JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE
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2024
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IF 3.3
Buqing Wang,Yasen Wang,Xiaoman Zhou,Xiao-Dong Gao,Morihisa Fujita,Zijie Li
BACKGROUND Acidic lipases with high catalytic activities under acidic conditions have important application values in the food, feed and pharmaceutical industries. However, the availability of acidic lipases is still the main obstacle to their industrial applications. Although a novel acidic lipase Rasamsonia emersonii (LIPR) was heterologously expressed in Escherichia coli , the expression level was unsatisfactory. RESULTS To achieve the high-efficiency expression and secretion of LIPR in Pichia pastoris GS115, the combinatorial optimization strategy was adopted including gene codon preference, signal peptide, molecular chaperone co-expression and disruption of vacuolar sorting receptor VPS10 . The activity of the combinatorial optimization engineered strain in a shake flask reached 1480 U mL −1 , which was 8.13 times greater than the P. pastoris GS115 parental strain. After high-density fermentation in a 5-L bioreactor, the highest enzyme activity reached as high as 11 820 U mL −1 . LIPR showed the highest activity at 40 °C and pH 4.0 in the presence of Ca 2+ ion. LIPR exhibited strong tolerance to methanol, indicating its potential application in biodiesel biosynthesis. Moreover, the gastrointestinal digestion simulation results demonstrated that LIPR was tolerant to pepsin and trypsin, but its activity was inhibited by sodium taurodeoxycholate. CONCLUSION This study provided an effective approach for the high expression of acidic lipase LIPR. LIPR was more appropriate for lipid digestion in the stomach than in intestine according to the gastrointestinal digestion simulation results. © 2024 Society of Chemical Industry.
研究领域:
生物技术
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酶工程
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食品科学
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DOI
PubMed
高影响因子
JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY
·
2011
·Vol. 87
·期 4
·553-558
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IF 1.818
Xiao-Feng Wang,Xu-Guang Shen,Yong-Chuan Sun,He-Yun Zhao,Li Xu,Yun Liu,Yun-Jun Yan
BACKGROUND: Yarrowia lipolytica lipase LIP2 (YlLIP2) is an important industrial enzyme that has many potential applications. Although it has been successfully expressed in Pichia pastoris under the control of the AOX1 promoter ( pAOX1 ), there have been many efforts to develop new alternative promoters to pAOX1 in order to avoid using methanol in the fermentation. Investigation of YlLIP2 production in P. pastoris using the formaldehyde dehydrogenase 1 promoter ( pFLD1 ) is especially attractive, since little is known about its application in methanol-free culture strategies. RESULTS: Three fed-batch cultivations were performed to investigate the production of YlLIP2 in a pFLD1 -based system. When methanol was used as the fed-batch feeding substrate, the maximum YlLIP2 activity obtained in a 10-L bioreactor was 30 000 U mL −1 after 143 h of culture, whereas the maximum YlLIP2 activity was further increased to 35 000 U mL −1 by adopting a co-induction strategy with methanol and methylamine as a mixed fed-batch substrate. Furthermore, the maximum YlLIP2 activity reached 13 000 U mL −1 after 80 h of cultivation in a methanol-free culture. CONCLUSION: The expression levels of YlLIP2 in the pFLD1 -based system were comparable with those in a pAOX1 -based system. The results suggest that pFLD1 is an attractive alternative to pAOX1 , and may make it feasible to induce high yields of protein expression. Copyright © 2011 Society of Chemical Industry
研究领域:
生物技术
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分子生物学
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DOI
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
·
2017
·
IF 2.502
Kun Zhang,Zhiyou Pan,Zhoujian Diao,Shuli Liang,Shuangyan Han,Suiping Zheng,Ying Lin
Kinetic resolution of sec -alcohols is a green process with biocatalyst. Candida antarctica lipase B (CALB) displayed on Pichia pastoris cell-surface (Pp-CALB) was characterized in kinetic resolution of sec -alcohols with different structures. The reaction parameters including acyl donors, molar ratio of substrates, solvents and temperatures were examined with 2-octanol as model substrate. 47.4% molar conversion of 2-octanol and 99.7% ee p were obtained after a 5 h reaction with Pp-CALB, and 90% of its original activity still remained after being reused for 10 cycles. Pp-CALB was then used to several sec -alcohols and it showed great enzymatic activity and enantioselectivity to all tested sec -alcohols, more than 93.1% of ee p . The enantioselective characteristics of Pp-CALB catalysed sec -alcohols with different structures were compared with Novozyme 435 which was almost the same. Solvent free system as one way of green chemistry was applied to Pp-CALB and Pp-CALB showed great catalytic activity and enantioselectivity. Pp-CALB was potential biocatalyst of high enzymatic activity and enantioselectivity using in resolution of sec -alcohols.
