来自热纤梭菌的家族11多糖裂解酶新成员——鼠李糖半乳糖醛酸裂解酶（CtRGLf）
A New Member of Family 11 Polysaccharide Lyase, Rhamnogalacturonan Lyase (CtRGLf) from Clostridium thermocellum.
作者信息
Dhillon Arun, Fernandes Vania O, Dias Fernando M V, Prates José A M, Ferreira Luis M A, Fontes Carlos M G A, Centeno M S J, Goyal Arun
机构信息
Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India.
CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa, Av. da Universidade de Técnica, 1300-477, Lisbon, Portugal.
出版信息
Mol Biotechnol. 2016 Apr;58(4):232-40. doi: 10.1007/s12033-016-9921-6.
A thermostable, alkaline rhamnogalacturonan lyase (RG lyase) CtRGLf, of family 11 polysaccharide lyase from Clostridium thermocellum was cloned, expressed, purified and biochemically characterised. Both, the full-length CtRGLf (80 kDa) protein and its truncated derivative CtRGL (63.9 kDa) were expressed as soluble proteins and displayed maximum activity against rhamnogalacturonan I (RG I). CtRGLf showed maximum activity at 70 °C, while CtRGL at 60 °C. Both enzymes showed maximum activity at pH 8.5. CtRGLf and CtRGL do not show higher activity on substrates with high β-D-galactopyranose (D-Galp) substitution, this catalytic property deviates from that of some earlier characterised RG lyases which prefer substrates with high D-Galp substitution. The enzyme activity of CtRGLf and CtRGL was enhanced by 1.5 and 1.3 fold, respectively, in the presence of 3 mM of Ca(2+) ions. The TLC analysis of the degraded products of RG I, released by the action of CtRGLf and CtRGL revealed the production of RG oligosaccharides as major products confirming their endolytic activity.
克隆、表达、纯化并对嗜热栖热放线菌（Clostridium thermocellum）11家族多糖裂解酶中的一种热稳定碱性鼠李糖半乳糖醛酸裂解酶（RG裂解酶）CtRGLf进行了生化表征。全长CtRGLf（80 kDa）蛋白及其截短衍生物CtRGL（63.9 kDa）均表达为可溶性蛋白，并对鼠李糖半乳糖醛酸聚糖I（RG I）表现出最大活性。CtRGLf在70℃时表现出最大活性，而CtRGL在60℃时表现出最大活性。两种酶在pH 8.5时均表现出最大活性。CtRGLf和CtRGL对具有高β-D-吡喃半乳糖（D-Galp）取代的底物没有更高的活性，这种催化特性不同于一些早期表征的RG裂解酶，后者更喜欢具有高D-Galp取代的底物。在存在3 mM Ca(2+)离子的情况下，CtRGLf和CtRGL的酶活性分别提高了1.5倍和1.3倍。对CtRGLf和CtRGL作用释放的RG I降解产物进行的TLC分析表明，主要产物为RG寡糖，证实了它们的内切活性。
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