CAJ | 학술논문

腈水合酶（ Nitirle hydratase，NHase）催化腈类物质转化为酰胺类物质，目前用于工业生产丙烯酰胺。但在催化过程中释放的热量易导致酶分子失活。研究通过蛋白质融合技术对腈水合酶进行分子改造，提高热稳定性。将2种双亲自组装肽（ self-assembling peptides，SAPs）EAK16和 ELK16分别融合至恶臭假单胞菌Pseudomonas putida NRRL-18668来源NHase非催化亚基β的N末端，构建出2种融合型NHase：EAK16-NHase和ELK16-NHase。经过表达、纯化后测定酶活力，发现EAK16-NHase和ELK16-NHase的酶活力分别为（426±14）U/mg和（372±12）U/mg，保留野生型酶活力的97%和85%。在50℃条件下孵育0~60 min，每5 min取样后测定残存酶活力，EAK16-NHase和 ELK16-NHase酶活力半衰期（ T50）分别为35 min 和40 min，野生型NHase为20 min。说明融合EAK16和ELK16均能提高NHase的热稳定性。研究表明融合SAPs能在不显著影响酶活力的条件下提高酶的热稳定性。
정수합매（ Nitirle hydratase，NHase）최화정류물질전화위선알류물질，목전용우공업생산병희선알。단재최화과정중석방적열량역도치매분자실활。연구통과단백질융합기술대정수합매진행분자개조，제고열은정성。장2충쌍친자조장태（ self-assembling peptides，SAPs）EAK16화 ELK16분별융합지악취가단포균Pseudomonas putida NRRL-18668래원NHase비최화아기β적N말단，구건출2충융합형NHase：EAK16-NHase화ELK16-NHase。경과표체、순화후측정매활력，발현EAK16-NHase화ELK16-NHase적매활력분별위（426±14）U/mg화（372±12）U/mg，보류야생형매활력적97%화85%。재50℃조건하부육0~60 min，매5 min취양후측정잔존매활력，EAK16-NHase화 ELK16-NHase매활력반쇠기（ T50）분별위35 min 화40 min，야생형NHase위20 min。설명융합EAK16화ELK16균능제고NHase적열은정성。연구표명융합SAPs능재불현저영향매활력적조건하제고매적열은정성。
Nitrilehydratase(NHase)catalyzesnitrilestotheamides,itisatpresentwidelyusedinindustrialpro-duction of acrylamide. However,the heat released during the catalysis process easily inactivated the enzyme molecule involved. In this study,protein engineering by translational fusion was employed to enhance the thermo-stability of the enzyme. Herein,two types of self-assembling peptides( SAPs)EAK16 and ELK16 was translationally fused to the non-catalytic subunit β of NHase from Pseudomonas putida NRRL-18668,yielding two chimeric fusions NHase EAK16-NHase and ELK16-NHase. Next,the two types of recombinant proteins were expressed in E. coli and purified before the enzyme activity were determined. It was found that the specific activity for EAK16-NHase and ELK16-NHase was(426 ± 14)U/mg and(372 ± 12)U/mg respectively,reserved 97% and 85% of that in wild-type NHase. Furthermore,after incubation of the wild-type NHase and chimeric NHase at 50℃ for a period of 0 min to 60 min and sampled at the interval of 5 min,the residual activity was determined. The data showed that the half-life of the enzymatic activity(T50)for EAK16-NHase and ELK16-NHase was 35 min and 40 min,respectively,while the wild-type enzyme was 20 min,indicating that translational fusion of SAPs was able to augment the thermo-stability of NHase. These results manifested that the thermo-stability was actually able to enhance that was not bound to at the cost of losing activity.