研究领域:
生物化学
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酶工程
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绿色化学
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产品关联
DOI
PubMed
高影响因子
PROCESS BIOCHEMISTRY
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2021
·
IF 3.757
Sai Ding,Deli Zhang,Yu Sha,Fangjuan Wang,Caice Liang,Tianpeng Chen,Wenjun Sun,Wei Zhuang,Bin Yu,Dong Liu,Ming Li,Hanjie Ying,Yong Chen
Compared with the traditional suspension fermentation, immobilised biofilm fermentation has advantages of strong resistance, high yield and continuous fermentation and is conducive to improving the industrial fermentation methods. The formation of a biofilm on correct supports is critical to immobilised fermentation. In this study, Pichia pastoris was used to understand and control the modification of surface properties of supports and cells. Following deletion of gene PAS_chr2-1_0773, in P. pastoris △0773, the cell-surface negative charge density decreased and hydrophobicity improved, which was conducive to adhesion. Therefore, according to the changing trend of cell surface properties, cotton fibre was used with succinic anhydride (SA) to modify the carrier for changing the surface properties for efficient conduction of immobilised fermentation. In the fermentation process, the adhesion between the modified cotton fibre and yeast was better than that between an unmodified cotton fibre and yeast, and the additive amount increased by ∼25 %. The enzyme activity of the △0773-cotton-SA fermentation system reached 302.7 U/mL, which was 39.6 % higher than that of the original fermentation system. Therefore, this study provides a guide for significant improvement of immobilised fermentation of fungi.
研究领域:
微生物学
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生物技术
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遗传工程
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DOI
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
·
2012
·Vol. 97
·期 3
·1113-1120
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IF 3.425
Jia Dongxu,Li Jianghua,Liu Long,Zhang Dongxu,Yang Yu,Du Guocheng,Chen Jian
A 1,965-bp fragment encoding a poly(vinyl alcohol) dehydrogenase (PVADH) from Sphingopyxis sp. 113P3 was synthesized based on the codon bias of the methylotrophic yeast Pichia pastoris . The fragment was then amplified by polymerase chain reaction and inserted into the site between Eco RI and Not I sites in pPIC9K, which was under the control of the AOX1 promoter and α-mating factor signal sequence from Saccharomyces cerevisiae . The recombinant plasmid, designated as pPIC9K-PVADH, was linearized using Sal I and transformed into P. pastoris GS115 by electroporation. The PVADH activity reached 55 U/mL in a shake flask and 902 U/mL in a 3-L bioreactor. Surprisingly, the sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and N-terminal sequencing indicated that the secreted PVADH was truncated, and it had only 548 amino acid residues (an 81-amino acid sequence from the secreted protein was cleaved). The optimum pH and temperature ranges for the truncated PVADH were 7.0–8.0 and 41–53 °C, respectively. The activation energy of the recombinant truncated PVADH was approximately 10.36 kcal/mol between 29 and 41 °C. Both Ca 2+ and Mg 2+ had stimulating effects on the activity of PVADH. With PVA1799 as the substrate, the truncated PVADH had a Michaelis constant ( K
m ) of 1.89 mg/mL and a maximum reaction rate ( V
max ) of 34.9 nmol/(min mg protein). To the best of our knowledge, this is the first report on the expression of PVADH in P. pastoris , and the achieved PVADH yield is the highest ever reported.
研究领域:
生物化学
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分子生物学
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微生物学
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产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2024
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IF 7.7
Buqing Wang,Xiaoman Zhou,Yasen Wang,Yahui Gao,Hideki Nakanishi,Morihisa Fujita,Zijie Li
The acidic lipase from Rasamsonia emersonii named LIPR has great potential for biodiesel synthesis due to its strong methanol tolerance. Nonetheless, the limited thermostability of LIPR and low expression level in Escherichia coli remain major obstacles to its use in biodiesel synthesis. To enhance the thermostability, the mutant LIPR harboring mutations A126C-P238C for the formation of a new disulfide bond and amino acid substitution D214L was obtained through rational design. To our delight, the thermostability of LIPR mutant was greatly improved. Moreover, a comprehensive optimization strategy, such as employing the Mss signal peptide, co-expressing the molecular chaperone protein disulfide isomerase (PDI), knocking out the vacuolar sorting receptor gene VPS10-01 , and overexpressing the dihydroxyacetone synthase gene DAS2 , was adopted to obtain the combination-optimized mutant Pichia pastoris strain GS54. Furthermore, the biodiesel synthetic capability with the mutant GS54-LIPR was verified and the production yield was 52.2 % after 24 h in a shake flask. Subsequently, a continuous flow system was adopted to increase the biodiesel yield to 73.6 % within 3 h, demonstrating its efficacy in enhancing enzyme biocatalysis. The engineered GS54-LIPR mutant lipase is an efficient and reusable biocatalyst for the sustained production of biodiesel in a continuous flow reaction.
