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Multifunctional elastin-like polypeptide renders ?-glucosidase enzyme phase transition and high stability.

ABSTRACT: Background:In the enzymatic conversion of biomass, it becomes an important issue to efficiently and cost-effectively degrade cellulose into fermentable glucose. ?-Glucosidase (Bgluc), an essential member of cellulases, plays a critical role in cellulosic biomass degradation. The difficulty in improving the stability of Bgluc has been a bottleneck in the enzyme-dependent cellulose degradation. The traditional method of protein purification, however, leads to higher production cost and a decrease in activity. To simplify and efficiently purify Bgluc with modified special properties, Bgluc-tagged ELP and His with defined phase transitions was designed to facilitate the process. Results:Here, a novel binary ELP and His tag was fused with Bgluc from termite Coptotermes formosanus to construct a Bgluc-linker-ELP-His recombinant fusion protein (BglucLEH). The recombinant plasmid Bgluc expressing a His tag (BglucH) was also constructed. The BglucLEH and BglucH were expressed in E. coli BL21 and purified using inverse transition cycling (ITC) or Ni-NTA resin. The optimum salt concentration for the ITC purification of BglucLEH was 0.5 M (NH4)2SO4 and the specific activity of BglucLEH purified by ITC was 75.5 U/mg for substrate p-NPG, which was slightly higher than that of BglucLEH purified by Ni-NTA (68.2 U/mg). The recovery rate and purification fold of BglucLEH purified by ITC and Ni-NTA were 77.8%, 79.1% and 12.60, 11.60, respectively. The results indicated that purification with ITC was superior to the traditional Ni-NTA. The K m of BglucLEH and BglucH for p-NPG was 5.27 and 5.73 mM, respectively. The K ca t/K m (14.79 S-1 mM-1) of BglucLEH was higher than that of BglucH (12.10 S-1 mM-1). The effects of ELP tag on the enzyme activity, secondary structure and protein stability were also studied. The results showed that ELP tag did not affect the secondary structure or enzyme activity of Bgluc. More importantly, ELP improved the protein stability in harsh conditions such as heating and exposure to denaturant. Conclusion:The Bgluc-linker-ELP-His system shows wide application prospect in maintaining the activity, efficient purification and improving the stability of Bgluc. These properties of BglucLEH make it an interesting tool to reduce cost, to improve the efficiency of biocatalyst and potentially to enhance the degradation of lignocellulosic biomass.

SUBMITTER: Zhou Y

PROVIDER: S-EPMC6589881 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Multifunctional elastin-like polypeptide renders β-glucosidase enzyme phase transition and high stability.

Zhou Yang Y Li Xiaofeng X Yan Dandan D Addai Peprah Frank F Ji Xingqi X Fletcher Emmanuella Esi EE Wang Yanwei Y Wang Yingying Y Gu Jie J Lin Feng F Shi Haifeng H

Biotechnology for biofuels 20190624

<h4>Background</h4>In the enzymatic conversion of biomass, it becomes an important issue to efficiently and cost-effectively degrade cellulose into fermentable glucose. β-Glucosidase (Bgluc), an essential member of cellulases, plays a critical role in cellulosic biomass degradation. The difficulty in improving the stability of Bgluc has been a bottleneck in the enzyme-dependent cellulose degradation. The traditional method of protein purification, however, leads to higher production cost and a d ...[more]

PMID: 31249620

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Project description:Elastin-Like Polypeptides (ELP) are environmentally responsive protein polymers which are easy to engineer and biocompatible, making them ideal candidates as drug carriers. Our team has recently utilized ELPs fused to FKBP12 to carry Rapamycin (Rapa), a potent immunosuppressant. Through high affinity binding to Rapa, FKBP carriers can yield beneficial therapeutic effects and reduce the off-site toxicity of Rapa. Since ICAM-1 is significantly elevated at sites of inflammation in diverse diseases, we hypothesized that a molecularly targeted ELP carrier capable of binding ICAM-1 might have advantageous properties. Here we report on the design, characterization, pharmacokinetics, and biodistribution of a new ICAM-1-targeted ELP Rapa carrier (IBPAF) and its preliminary characterization in a murine model exhibiting elevated ICAM-1. Lacrimal glands (LG) of male NOD mice, a disease model recapitulating the autoimmune dacryoadenitis seen in Sjögren's Syndrome patients, were analyzed to confirm that ICAM-1 was significantly elevated in the LG relative to control male BALB/c mice (3.5-fold, p < 0.05, n = 6). In vitro studies showed that IBPAF had significantly higher binding to TNF-α-stimulated bEnd.3 cells which overexpress surface ICAM-1, relative to nontargeted control ELP (AF)(4.0-fold, p < 0.05). A pharmacokinetics study in male NOD mice showed no significant differences between AF and IBPAF for plasma half-life, clearance, and volume of distribution. However, both constructs maintained a higher level of Rapa in systemic circulation compared to free Rapa. Interestingly, in the male NOD mouse, the accumulation of IBPAF was significantly higher in homogenized LG extracts compared to AF at 2 h (8.6 ± 6.6% versus 1.3 ± 1.3%, respectively, n = 5, p < 0.05). This accumulation was transient with no differences detected at 8 or 24 h. This study describes the first ICAM-1 targeted protein-polymer carrier for Rapa that specifically binds to ICAM-1 in vitro and accumulates in ICAM-1 overexpressing tissue in vivo, which may be useful for molecular targeting in diverse inflammatory diseases where ICAM-1 is elevated.

Dataset Information

Multifunctional elastin-like polypeptide renders ?-glucosidase enzyme phase transition and high stability.

Publications

Multifunctional elastin-like polypeptide renders β-glucosidase enzyme phase transition and high stability.

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