一个来自节丛孢菌的多糖单加氧酶的表达与表征
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摘要
从一株具有降解纤维素能力的Arthrobotrys sp.CX1菌株中成功克隆得到编码LPMO蛋白的基因,命名为LPMO10331。连接到pPICZαA表达载体上并转化至Pichia pastoris X-33菌株中进行诱导表达,获得一个新的多糖单加氧酶,命名为LPMO10331。应用SEM和XRD等方法对该重组酶进行酶活表征,结果表明,在1 mmol/L维生素C条件下,滤纸纤维素结晶度下降了8%,链内和链间的氢键发生断裂,并且使滤纸表面纤维素多糖链发生氧化还原反应产生了许多断裂端口。
A gene encoding LPMO protein was cloned successfully from a strain of Arthrobotrys sp. CX1 which could degrade cellulose efficiently. It was named LPMO10331, and constructed into pPICZαA expression vector, then transformed into Pichia pastoris X-33 strain for inducing expression. A new lytic polysaccharide monooxygenase named LPMO10331 was obtained, and it was characterized by SEM and XRD. The results showed that under the condition of 1 mmol/L vitamin C, the crystallinity of cellulose in filter paper decreased by 8%. The hydrogen bonds in and between the chains were broken, and the redox reaction of cellulose polysaccharide chains on the surface of filter paper resulted in many breaking ports.
A gene encoding LPMO protein was cloned successfully from a strain of Arthrobotrys sp. CX1 which could degrade cellulose efficiently. It was named LPMO10331, and constructed into pPICZαA expression vector, then transformed into Pichia pastoris X-33 strain for inducing expression. A new lytic polysaccharide monooxygenase named LPMO10331 was obtained, and it was characterized by SEM and XRD. The results showed that under the condition of 1 mmol/L vitamin C, the crystallinity of cellulose in filter paper decreased by 8%. The hydrogen bonds in and between the chains were broken, and the redox reaction of cellulose polysaccharide chains on the surface of filter paper resulted in many breaking ports.
引文
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[2] JAGTAP S,RAO M.Purification and properties of a low molecular weight 1,4-β-D-glucan glucohydrolase having one active site for carboxymethyl cellulose and xylan from an alkalothermophilic Thermomonospora sp.[J].Biochemical and Biophysical Research Communications,2005,329(1):111-116.
[3] KLEMM D.Polysaccharides Ⅱ[M].Berlin:Springer,2006.
[4] BEESON W T,PHILLIPS C M,CATE J H D,et al.Oxidative cleavage of cellulose by fungal copper-dependent polysaccharide monooxygenases[J].Journal of the American Chemical Society,2012,134(2):890-892.
[5] HARRIS P V,WELNER D,MCFARLAND K C,et al.Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61:structure and function of a large,enigmatic family[J].Biochemistry,2010,49(15):3305-3316.
[6] LAN W,ZHANG H,CHEN X,et al.Gelatinization and decrystallization of cellulose by newly isolated Arthrobotrys sp.CX1 to facilitate cellulose degradability[J].Cellulose,2016,23(6):3543-3554.
[7] UNGER T,JACOBOVITCH Y,DANTES A,et al.Applications of the restriction free (RF) cloning procedure for molecular manipulations and protein expression[J].Journal of Structural Biology,2010,172(1):34-35.
[8] 徐志武,张颖,王智学,等.一种高效的酿酒酵母和毕赤酵母电击转化法[J].中山大学学报(自然科学版),2010,49(3):98-101.