研究领域:
生物化学
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分子生物学
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工业微生物学
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DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2025
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IF 8.5
Ziyang Ye,Shuiyuan Zhou,Xiangzhao Mao,Yin Li,Taicheng Zhu
Chondroitin sulfate (CS) is widely used in dietary supplements and pharmaceuticals due to its biological activities. However, the large molecular size of high-molecular-weight CS limits its bioavailability and therapeutic efficacy, necessitating the production of low-molecular-weight CS or oligosaccharides to enhance absorption and bioactivity. Here, we report the heterologous expression of recombinant chondroitin hydrolase (rCeChnase) from Caenorhabditis elegans in the methylotrophic yeast Pichia pastoris . This expression system offers an environmentally friendly and cost-effective alternative to animal-derived hyaluronidases, with rCeChnase demonstrating significantly higher catalytic efficiency. The enzyme exhibited optimal activity at 37 °C and pH 5.0, with the highest stability at 25 °C and pH 5.0. A five-fold increase in enzymatic activity production, reaching over 40,000 U/mL, was achieved by lowering the culture temperature to 25 °C. rCeChnase showed a strong preference for non-sulfated chondroitin, with minimal activity towards CS and hyaluronic acid. The enzyme was used to efficiently degrade high-molecular-weight chondroitin into low-molecular-weight chondroitin and oligosaccharides, which were verified by agarose gel electrophoresis and LC-MS. rCeChnase's endo -β1,4-hydrolysis activity was confirmed, and the major product was chondroitin tetrasaccharides. This study highlights the industrial potential of rCeChnase as a promising tool for the production of native low-molecular-weight chondroitin and oligosaccharides.
研究领域:
生物工程
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酶学
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糖生物学
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分子生物学
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发酵工程
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生物制药
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产品关联
DOI
PubMed
高影响因子
PROTEIN EXPRESSION AND PURIFICATION
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2020
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IF 1.513
Jie Zhu,Kun Yang,Aijie Liu,Xiaoxue Lu,Linsong Yang,Qinghuan Zhao
The applications of viral protein cages have expanded rapidly into the fields of bionanotechnology and materials science. However, the low-cost production of viral capsid proteins (CPs) on a large scale is always a challenge. Herein, we develop a highly efficient expression system by constructing recombinant Pichia pastoris cells as a “factory” for the secretion of soluble cowpea chlorotic mottle virus (CCMV) CPs. Under optimal induction conditions (0.9 mg/mL of methanol concentration at 30 °C for 96 h), a high yield of approximately 95 mg/L of CCMV CPs was harvested from the fermentation supernatant with CPs purity >90%, which has significantly simplified the rest of the purification process. The resultant CPs are employed to encapsulate Ruthenium (Ru) nanoparticles (NPs) via in-vitro self-assembly to prepare hybrid nanocatalyst , i.e. [email protected] particles (VLPs). The catalytic activity over [email protected] was evaluated by reducing 4-nitrophenol (4-NP) to 4-aminophenol (4-AP). The results indicate that, with the protection of protein cages, Ru NPs were highly stabilized during the catalytic reaction. This results in enhanced catalytic activity (reaction rate constant k = 0.14 min −1 ) in comparison with unsupported citrate-stabilized Ru NPs (Ru-CA) ( k = 0.08 min −1 ). Additionally, comparatively lower activation energy over [email protected] (approximately 32 kJ/mol) than that over Ru-CA (approximately 39 kJ/mol) could be attributed to the synergistic effect between Ru NPs and some functional groups such as amino groups (–NH 2 ) on CPs that weakened the activation barrier of 4-NP reduction. Therefore, enhanced activity and decreased activation energy over [email protected] demonstrated the superiority of [email protected] to unsupported Ru-CA.
研究领域:
生物技术
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材料科学
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产品关联
DOI
PubMed
高影响因子
Fermentation-Basel
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2024
·Vol. 10
·期 4
·208
·
IF 3.3
Chenyang Li,Tao Zhang,Zhengshan Luo,Jingwen Zhou,Jianghua Li,Jian Chen,Guocheng Du,Xinrui Zhao
Mammalian hemoglobins (HB) are a kind of heme-binding proteins that play crucial physiological roles in various organisms. The traditional techniques employed for the extraction of HB are expensive and time-consuming, while the yields of mammalian HB in previous reports were quite low. The industrialPichia pastorisis a highly effective platform for the secretory expression of heterologous proteins. To achieve efficient secretory expression of HB inP. pastoris, multiple strategies were applied, including the selection of a suitable host, the screening of optimal endogenous signal peptides, the knockout ofVPS10,VTH1, andPEP5, and the co-expression of Alpha-Hemoglobin Stabilizing Protein (AHSP). In addition, the conditions for producing HB were optimized at shaking-flask level (BMMY medium with 100 mg/L of hemin, 2% methanol, and 24 °C). Based on these conditions, the higher titers of bovine hemoglobin (bHB, 376.9 ± 13.3 mg/L), porcine hemoglobin (pHB, 119.2 ± 7.3 mg/L), and human hemoglobin (hHB, 101.1 ± 6.7 mg/L) were achieved at fermenter level. The engineeredP. pastorisstrain and comprehensive strategies can also be applied to facilitate the synthesis of other high-value-added hemoproteins or hemoenzymes.
