毕赤酵母中糖基化修饰对持续性内切葡聚糖酶SmCel5A的影响OA
Impact of glycosylation on processive endoglucanase SmCel5A in Pichia pastoris
将来源于生孢噬纤维菌Sporocytophaga sp.CX11的持续性内切葡聚糖酶SmCel5A在毕赤酵母X-33中异源表达,质谱鉴定出其Asn258位点存在N-糖基化修饰.为研究糖基化修饰对SmCel5A-pPICZαA-X33催化性能的影响,构建了突变体SmCel5A-N258D-pPICZαA-X33,并在毕赤酵母中进行表达.酶学性质表征结果表明,与SmCel5A-pPICZαA-X33相比,去糖基化修饰后的SmCel5A-N258D-pPICZαA-X33对羧甲基纤维素钠的酶活力下降了 95％,对微晶纤维素和滤纸的酶活力也分别下降了75％和50％;最适pH由7.0变为6.0,在碱性条件下稳定性下降;最适温度由40 ℃变为35 ℃且温度稳定性较差,说明Asn258位点的糖基化修饰在维持SmCel5A的催化活性、温度稳定性及耐碱性等方面具有重要作用.
The endoglucanase SmCel5A from Sporocytophaga sp.CX11 was heterologously expressed in Pichia pastoris X-33,and the N-glycosylation modification was identified at its Asn258 site by mass spectrometry.To investigate the effect of glycosylation modification in Sm Cel5A-pPICZaA-X33,the mutant Sm Cel5A-N258D-pPICZaA-X33 was constructed and expressed in P.pastoris.Characterization of enzymatic properties revealed the activity of deglycosylated Sm Cel5A-N258D-pPICZaA-X33 against sodium carboxymethyl cellulose 95％decreasd compared with Sm Cel5 A-pPICZaA-X33,and its activities against microcrystalline cellulose and filter paper decreased by 75％and 50％,respectively.The optimal pH shifted from 7.0 to 6.0,with reduced stability under alkaline conditions.The optimal temperature decreased from 40 ℃ to 35 ℃,accompanied by poorer thermal stability.The results indicate that the glycosylation modification at the Asn258 site plays a crucial role in maintaining the catalytic activity,thermal stability,and alkali tolerance of SmCel5A.
王曦泽;王梓懿;陈晓艺
大连工业大学 生物工程学院,辽宁 大连 116034大连工业大学 生物工程学院,辽宁 大连 116034大连工业大学 生物工程学院,辽宁 大连 116034
生物科学
内切葡聚糖酶生孢噬纤维菌N-糖基化修饰毕赤酵母
endoglucanaseSporocytophaga sp.N-glycosylation modificationPichia pastoris
《大连工业大学学报》 2026 (1)
27-34,8
辽宁省教育厅重点攻关项目(LJKZZ20220061).
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