Bai Xi, Yuan Xianjun, Wen Aiyou, Li Junfeng, Bai Yunfeng, Shao Tao
Institute of Ensiling and Processing of Grass, Nanjing Agricultural University , Nanjing , China.
College of Animal Science, University of Science and Technology of Anhui , Fengyang , China.
PeerJ. 2016 Nov 8;4:e2679. doi: 10.7717/peerj.2679. eCollection 2016.
Many biotechnological and industrial applications can benefit from cold-adapted EglCs through increased efficiency of catalytic processes at low temperature. In our previous study, A1 which was isolated from a wood-inhabiting termite could secrete a cold-adapted EglC. However, its EglC was difficult to purify for enzymatic properties detection because of its low activity (0.8 U/ml). The objective of the present study was to clone and express the gene in to improve production level and determine the enzymatic properties of the recombinant enzyme.
The gene was cloned from A1 by thermal asymmetric interlaced PCR. was transformed into vector pET22b and functionally expressed in . The recombination protein EglC22b was purified for properties detection.
SDS-PAGE revealed that the molecular mass of the recombinant endoglucanase was approximately 42 kDa. The activity of the pET22b-EglC crude extract was 9.5 U/ml. Additionally, it was active at pH 6.5-8.0 with an optimum pH of 7.0. The recombinant enzyme had an optimal temperature of 30-40 °C and exhibited >50% relative activity even at 5 °C, whereas it lost approximately 90% of its activity after incubation at 60 °C for 30 min. Its activity was enhanced by Co and Fe, but inhibited by Cd, Zn, Li, Triton X-100, DMSO, acetonitrile, Tween 80, SDS, and EDTA.
These biochemical properties indicate that the recombinant enzyme is a cold-adapted endoglucanase that can be used for various industrial applications.
许多生物技术和工业应用可通过提高低温催化过程的效率而从冷适应内切葡聚糖酶（EglCs）中受益。在我们之前的研究中，从栖息于木材的白蚁中分离出的A1能够分泌一种冷适应内切葡聚糖酶。然而，由于其活性较低（0.8 U/ml），其内切葡聚糖酶难以纯化用于酶学性质检测。本研究的目的是在大肠杆菌中克隆并表达该基因，以提高产量水平并确定重组酶的酶学性质。
通过热不对称交错PCR从A1中克隆该基因。将其转化到载体pET22b中并在大肠杆菌中进行功能表达。纯化重组蛋白EglC22b用于性质检测。
SDS-PAGE显示重组内切葡聚糖酶的分子量约为42 kDa。pET22b-EglC粗提物的活性为9.5 U/ml。此外，它在pH 6.5-8.0时具有活性，最适pH为7.0。重组酶的最适温度为30-40℃，即使在5℃时仍表现出>50%的相对活性，而在60℃孵育30分钟后其活性丧失约90%。其活性受到Co和Fe的增强，但受到Cd、Zn、Li、Triton X-100、DMSO、乙腈、吐温80、SDS和EDTA的抑制。
这些生化性质表明该重组酶是一种可用于各种工业应用的冷适应内切葡聚糖酶。
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