研究领域:
生物技术
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蛋白质工程
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微生物生物技术
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产品关联
DOI
高影响因子
Bioresources and Bioprocessing
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2024
·Vol. 11
·期 1
·1-12
·
IF 4.3
Fu Xin,Lin Kexin,Zhang Xiaodong,Guo Zhiyong,Kang Lixin,Li Aitao
Unspecific peroxygenases (UPOs) are glycosylated enzymes that provide an efficient method for oxyfunctionalizing a variety of substrates using only hydrogen peroxide (H2O2) as the oxygen donor. However, their poor heterologous expression has hindered their practical application. Here, a novel UPO from Marasmius fiardii PR910 (MfiUPO) was identified and heterologously expressed in Pichia pastoris. By employing a two-copy expression cassette, the protein titer reached 1.18 g L−1 in a 5 L bioreactor, marking the highest record. The glycoprotein rMfiUPO exhibited a smeared band in the 40 to 55 kDa range and demonstrated hydroxylation, epoxidation and alcohol oxidation. Moreover, the peroxidative activity was enhanced by 150% after exposure to 50% (v/v) acetone for 40 h. A semi-preparative production of 4-OH-β-ionone on a 100 mL scale resulted in a 54.2% isolated yield with 95% purity. With its high expression level, rMfiUPO is a promising candidate as an excellent parental template for enhancing desirable traits such as increased stability and selectivity through directed evolution, thereby meeting the necessary criteria for practical application.
研究领域:
生物化学
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酶工程
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合成生物学
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DOI
PubMed
高影响因子
Food Bioengineering
·
2025
·
IF 2.3
Yi Liu,Yu Xie,Xingyu Shen,Shihui Wang,Yi Xiong,Guimin Zhang
Chymotrypsin C (CTRC) is a multifunctional serine protease that have shown great application potential in the field of food processing. This study aims to efficiently express CTRC in Pichia pastoris and provide a safe and highly effective new enzyme formulation for the food industry. Here, we successfully expressed and secreted human chymotrypsinogen C (proCTRC) in P. pastoris through multiple strategies. First, we replaced the native AOX1 promoter with the stronger HpFMD promoter and used ribosomal DNA (rDNA) loci for gene integration to achieve significant expression and secretion of proCTRC. In addition, we designed a mutant pro2M that prevented host protease cleavage by replacing the dipeptide motif “KR” with “EK”. The enzyme activity of wild type and mutant pro2M in the fermentation supernatant reached 0.59 ± 0.003 U/mL and 3.94 ± 0.27 U/mL, respectively. Meanwhile, the mutant showed a specific activity of 54.84 U/mg, 3.3-fold higher than the wild type's 16.6 U/mg. Further, the recombinant pro2M and proCTRC can exhibite good stability under neutral pH conditions. In summary, we have established P. pastoris as a powerful platform for high-yield CTRC production and highlighted the importance of engineering protease-resistant variants to optimize enzyme secretion and activity.
研究领域:
分子生物学
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生物过程工程
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酶技术
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重组蛋白表达
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食品生物技术
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产品关联
DOI
高影响因子
Food Bioscience
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2025
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IF 5.9
Zewei Lu,Changhui Lv,Zhuoer Chen,Dianqi Yang,Renyou Gan,Xiaoqiang Ma
Hemoglobin (HB) is a valuable additive for enhancing the taste, flavor, and nutrition of plant-based protein products. With rising demand for sustainable and health-conscious food options, the exploration of novel HB sources has become increasingly important to meet consumer needs. In this study, a plant-derived HB from Vigna angularis (VaHB) was heterologously expressed and characterized in Pichia pastoris . The data indicated that VaHB exhibited prominent absorption peaks at 415 and 540 nm, a secondary structure comparable to reference standards, high peroxidase activity (123.6 U/mg), and remarkable thermal stability (Tm = 79.6 °C). These attributes were found to differ from those of Glycine a HB (GmHB), an approved color additive in plant-based protein products. To optimize its production, the copy number of the globin-coding gene was enhanced and the entire heme biosynthesis pathway was reengineered, achieving a titer of 117.7 mg/L. These findings indicate that VaHB could be considered a comparable alternative to GmHB.
研究领域:
生物化学
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分子生物学
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生物技术
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食品科学
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蛋白质工程
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产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）