，其中AuMan5AY111F的ΔGbind为-237.7 kJ/mol ，较其他酶的ΔGbind均低.基于该理性设计采用大引物PCR技术将AuMan5A基因(Auman5A)中编码Tyr111的密码子TAC突变为编码Phe111的TTC ，构建出突变酶基因( A uman5A Y111F ).分别将 A uman5A Y111F和 A uman5A 在毕赤酵母GS115中进行表达，并对重组表达产物reAuMan5AY111F和reAuMan5A进行分离纯化和动力学常数测定.结果表明：reAuMan5AY111F对瓜尔豆胶的 Km 值由突变前的3.9 mg/mL降低为2.5 mg/mL ；而突变前后酶的Vmax值变化不大.该研究运用多种生物信息学软件对提高AuMan5A底物亲和力进行了理性设计，并通过定点突变证实之，这为β-甘露聚糖酶乃至其他各种酶底物亲和力的定向改造提供了新的技术策略.
이우좌미곡매(Aspergillus usamii)YL-01-78적5가족β-감로취당매(AuMan5A)위연구대상，대기저물친화력적정향개조진행이성설계급정점돌변이획취미씨상수 Km 치교저적돌변매AuMan5AY111F .수선채용동원건모화분자대접모의등방법예측AuMan5A여감로이당대접복합물적공간결구；재차결구상사용PyMol연건통계도거감로이당8?이내적38개안기산위점.기차대불동래원적、여AuMan5A일급결구서렬전동솔대우50％적β-감로취당매진행다서렬비대；배제21개보수안기산위점，의거17개비보수위점상적안기산성질급기소처공간위치，선택AuMan5A중적Tyr111、Phe206화Tyr243작위의돌변안기산，장타문분별체환위성질상사적혹재기타β-감로취당매서렬중출현빈솔고적안기산，형성일계렬의돌변매.최후채용MM-PBSA법계산 AuMan5A급기의돌변매여감로이당적결합자유능(ΔGbind )，기중AuMan5AY111F적ΔGbind위-237.7 kJ/mol ，교기타매적ΔGbind균저.기우해이성설계채용대인물PCR기술장AuMan5A기인(Auman5A)중편마Tyr111적밀마자TAC돌변위편마Phe111적TTC ，구건출돌변매기인( A uman5A Y111F ).분별장 A uman5A Y111F화 A uman5A 재필적효모GS115중진행표체，병대중조표체산물reAuMan5AY111F화reAuMan5A진행분리순화화동역학상수측정.결과표명：reAuMan5AY111F대과이두효적 Km 치유돌변전적3.9 mg/mL강저위2.5 mg/mL ；이돌변전후매적Vmax치변화불대.해연구운용다충생물신식학연건대제고AuMan5A저물친화력진행료이성설계，병통과정점돌변증실지，저위β-감로취당매내지기타각충매저물친화력적정향개조제공료신적기술책략.
β-mannanases ( endo-β-1 ,4-D-mannanases , EC 3 .2 .1 .78) , which exist widely in various organisms especially in microorganisms , can catalyze the cleavage of internal β-1 ,4-D-mannosidic linkages of mannan backbones . To date , almost all known β-mannanases have been classified into glycoside hydrolase ( GH) families 5 , 26 and 113 based on their amino acid sequence alignment and hydrophobic cluster analysis . Recently , they have attracted much attention due to their great potential applications in industrial processes , such as bioleaching pulps , depolymerizing anti-nutritional factors in feedstuffs , extracting oils from leguminous seeds , hydrolyzing mannan-based polymers in hydraulic fracturing of oil and gas wells , and producing mannooligosaccharides . However , most of commercialβ-mannanases had some shortages in enzymatic properties , such as lower substrate affinity and poorer tolerance to extreme environments , which hindered the development of β-mannanases . A GH family 5 β-mannanase ( AuMan5A) from Aspergillus usamii YL-01-78 was used as the object of this study . The directed modification for its substrate affinity was subjected to the rational design and site-directed mutagenesis to gain a mutant enzyme AuMan5AY111F with higher affinity . Firstly , the three-dimensional ( 3-D) structure of a docked complex of Auman5A with mannobiose was predicted through homology modeling and molecular docking simulation . On the basis of this 3-D structure , 38 amino acid sites in proximity to mannobiose within 8 ? were located by using the PyMol software . Secondly , the multiple alignment of various β-mannanase sequences was performed , among which each sequence shared more than 50% identity with AuMan5A . According to the properties of amino acids at 17 non-conserved sites and their locations on the 3-D structure of AuMan5A , Tyr111 , Phe206 and Tyr243 were selected to be substituted with the similar amino acids and/or high frequency ones in otherβ-mannanase sequences , respectively , forming a series of mutant enzymes . Lastly , binding free energies (ΔGbind ) of various β-mannanases with mannobiose were calculated by using the molecular mechanics Poisson-Boltzmann surface area ( MM-PBSA) method , respectively . The ΔGbind of AuMan5AY111F was -237.7 kJ/mol , lower than those of other enzymes .Based on the rational design ,an AuMan5AY111F-encoding gene , Auman5AY111F , was constructed by mutating a Tyr111-encoding codon ( TAC) of the Auman5A into a Phe111-encoding TTC by megaprimer PCR . Then , the Auman5AY111F and Auman5A were expressed in Pichia pastoris GS115 , and kinetic parameters of the purified recombinant AuMan5AY111F and AuMan5A ( reAuMan5AY111F and reAuMan5A ) were determined , respectively . The results displayed that the Km value of reAuMan5AY111F , towards guar gum , dropped to 2 .5 mg/mL from 3 .9 mg/mL of reAuMan5A , indicating the substrate affinity of reAuMan5A increased correspondingly . While , the Vmax value of reAuMan5A kept almost unchanged after site-directed mutagenesis . The directed modification of AuMan5A based on the rational design for enhancing its substrate affinity was firstly predicted by using various bioinformatics softwares , and then was confirmed by site-directed mutagenesis . This work provides a novel technology strategy for the directed modification of substrate affinities of β-mannanases and other enzymes .