INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2025
·
IF 8.5
Qihui Lu,Hui Liao,Zedong Jiang,Yanbing Zhu,Yijuan Han,Lijun Li,Hui Ni,Qingbiao Li
Glycosylation plays a critical role in regulating activity, stability, and correct folding of enzymes. In this study, recombinant Aspergillus niger α-L-rhamnosidase (r-Rha1) was employed to explore the impact of glycosylation in Pichia pastoris on the enzymatic properties and protein folding . β-elimination reaction and deglycosylase treatment assays demonstrated that r-Rha1 undergoes primarily N-glycosylation. The deglycosylated r-Rha1 was prepared in two ways: treating with Endoglycosidase F 1 after expression (referred to as r-Rha1- vitro ), or inhibiting intracellular glycosylation using tunicamycin (referred to as r-Rha1- vivo ). Deglycosylation resulted in a 0.22-fold decrease in activity for r-Rha1- vitro and due to its slower turnover rate, r-Rha1- vivo showed a 0.73-fold decrease in activity. r-Rha1- vitro maintained the similar optimal temperature as r-Rha1, r-Rha1- vivo displayed a 10 °C lower optimal temperature. Compared to the decreased extent of r-Rha1- vitro in t 1/2 at 55 °C, 60 °C, and 65 °C and T m , chemical interferent deglycosylation in vivo showed a more profound impact on r-Rha1. Analyses based on circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry revealed significant changes in the structure and thermodynamic stability of r-Rha1- vivo , accounting for its marked decline in activity and stability. The significant and unpredictable structure changes of r-Rha1- vivo proved the essential role of glycosylation for appropriate folding in P. pastoris .
研究领域:
生物化学
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2020
·
IF 5.162
Sa Luo,Zhen Qin,Qiming Chen,Liqiang Fan,Lihua Jiang,Liming Zhao
Chitooligosaccharides (COS) are hydrolytic products of chitosan that are essential in functional food, medicine, and other fields due to their biological activities. Commercial COS are often prepared by the hydrolysis of chitosan by chitosanase. In this study, a glycoside hydrolase family 46 cluster B chitosanase from Bacillus amyloliquefaciens (BaCsn46B) was efficiently expressed in Pichia pastoris . The recombinant enzyme was secreted into the culture medium that reached a total extracellular protein concentration of 4.5 g/L with an activity of 8907.2 U/mL in a high cell density fermenter (5 L). The molecular mass of deglycosylated BaCsn46B was 29.0 kDa. Purified BaCsn46B exhibited excellent enzymatic properties, which had high specific activity (2380.5 U/mg) under optimal reaction conditions (55 °C and pH 6.5). BaCsn46B hydrolyzed chitosan yielded a series of COS with different degrees of polymerization by endo-type cleavage. The end hydrolytic products of BaCsn46B were chitobiose and chitotriose, while no monosaccharide yield was evident in the hydrolytic reaction. The excellent secreted expression level and hydrolytic performance make the enzyme a desirable biocatalyst for the industrial preparation of COS.
