1.Characterization of Humicola insolens cutinase-tachystatin A2 fusion protein and its application in treatment of recycled paper stickies.
Guangyao LI ; Zhanzhi LIU ; Ying ZHANG ; Jing WU
Chinese Journal of Biotechnology 2022;38(1):207-216
With the decrease of forest timber resources, the recycling of waste paper has received increasing attention. However, the stickies produced in the process of waste paper recycling may negatively affect the production of recycled paper. The biological decomposition of stickies, which has the advantages of high efficiency, high specificity and pollution-free, is achieved mainly through the enzymatic cleavage of the ester bond in the stickies components to prevent flocculation. Cutinase is a serine esterase that can degrade some components of the stickies. Previous research indicated that the anchor peptide tachystatin A2 (TA2) is able to bind polyurethane. In this study, the cutinase HiC derived from Humicola insolens was used to construct a fusion protein HiC-TA2 by megaprimer PCR of the whole plasmid (MEGAWHOP). The enzymatic properties and the degradation efficiency of the fusion protein on poly(ethyl acrylate) (PEA), a model substrate of stickies component, were determined. The results showed that the degradation efficiency, the size decrease of PEA particle, and the amount of ethanol produced by HiC-TA2 were 1.5 times, 6.8 times, and 1.4 times of that by HiC, respectively. These results demonstrated that TA2 improved the degradation efficiency of HiC on PEA. This study provides a useful reference for biological decomposition of stickies produced in the process of recycled paper production.
Carboxylic Ester Hydrolases/genetics*
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Fungal Genus Humicola
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Polyurethanes
2.Cloning, expression and activity analysis of cutinase from Sclerotinia sclerotiorum.
Ruihua LÜ ; Linna SHI ; Xirong ZHANG ; Zhao FENG
Chinese Journal of Biotechnology 2022;38(1):386-395
Cutinase can degrade aliphatic and aromatic polyesters, as well as polyethylene terephthalate. Lack of commercially available cutinase calls for development of cost-effective production of efficient cutinase. In this study, eight cutinase genes were cloned from Sclerotinia sclerotiorum. The most active gene SsCut-52 was obtained by PCR combined with RT-PCR, expressed in Escherichia coli BL21 and purified by Ni-NTA affinity chromatography to study its characteristics and pathogenicity. Sscut-52 had a total length of 768 bp and 17 signal peptides at the N terminals. Phylogenetic analysis showed that its amino acid sequence had the highest homology with Botrytis keratinase cutinase and was closely related to Rutstroemia cutinase. Sscut-52 was highly expressed during the process of infecting plants by Sclerotinia sclerotiorum. Moreover, the expression level of Sscut-52 was higher than those of other cutinase genes in the process of sclerotia formation from mycelium. The heterologously expressed cutinase existed in the form of inclusion body. The renatured SsCut-52 was active at pH 4.0-10.0, and mostly active at pH 6.0, with a specific activity of 3.45 U/mg achieved. The optimum temperature of SsCut-52 was 20-30 ℃, and less than 60% of the activity could be retained at temperatures higher than 50 ℃. Plant leaf infection showed that SsCut-52 may promote the infection of Banlangen leaves by Sclerotinia sclerotiorum.
Ascomycota/genetics*
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Carboxylic Ester Hydrolases
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Cloning, Molecular
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Phylogeny
3.Oligomerization triggered by foldon to enhance the catalytic efficiency of feruloyl esterase.
Lei ZHANG ; Linchao LEI ; Guangya ZHANG ; Xialan LI
Chinese Journal of Biotechnology 2019;35(5):816-826
A new method to express oligomerized feruloyl esterase (FAE) in Pichia pastoris GS115 to improve the catalytic efficiency was developed. It was realized by fusing the foldon domain at the C-terminus of FAE, and the fusion protein was purified by histidine tag. Fusion of the feruloyl esterase with the foldon domain resulted spontaneously forming a trimer FAE to improve the catalytic performance. The oligomerized FAE and monomeric FAE were obtained by purification. The apparent molecular weight of the oligomerized FAE was about 110 kDa, while the monomeric FAE about 40 kDa, and the optimum temperature of the oligomerized FAE was 50 °C, which is the same as the monomeric one. The optimal pH of the oligomerized FAE is 5.0, while the optimal pH of the monomer FAE is 6.0. When compared with the monomeric ones, the catalytic efficiency (kcat/Km) of the oligomerized FAE increased 7.57-folds. The catalytic constant (kcat) of the oligomerized FAE increased 3.42-folds. The oligomerized FAE induced by foldon have advantages in the catalytic performances, which represents a simple and effective enzyme-engineering tool. The method proposed here for improving the catalytic efficiency of FAE would have great potentials for improving the catalytic efficiency of other enzymes.
Carboxylic Ester Hydrolases
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metabolism
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Catalysis
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Molecular Weight
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Pichia
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genetics
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metabolism
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Polymerization
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Protein Engineering
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Substrate Specificity
4.Two cases of MEGDEL syndrome due to variants of SERAC1 gene and a literature review.
Xiaoxia LIN ; Xi LIN ; Zheng YAN ; Yanhui CHEN ; Shan CHEN
Chinese Journal of Medical Genetics 2023;40(9):1100-1106
OBJECTIVE:
To explore the clinical phenotype and genetic features of two children with MEGDEL syndrome due to variants of the SERAC1 gene.
