INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2014
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IF 3.096
Zhi-Jiang Wang,Jinhyuk Lee,Yue-Xiu Si,Wei Wang,Jun-Mo Yang,Shang-Jun Yin,Guo-Ying Qian,Yong-Doo Park
To gain insight into the structural and folding mechanisms of Antarctic krill alkaline phosphatase (ALP), the enzyme was properly purified by (NH 4 ) 2 SO 4 fractionation and by both Sephadex G-75 and DEAE anion exchange chromatography . The purified enzyme (62.6 kDa; 2.62 unit/mg) was unstable at temperatures exceeding 30 °C. Denaturants, such as sodium dodecyl sulfate (SDS), guanidine HCl, and urea, were applied to evaluate the folding mechanism, including kinetics and thermodynamics, of krill ALP. Sodium dodecyl sulfate elicited no significant effect on ALP activity even at excessively high concentrations (300 mM), whereas guanidine HCl and urea effectively inactivated the enzyme at concentrations of 2 and 3.5 M, respectively. Kinetic studies showed that the enzymatic inhibition by guanidine HCl and urea represented a first-order reaction that was a monophasic unfolding process. This process was found to be associated with conformational changes without significant transient free-energy changes. Additionally, the overall structural changes occurred proximally to the active site pocket. Our study provides new insight into ALP of the Antarctic krill, which lives in extreme environmental conditions.
研究领域:
生物化学
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海洋生物学
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PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2025
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IF 8.5
Bo Zhang,Tong Wang,Siyu Ni,Yaping Zong,Nan Wang,Yunlong Si,Yong Li
Recovery of bioactive proteins from insoluble inclusion bodies (IB) is a critical challenge when purifying recombinant proteins expressed in E. coli . One practical solution is to enhance the renaturability or refolding capability of the target protein after the insoluble proteins are solubilized with denaturants such as urea. While some methods, such as using chemical additives and manipulating the physical environment, have been reported to assist in protein refolding, their effectiveness remains limited. This work evaluated the potential of a highly conserved domain found in five biotin-dependent carboxylases as a fusion tag for purifying IB proteins. We found that this tag, comprised of 67 amino acids (named P67) as eight anti-parallel β-sheets that collectively form a flattened barrel structure, significantly improved the refolding process of its IB protein partner. The yield of bioactive proteins fused with this tag recovered from the dialysis stage was markedly higher than untagged ones. We further proved that this effect relies on the structural integrity of the tag's spatial conformations. Under natural conditions, soluble proteins can be purified using the P67 tag via the biotin/streptavidin affinity system. In conclusion, this tag offers a promising methodology for purifying both insoluble and soluble recombinant proteins.
研究领域:
分子生物学
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蛋白质科学
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生物技术
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生物化学
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高影响因子
JOURNAL OF SEPARATION SCIENCE
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2025
·Vol. 48
·期 8
·e70242
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IF 2.8
TingTing Yu,FuRong Yang,Fang Wang,YongMei Chen,Yi Liu,Nan Hu
This study investigated the chain length-dependent extraction efficiency of two cationic surfactants—dodecyltrimethylammonium bromide (C12, 12-carbon chain) and cetyltrimethylammonium bromide (C16, 16-carbon chain)—for reverse micelle extraction of prolamins from Baijiu Jiuzao (Chinese Baijiu distillers' grains). Although pH variations (4.0–9.0) had negligible effects, ethanol, ionic strength, and chaotropic agent demonstrated distinct chain length-dependent behaviors: ethanol increased C12's forward extraction efficiency by 10.6% (absolute) while enhancing C16's backward efficiency by 8.7%. Potassium chloride enhanced C16's forward efficiency by 4.7%, while potassium bromide significantly improved C12's backward efficiency by 15.9%. Guanidine hydrochloride consistently promoted C16 in both extraction phases. Dynamic light scattering demonstrated significant differences ( p < 0.05) in reverse micelle sizes: C12 formed smaller empty reverse micelles (1.5 ± 0.2 nm) compared to C16 (2.5 ± 0.5 nm). During forward extraction, reverse micelles expanded significantly to 217.9 ± 6.3 nm (C12) and 311.2 ± 43.2 nm (C16). Backward extraction released prolamin particles of 449.0 ± 16.2 nm (C12) and 650.4 ± 29.7 nm (C16), confirming the size-exclusion-driven release mechanism. The more compact C12 reverse micelles correlated with lower total prolamin yield (26.6 ± 0.1% vs. 32.2 ± 0.1% for C16, p < 0.01) and substantially diminished bovine serum albumin recovery (5.2 ± 0.2% vs. 45.3 ± 2.5%, p < 0.01). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis corroborated these findings, further demonstrating that reverse micelle-extracted prolamins formed thermally stable dimers resistant to reductive cleavage. Combined with dynamic light scattering data (particle size range: 400–700 nm in the backward-extraction aqueous phase), these findings demonstrate that prolamin aggregates after reverse micelle extraction. These findings demonstrate that surfactant chain length as a key parameter for optimizing reverse micellar systems, where shorter-chain surfactants facilitate selective extraction, whereas longer chains enhance yields.
研究领域:
食品科学
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胶体与界面科学
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蛋白质提取
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生物技术
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化学工程
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高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2024
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IF 7.7
Hao Liu,Min Huang,Shuxin Wei,Xiaowen Wang,Yaqin Zhao,Zhengyang Han,Xianfeng Ye,Zhoukun Li,Yanling Ji,Zhongli Cui,Yan Huang
β-1,3-Galactanases selectively degrade β-1,3-galactan, thus it is an attractive enzyme technique to map high-galactan structure and prepare galactooligosaccharides. In this work, a gene encoding exo -β-1,3-galactanase (PxGal43) was screened form Paenibacillus xylanexedens , consisting of a GH43 domain, a CBM32 domain and α-L-arabinofuranosidase B (AbfB) domain. Using β-1,3-galactan (AG-II-P) as substrate, the recombined enzyme expressed in Escherichia coli BL21 (DE3) exhibited an optimal activity at pH 7.0 and 30 °C. The enzyme was thermostable, retaining >70 % activity after incubating at 50 °C for 2 h. In addition, it showed high tolerance to various metal ions, denaturants and detergents. Substrate specificity indicated that PxGal43 hydrolysis only β-1,3-linked galactosyl oligosaccharides and polysaccharides, releasing galactose as an exo -acting manner. The function of the CBM32 and AbfB domain was revealed by their sequential deletion and suggested that their connection to the catalytic domain was crucial for the oligomerization, catalytic activity, substrate binding and thermal stability of PxGal43. The substrate docking and site-directed mutagenesis proposed that Glu191, Gln244, Asp138 and Glu81 served as the catalytic acid, catalytic base, pKa modulator, and substrate identifier in PxGal43, respectively. These results provide a better understanding and optimization of multi-domain bacterial GH43 β-1,3-galactanase for the degradation of arabinogalactan.
研究领域:
生物化学
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酶学
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微生物学
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PubMed
高影响因子
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