研究领域:
生物化学
·
食品科学
·
医学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Microorganisms
·
2023
·Vol. 12
·期 1
·38
·
IF 4.5
Kun Geng,Ying Lin,Xueyun Zheng,Cheng Li,Shuting Chen,He Ling,Jun Yang,Xiangyu Zhu,Shuli Liang
Acetaldehyde is an important carbonyl compound commonly detected in wines. A high concentration of acetaldehyde can affect the flavor of wines and result in adverse effects on human health. Alcohol dehydrogenase I (ADH1) inSaccharomyces cerevisiaecatalyzes the reduction reaction of acetaldehyde into ethanol in the presence of cofactors, showing the potential to reduce the content of acetaldehyde in wines. In this study,ADH1was successfully expressed inPichia pastorisGS115 based on codon optimization. Then, the expression level ofADH1was enhanced by replacing its promoter with optimized promoters and increasing the copy number of the expression cassette, with ADH1 being purified using nickel column affinity chromatography. The enzymatic activity of purified ADH1 reached 605.44 ± 44.30 U/mg. The results of the effect of ADH1 on the content of acetaldehyde in wine revealed that the acetaldehyde content of wine samples was reduced from 168.05 ± 0.55 to 113.17 ± 6.08 mg/L with the addition of 5 mM NADH and the catalysis of ADH1, and from 135.53 ± 4.08 to 52.89 ± 2.20 mg/L through cofactor regeneration. Our study provides a novel approach to reducing the content of acetaldehyde in wines through enzymatic catalysis.Keywords:alcohol dehydrogenase I;Pichia pastoris;cofactor regeneration;multicopy;acetaldehyde;wine
研究领域:
微生物学
·
生物技术
·
食品科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
PROTEIN EXPRESSION AND PURIFICATION
·
2015
·
IF 1.695
Xin Chen,Yong-hao Hu,Wei-di Chen,Wen-di Li,Zi-chao Huang,Yun Li,Yu-wen Luo,Yu-xia Huang,Yi-tai Chen,Kai Wang,Ling Li
Background Plectasin might serve as a substitute for traditional antibiotics, but its yields and antimicrobial activities warrant further investigation. Objective To identify the influence of inducible versus constitutive expression of plectasin on yields and antimicrobial activities. Methods Through SOE-PCR, a recombinant plectasin gene was generated and inserted into inducible (pPICZαA) and constitutive (pGAPZαA) vectors in order to create Pichia pastori s GS115 strains. After 120 h of fermentation, supernatants were purified by an AKTA purifier using nickel columns. Minimal inhibitory concentration (MIC) and inhibition zone assays were performed after Tricine-SDS-PAGE. Results After 120 h of fermentation, the yield of constitutive plectasin (370 μg/ml) was much lower than that from inducible vector (880 μg/ml) ( P < 0.05). However, constitutive strain reached its plateau phase faster and keep more consistent yield ( P < 0.05). The MICs of inducible plectasin against Methicillin-resistant Staphylococcus aureus (MRSA) 15471118, vancomycin-resistant Enterococcus feces (VREF), and penicillin-resistant Streptococcus pneumonia (PRSP) 31355 were 64, 32, and 64 μg/ml, respectively, while those of constitutive plectasin were 4, 4, and 16 μg/ml. No significant differences were observed in antimicrobial activities between inducible and constitutive plectasin for MRSA 15471118, VREF and PRSP 31355 (all P ＞ 0.05). However, constitutive plectasin had a larger inhibition zone than inducible plectasin with the same mass. Conclusions Although P. pastoris GS115 (pGAPZαA-Plectasin-GS115) had lower expression than P. pastoris GS115 (pPICZαA-plectasin-GS115), it reached the plateau phase faster, had steadier yields and showed superiority in antimicrobial activities. Therefore, pGAPZαA might be more suitable for expression of plectasin in GS115 compared with pPICZαA.
研究领域:
分子生物学
·
微生物学
·
生物技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2022
·
IF 5.005
Qi Shen,Xiao-Ting Zhou,Qian Guo,Yu-Zhen Xue,Ya-Ping Xue,Yu-Guo Zheng
Pichia pastoris is widely used for the production of valuable recombinant proteins. An advantage of P. pastoris over other expression systems is that it secretes low levels of endogenous proteins, which facilitates the purification processes if the desired recombinant proteins are efficiently secreted into the culture medium. However, not all recombinant proteins can be successfully secreted by P. pastoris, especially enzymes that are located in intracellular compartments in their native hosts. Few studies have reported strategies for releasing recombinant proteins which cannot be secreted by standard protocols. Here, we investigated whether this challenge can be addressed using novel secretion leaders. Analysis of the secretome and transcriptome of P. pastoris indicated that the four genes with the highest protein-to-transcript ratios were EPX1, PAS_chr3_0030, SCW10, and UTH1, suggesting that their gene products contain efficient secretion leaders. Our data revealed that the signal peptide derived from the PAS_chr3_0030 gene product conferred secretion competence to certain industrial enzymes, e.g., a nitrilase of Alcaligenes faecalis ZJUTB10, a ribosylnicotinamide kinase of P. pastoris, and a glucose dehydrogenase of Exiguobacterium sibiricum. Therefore, the signal peptide derived from the PAS_chr3_0030 gene product represents a novel secretion sequence for the secretory expression of recombinant enzymes in P. pastoris.IMPORTANCE Although P. pastoris is widely used for the secretory production of pharmaceutical proteins, its successful applications in the secretory production of industrial enzymes are limited. The α-mating factor pre-pro leader is the most widely used secretion signal in P. pastoris, but numerous industrial enzymes cannot be secreted using it. The importance of this study is that we identified a signal peptide derived from the PAS_chr3_0030 gene product which conferred secretion competence to three-quarters of the enzymes tested. This signal peptide derived from the PAS_chr3_0030 gene product may facilitate the application of P. pastoris in industrial biocatalysis.
