红灰链霉菌硫氧还蛋白还原酶的异源表达及其酶学性质分析

Heterologous Expression and Enzymatic Characterization of a Thioredoxin Reductase from Streptomyces rubrogriseus

关键词:  二硫键还原酶  红灰链霉菌  异源表达  表达优化  酶学性质

Key words:  disulfide bond reductase  Streptomyces rubrogriseus  heterologous expression  expression optimization  enzymatic properties

摘要:

为充分利用废弃羽毛中的角蛋白资源，本研究利用聚合酶链式反应(Polymerase Chain Reaction,PCR)扩增得到红灰链霉菌(Streptomyces rubrogriseus)硫氧还蛋白还原酶(Thioredoxin Reductase，TrxR)基因trxB，通过构建重组质粒并将其转化到大肠杆菌[Escherichia coli BL21(DE3)]中进行表达，得到的TrxR经镍离子亲和层析纯化后测定酶学性质。结果表明：经PCR扩增得到大小为969 bp的trxB，并成功构建重组质粒pET22b-trxB。在最优诱导表达条件[异丙基-β-D-硫代吡喃半乳糖苷(IPTG)浓度为0.5 mmol/L，光密度(OD₆₀₀)为0.8，诱导温度为30 ℃，诱导时间为4 h]下，纯化后的TrxR大小为38.5 kDa。以羽毛粉为底物进行酶促反应时，TrxR的最适pH值为8.0，最适温度为60 ℃；K⁺、Mg²⁺对TrxR酶活力有明显的激活作用,而Fe²⁺、Fe³⁺、Cu²⁺对其有明显的抑制作用；异丙醇、苯甲基磺酰氟(PMSF)、二甲基亚砜(DMSO)均对TrxR酶活力有抑制作用,而二硫苏糖醇(DTT)对TrxR酶活力有促进作用；TrxR酶促反应的米氏常数(K_m)为64.04 mg/mL，最大反应速率(V_max)为3.70 U/min。TrxR具有较高的酶活力和回收率，在降解羽毛角蛋白方面有一定的应用价值。

Abstract:

To make full use of the keratin resources in waste feathers,this study employed Polymerase Chain Reaction (PCR) to amplify the Thioredoxin Reductase (TrxR) gene trxB of Streptomyces rubrogriseus.The recombinant plasmid was constructed and transformed into Escherichia coli BL21 (DE3) for expression.The obtained protein was purified by nickel ion affinity chromatography and its enzymatic properties were determined.The results showed that trxB with a size of 969 bp was cloned,and the recombinant expression plasmid pET22b-trxB was successfully constructed.The optimal induction conditions for expression of the recombinant enzyme TrxR were as follows:Isopropyl-β-D-thiogalactopyranoside (IPTG) concentration of 0.5 mmol/L,Optical Density (OD₆₀₀) of 0.8,induction temperature of 30 ℃,and induction time of 4 h.The purified TrxR had a molecular weight of 38.5 kDa.The optimal pH value and temperature of the recombinant enzyme were 8.0 and 60 ℃,respectively,when feather powder was used as the substrate.K⁺ and Mg²⁺ had obvious activating effects on the enzyme,while Fe²⁺,Fe³⁺,and Cu²⁺ had obvious inhibitory effects.Isopropanol,Phenylmethylsulfonyl Fluoride (PMSF) and Dimethyl Sulfoxide (DMSO) all inhibited the enzyme activity of TrxR,while Dithiothreitol (DTT) had a promoting effect on the enzyme activity of TrxR.The K_m and V_max of TrxR were 64.04 mg/mL and 3.70 U/min,respectively.The TrxR has high enzyme activity and good recovery yield,demonstrating certain application value in the degradation of keratin.