Food Bioscience
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2025
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IF 5.9
Tingting Li,Hongkai Tan,Tianliang Bai,Yong Wu,Xuanyi Meng,Yanhai Xie,Xin Li,Hongbing Chen
Pitcher fluid of Nepenthes × miranda is usually discarded before transportation despite containing many functional materials, in which hydrolytic proteases are particularly attractive to us. In our study, we collected the pitcher fluid of Nepenthes × miranda and obtained pitcher fluid proteases with a proteolytic activity of 6.658 U/mL. Then the optimal pH and temperature were detected, and the activators, denaturants and inhibitors were screened respectively. Finally, Michaelis constants were calculated to quantify the affinity with different substrates. The results showed peak activity at pH 2.05 to 2.50 and temperatures between 50 °C and 55 °C, which indicated an advantage of exerting proteolytic activity at lower pH levels and higher temperature compared to nepenthesins from other Nepenthes species. Moreover, the pitcher fluid proteases could be activated by Mn 2+ significantly and tolerated glycerol of high content. We discover a way of the new protease resources, a rational use for biochemistry and a direction for the reuse of agricultural waste.
研究领域:
植物学
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生物化学
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DOI
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
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2018
·Vol. 103
·期 3
·1289-1298
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IF 3.34
Zhang Pujuan,Zhang Jinru,Zhang Lujia,Sun Jianan,Li Yuan,Wu Lian,Zhou Jiahai,Xue Changhu,Mao Xiangzhao
AgWH50C, an exo-β-agarase of GH50 isolated from Agarivorans gilvus WH0801, plays a key role in the enzymatic production of neoagarobiose, which has great application prospect in the cosmetics and pharmaceutical industry. In contrast, the poor thermostability becomes the main obstructive factor of glycoside hydrolase (GH) family 50 agarases, including AgWH50C. Herein, based on the AgWH50C crystal structure, we designed several mutants by a multiple cross-linked rational design protocol used thermostability predicting softwares ETSS, PoPMuSiC, and HotMuSiC. To our surprise, the mutant K621F increased its relative activity by as much as 45% and the optimal temperature increased to 38 °C compared to that of wild-type, AgWH50C (30 °C). The thermostability of K621F also exhibited a substantial improvement. Considering that the gelling temperature of the agarose is higher than 35 °C, K621F can be used to hydrolyze agarose for neoagarobiose production.
研究领域:
生物化学
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酶学
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生物技术
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结构生物学
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DOI
PubMed
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
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2020
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IF 3.475
Hong-Yan Wang,Yu-Li Xie,Xin Shi,Hong-Ling Shi,Jian-He Xu,Cun-Duo Tang,Lun-Guang Yao,Yun-Chao Kan
Halogenated mandelate and its derivatives are important scaffolds in chemical industry. However, the natural D -mandelate dehydrogenase (DMDH) showed relatively low catalytic activity toward D -o-chloromandelic acid, which largely limits the efficiency of dehydrogenation processes. To improve catalytic activity of Lactobacillus brevis D -mandelate dehydrogenase ( Lb DMDH) toward halogenated substrate, a D -mandelate dehydrogenase mutant Lb DMDH N253S with higher catalytic activity toward D -o-chloromandelic acid was successfully obtained by directed evolution, which was about 29 times of Lb DMDH. In addition, the temperature optimum of Lb DMDH N253S is 50 °C, and closer to the optimal growth temperature for Escherichia coli , indicating that it may be more advantageous in whole cell catalysis using living cells as catalysts. Meanwhile, the K cat value toward D -o-chloromandelic acid of Lb DMDH N253S is 2.08 s −1 , which is significantly higher than the one (0.09 s −1 ) of Lb DMDH, indicating that Lb DMDH N253S displayed higher catalytic efficiency toward D -o-chloromandelic acid. Then, we preliminarily explained the molecular basis for its catalytic performance by in silico design methods. All of these results established a solid foundation for molecular modification of DMDHs and the other industrial enzymes.