研究领域:
分子生物学
·
生物技术
·
蛋白质工程
·
酶学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Frontiers in Bioengineering and Biotechnology
·
2021
·
IF 5.89
Miao Ting,Basit Abdul,Liu Junquan,Zheng Fengzhen,Rahim Kashif,Lou Huiqiang,Jiang Wei
Xylanases with high specific activity has been focused with great interest as a useful enzyme in biomass utilization. The production of recombinant GH11 xylanase (MYCTH_56237) from Myceliophthora thermophila has been improved through N-terminal signal peptide engineering in P. pastoris. The production of newly recombinant xylanase (termed Mtxyn11C) was improved from 442.53 to 490.7 U/mL, through a replacement of α-factor signal peptide with the native xylanase signal peptide segment (MVSVKAVLLLGAAGTTLA) in P. pastoris. Scaling up of Mtxyn11C production in a 7.5 L fermentor was improved to the maximal production rate of 2503 U/mL. In this study, the degradation efficiency of Mtxyn11C was further examined. Analysis of the hydrolytic mode of action towards the birchwood xylan (BWX) revealed that Mtxyn11C was clearly more effective than commercial xylanase and degrades xylan into xylooligosaccharides (xylobiose, xylotriose, xylotetraose). More importantly, Mtxyn11C in combination with a single multifunctional xylanolytic enzyme, improved the hydrolysis of BWX into single xylose by 40%. Altogether, this study provided strategies for improved production of xylanase together with rapid conversion of xylose from BWX, which provides sustainable, cost-effective and environmental friendly approaches to produce xylose/XOSs for biomass energy or biofuels production.
研究领域:
生物技术
·
酶学
·
生物化学工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Bioresources and Bioprocessing
·
2024
·Vol. 11
·期 1
·1-10
·
IF 4.3
Mei Jianfeng,Han Yanchao,Zhuang Shihang,Yang Zhikai,Yi Yu,Ying Guoqing
Biliverdin, a bile pigment hydrolyzed from heme by heme oxygenase (HO), serves multiple functions in the human body, including antioxidant, anti-inflammatory, and immune response inhibitory activities. Biliverdin has great potential as a clinical drug; however, no economic and efficient production method is available currently. Therefore, the production of biliverdin by the biotransformation of exogenous heme using recombinant HO-expressing yeast cells was studied in this research. First, the heme oxygenase-1 gene ( HO1 ) encoding the inducible plastidic isozyme from Arabidopsis thaliana , with the plastid transport peptide sequence removed, was recombined into Pichia pastoris GS115 cells. This resulted in the construction of a recombinant P. pastoris GS115-HO1 strain that expressed active HO1 in the cytoplasm. After that, the concentration of the inducer methanol, the induction culture time, the pH of the medium, and the concentration of sorbitol supplied in the medium were optimized, resulting in a significant improvement in the yield of HO1. Subsequently, the whole cells of GS115-HO1 were employed as catalysts to convert heme chloride (hemin) into biliverdin. The results showed that the yield of biliverdin was 132 mg/L when hemin was added to the culture of GS115-HO1 and incubated for 4 h at 30 °C. The findings of this study have laid a good foundation for future applications of this method for the economical production of biliverdin. Graphical Abstract
研究领域:
生物技术
·
药学
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
·
2023
·
IF 3.9
Qing-Yun Cai,Cheng-Tong Ge,Shan-Jing Yao,Dong-Qiang Lin
Mixed-mode chromatography (MMC) with the specially-designed ligands shows good adsorption selectivity for protein separation. In this study MMC was explored to purify recombinant human serum albumin (rHSA) from Pichia pastoris culture broth, which combined bind-elute and flow-through modes. Four mixed-mode resins were evaluated for their adsorption capacities of HSA. It was found that two home-made MMC resins showed special performances. IC-B-6FF resin with indole-3-acetic acid-cysteine ligand had high adsorption and selectivity towards HSA, and SFA-B-6FF resin with sulfapyridine ligand showed minimal binding to HSA. Therefore, a two-step MMC separation strategy was designed that IC-B-6FF was used to capture rHSA directly from yeast culture broth and then SFA-B-6FF resin was used to polish the remaining impurities by flow-through mode. The results revealed that the purity of rHSA reached 95.1% with a yield of 81.2% after the two-step separation. The removal rate of host cell proteins (HCPs) was 99.8% with final HCP level as low as 50 ppm. The results demonstrate that MMC combining bind-elute and flow-through modes has a promising potential to separate protein with high selectivity and process efficiency.
研究领域:
生物技术
·
蛋白质工程
✓ 已验证
产品关联
DOI
高影响因子
Food Bioscience
·
2023
·
IF 5.2
Yanru Chen,Yin Wan,Wenqin Cai,Xiaming Che,Xuehui Sun,Hong Peng,Huibo Luo,Dan Huang,Guiming Fu
Ethanol stress is an important environmental factor that affects physiological metabolism and aroma-producing capacity of yeast during the brewing process of alcoholic products. To explore the mechanisms of the growth and aroma-producing metabolism of Pichia anomala under ethanol stress, transcriptomic and metabolomic results were collected from different concentrations (3%, 6% and 9%) of the ethanol stressor, and genes, metabolites and metabolic pathways that relate to growth and aroma-producing were screened. Integrated transcriptomic and metabolomic results showed that P. anomala could respond to ethanol stress by activating purine and pyrimidine , glycerophospholipid and antioxidant metabolic pathways. However, the damage to the growth of P. anomala under ethanol stress was closely related to the inhibition of carbohydrate metabolism, ABC transporter , energy metabolism and amino acid pathways, the influence on these pathways became more serious with the increase of ethanol concentrations. Meantime, the decrease in production of phenethyl alcohol and isoamyl alcohol was related to the significantly down-regulated of ARO9 , ARO10 , ADH1 and ADH2 genes expression in phenylalanine , tyrosine and tryptophan , and glycolysis pathways of P. anomala under ethanol stress. The enhanced expression of ATF1 , EAT1 , EEB1 and TGL2 genes in P. anomala was the main reason for the high yield of ethyl caproate and ethyl acetate under ethanol stress. These results revealed a theoretical basis for further study of the effect mechanisms of ethanol stress on P. anomala and molecular targets for the construction of genetic engineering strains with high ethanol tolerance and high aroma production.