研究领域:
生物化学
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酶工程
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分子生物学
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DOI
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2018
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IF 3.909
Di Xu,Mengyun Wu,Xue Li,Mingyu Xia,Dongchun Liu,Yinghui Dai,Qing Yu,Bin Wu,Dong Wang
Bufadienolides, one kind of steroids, are the major active component secreted by ear-side gland of Bufo species. Preliminary studies on high-throughput transcriptome sequencing about B. bufo gargarizans showed that the expression of 3 β -Hydroxysteroid dehydrogenase (3 β HSD) in ear-side gland was nearly 20 times higher than that in liver. The enzyme 3 β HSD is an essential step in the biosynthesis of steroid such as progesterone, estrogens and androgens in steroidogenic tissues. Accordingly, 3 β HSD is probably an important enzyme involved in the biosynthesis of bufadienolides. In this study, Bbg-3βHSD cDNA was cloned from the ear-side gland of B. bufo gargarizans . Genetic engineering techniques were used to construct a recombinant prokaryotic fusion expression plasmid pCOLD- Bbg3βHSD which was introduced into E. coli BL21 for prokaryotic expression. Bbg- 3 β HSD has an open reading frame (ORF) of 1134 bp and encodes 377 amino acid residues. The speculated protein molecular weight is 42.8 kDa and its theoretical isoelectric point is 8.68. Amino acid sequence homologous analysis showed that Bbg -3 β HSD was highly homologous to the 3 β HSD protein of other species. Phylogenetic tree showed the highest similarity between Bbg -3 β HSD and 3 β HSD from Rana rugosa . The optimized expression of recombinant Bbg -3 β HSD were achieved by inducing with 0.1 mmol L −1 IPTG at 15 °C for 20 h. Enzymatic activity in vitro shows that pregnenolone and dehydroepiandroesterone could be 3 β -oxidized by Bbg -3 β HSD when NAD + was used as the coenzyme. Enzymatic properties showed that the optimum reaction temperature of recombinant Bbg- 3 β HSD was 40 °C, the optimum pH was 8.5, and the optimum coenzyme concentration was 1.5 mmol L −1 .
研究领域:
生物化学
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分子生物学
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酶学
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药理学
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产品关联
DOI
PubMed
高影响因子
ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY
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2021
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IF 6.291
Zhijian Ke,Qian Zhu,Wankui Jiang,Yidong Zhou,Mingliang Zhang,Mingli Jiang,Qing Hong
Carbaryl is the representative of carbamate insecticide. As an acetylcholinesterase inhibitor, it poses potential threat to humans and other non-target organisms. Agrobacterium sp. XWY-2, which could grow with carbaryl as the sole carbon source, was isolated and characterized. The carH gene, encoding a carbaryl hydrolase, was cloned from strain XWY-2 and expressed in Escherichia coli BL21 (DE3). CarH was able to hydrolyze carbamate pesticides including carbaryl, carbofuran, isoprocarb, propoxur and fenobucarb efficiently, while it hydrolyzed oxamyl and aldicarb poorly. The optimal pH of CarH was 8.0 and the optimal temperature was 30 ℃. The apparent K m and k cat values of CarH for carbaryl were 38.01 ± 2.81 μM and 0.33 ± 0.01 s −1 , respectively. The point mutation experiment demonstrated that His341, His343, His346, His416 and D437 are the key sites for CarH to hydrolyze carbaryl.
研究领域:
环境科学
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微生物学
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生物化学
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酶学
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DOI
PubMed
高影响因子
Frontiers in Microbiology
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2022
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IF 6.064
Wang Chengyong,Chen Xueman,Zhou Ning,Chen Yan,Zhang Alei,Chen Kequan,Ouyang Pingkai
A novel multifunctional chitinase (CmChi3)-encoding gene was cloned from Chitinolyticbacter meiyuanensis and actively expressed in Escherichia coli. Sequence analysis showed that CmChi3 contains two glycoside hydrolase family 18 (GH18) catalytic domains and exhibited low identity with well-characterized chitinases. The optimum pH and temperature of purified recombinant CmChi3 were 6.0 and 50°C, respectively. CmChi3 exhibited strict substrate specificity of 4.1 U/mg toward colloidal chitin (CC) and hydrolyzed it to yield N-acetyl-D-glucosamine (GlcNAc) as the sole end product. An analysis of the hydrolysis products toward N-acetyl chitooligosaccharides (N-acetyl COSs) and CC substrates revealed that CmChi3 exhibits endochitinase, N-acetyl-β-d-glucosaminidase (NAGase), and transglycosylase (TGase) activities. Further studies revealed that the N-terminal catalytic domain of CmChi3 exhibited endo-acting and NAGase activities, while the C-terminal catalytic domain showed exo-acting and TGase activities. The hydrolytic properties and favorable environmental adaptations indicate that CmChi3 holds potential for commercial GlcNAc production from chitin.
研究领域:
生物化学
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酶学
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生物技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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