研究领域:
微生物学
·
分子生物学
·
食品科学
✓ 已验证
DOI
高影响因子
TRANSACTIONS OF TIANJIN UNIVERSITY
·
2018
·Vol. 24
·期 6
·513-521
·
IF 0
Shan Yuyao,Qi Wei,Wang Mengfan,Su Rongxin,He Zhimin
Recently, enzymatic peptide synthesis has drawn increasing attention due to its eco-friendly reagents and mild conditions, as compared to traditional chemical peptide synthesis. In this study, we successfully produced an important antioxidant dipeptide precursor, BOC-Tyr-Ala, via a kinetically controlled enzymatic peptide synthesis reaction, catalyzed by the recombinant carboxypeptidase Y (CPY) expressed in P. pastoris GS115. In this reaction, the enzyme activity was 95.043 U/mL, and we used t -butyloxycarbonyl- l -tyrosine-methyl ester (BOC-Tyr-OMe) as the acyl donor and l -alanine ( l -Ala) was the amino donor. We optimized the reaction conditions to be: 30 °C, pH 9.5, organic phase (methanol)/aqueous phase = 1:20, BOC-Tyr-OMe 0.05 mol/L, Ala 0.5 mol/L, and a reaction time of 12 h. Under these conditions, the dipeptide yield reached 49.84%. Then, we established the kinetic model of the synthesis reaction in the form of Michaelis–Menten equation according to the concentration–time curve during the process and the transpeptidation mechanism. We calculated the apparent Michaelis constant \(K_{\text{m}}^{\text{app}}\) and the apparent maximum reaction rate \(r_{\hbox{max} }^{\text{app}}\) to be \(2.9946 \times 10^{{^{ - 2} }} {\text{ mol/L}}\) and \(2.0406 \times 10^{ - 2} {\text{ mmol/(mL}}\,{\text{h)}}\) , respectively.
研究领域:
生物技术
·
酶学
·
肽化学
✓ 已验证
产品关联
DOI
Nanomaterials
·
2018
·Vol. 8
·期 6
·376
·
IF 3.504
Luhang Xu,Dengpo Huang,Huimei Chen,Xiaoling Jing,Jiale Huang,Tareque Odoom-Wubah,Qingbiao Li
Synthesis and applications of one dimensional (1D) metal nanostructures have attracted much attention. However, one-step synthesis of bimetallic nanowires (NWs) has remained challenging. In this work, we developed a microorganism-mediated, hexadecyltrimethylammonium bromide (CTAB)-directed (MCD) approach to synthesize closely packed and long Au-Ag NWs with the assistance of a continuous injection pump. Characterization results confirmed that the branched Au-Ag alloy NWs was polycrystalline. And the Au-Ag NWs exhibited a strong absorbance at around 1950 nm in the near-infrared (NIR) region, which can find potential application in NIR absorption. In addition, the Au-Ag NWs showed excellent surface-enhanced Raman scattering (SERS) enhancement when 4-mercaptobenzoic acid (MBA) and rhodamine 6G (R6G) were used as probe molecules.Keywords:microorganism-mediated;Au-Ag NWs;SERS enhancement;Pichia pastoriscells;CTAB
研究领域:
材料科学
·
纳米技术
·
生物化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2022
·
IF 5.895
Fenghua Wang,Zehui Guo,Zixuan Yang,Xueying Li,Xue Zhang,Xiangyang Ma,Zhuoxuan Han,Fuping Lu,Yihan Liu
Ceramide is a natural functional ingredient as food additive and medicine that has attracted extensive attention in the food, medical, and cosmetic industries. Here, we developed a biotechnological strategy based on a recombinant whole-cell biocatalyst for efficiently producing ceramide from crude soybean oil sediment (CSOS) waste. A novel phospholipase C (PLCac) from Acinetobacter calcoaceticus isolated from soil samples was identified and characterized. Furthermore, recombinant Komagataella phaffii displaying PLCac (dPLCac) on the cell surface was constructed as a whole-cell biocatalyst with better thermostability (30–60 °C) and pH stability (8.0–10.0) to successfully produce ceramide. After synergistical optimization of reaction time and dPLCac dose, the ceramide yield of hydrolyzing from CSOS using dPLCac was 51% (the theoretical maximum yield of converting sphingomyelin, ∼70%) and the relative yield was over 50% after seven consecutive 4 h batches under the optimized conditions. Our study provides a potentially promising strategy for the commercial production of ceramide.
研究领域:
生物技术
·
生物化学
·
环境科学
·
食品科学
·
制药科学
·
化妆品科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）