METHODS:
Two children who had presented at the Fujian Medical University Union Hospital respectively on July 14, 2020 and July 28, 2018 were selected as the study subjects. Clinical features and results of genetic testing were retrospectively analyzed.
RESULTS:
Both children had featured developmental delay, dystonia and sensorineural deafness, along with increased urine 3-methylglutaric acid levels. Magnetic resonance imaging revealed changes similar to Leigh-like syndrome. Gene sequencing revealed that both children have harbored pathogenic compound heterozygous variants of the SERAC1 gene, including c.1159C>T and c.442C>T in child 1, and c.1168C>T and exons 4~9 deletion in child 2.
CONCLUSION
Children with MEGDEL syndrome due to SERAC1 gene variants have variable clinical genotypes. Delineation of its clinical characteristics and typical imaging changes can facilitate early diagnosis and treatment. Discovery of the novel variants has also enriched the spectrum of SERAC1 gene variants.
Humans
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Retrospective Studies
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Metabolism, Inborn Errors
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Hearing Loss, Sensorineural/genetics*
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Dystonia
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Carboxylic Ester Hydrolases
5.Advances in cutinase research.
Jianghua LI ; Long LIU ; Sheng CHEN ; Guocheng DU ; Jian CHEN
Chinese Journal of Biotechnology 2009;25(12):1829-1837
Cutinase (EC 3.1.1.74) is a kind of hydrolase capable of catalyzing the cleavage of ester bonds of cutin to release fatty acids. Cutinase displayed hydrolytic activity not only toward cutin but also a variety of soluble synthetic esters, insoluble triglycerides and polyesters. Besides its hydrolytic activity, cutinase also showed synthetic activity and transester activity. Therefore, cutinase was evaluated as a versatile lipolytic enzyme used in food and chemical industry. Recently, it is found that cutinase has potential use in cotton bio-scouring and synthetic fibers modification. Cutinase is the most important enzyme in clean production of textile industry.
Amino Acid Sequence
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Carboxylic Ester Hydrolases
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chemistry
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genetics
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metabolism
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Catalysis
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Environmental Pollution
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prevention & control
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Molecular Sequence Data
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Textile Industry
6.Construct a molecular switch based on bacterial quorum sensing.
Chinese Journal of Biotechnology 2013;29(9):1301-1312
Engineering the existing or manual assembling biosynthetic pathways involves two important issues: the activity and expression level of key enzymes in the pathway. Concerning the enzyme expression study, the conventional approach is to use strong promoter to initiate the overexpression of the target protein. The excessive expression of the target protein usually result in the intracellular accumulation of a large number of inactive inclusion bodies, thereby seriously affect the physiological state of the cell and the effective functioning of the relevant biological pathways. To solve this problem, we would like to design a molecular switch to precisely manipulate the expression level of key enzymes in the biosynthetic process, which has important practical value for the study of metabolic rhythm of the biosynthetic pathway and to promote the efficiency of the biosynthetic pathway. Based on the basic principles of quorum sensing existing in the bacterial community and combining the dynamic characteristics of the enzymatic catalysis, we first established cell-cell communication mechanisms mediated by signal molecule homoserine lactone (AHL) in the E. coli community and target protein EGFP was expressed under the control of the promoter P(lux1). In the process of cell growth, AHL accumulated to a certain concentration to start the expression of target gene egfp. At the different cell growth stages, AHL-degrading enzyme AiiA was induced and resulted in the degradation of AHL molecule in a controlled environment, thereby controlling the transcription efficiency of target gene egfp and ultimately achieve the precise control of the level of expression of the target protein EGFP. The detection of cell growth state, the mRNA level and protein expression level of the target gene showed the artificially designed molecular switch can control the level of expression of a target gene in a convenient and efficient manner with a spatial and temporal regulation of rigor. The molecular switch is expected to be widely used in the field of metabolic engineering and synthetic biology research areas.
Carboxylic Ester Hydrolases
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genetics
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Escherichia coli
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enzymology
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genetics
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physiology
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Gene Expression Regulation, Bacterial
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Green Fluorescent Proteins
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biosynthesis
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genetics
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Metalloendopeptidases
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genetics
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Quorum Sensing
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genetics
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physiology
7.Preparation of recombinant cutinase and its application in surface modification of poly (ethylene terephthalate).
Yao ZHANG ; Sheng CHEN ; Dan WU ; Miao HE ; Kongliang ZHU ; Jian CHEN ; Jing WU
Chinese Journal of Biotechnology 2011;27(7):1057-1064
Fermentation and induction conditions for recombinant Escherichia coli expressing Thermobifida fusca cutinase were optimized in flasks and 3L fermenter. Surface modification of poly (ethylene terephthalate) fibers with cutinase was also discussed. The results showed that, cutinase yield reached 128 U/mL by adding 2 g/L inducer lactose and 0.5% glycine. In the fed-batch culture in a 3 L fermenter, the maximum biomass cutinase activity was up to 506 U/mL, which is the highest bacterial cutinase activity reported by far. Recombinant cutinase was used to modify polyester fibers and terephthalic acid substance was detected by using UV analysis. The dyeing and wetting properties of cutinase treated fibers were higher than untreated fibers. Combined utilization of cutinase and Triton X-100 can significantly improve the hydrophilicity of polyester. This is the first report of surface modification on polyester fibers by bacterial cutinase.
Actinomycetales
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enzymology
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genetics
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Carboxylic Ester Hydrolases
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biosynthesis
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chemistry
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genetics
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Escherichia coli
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genetics
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metabolism
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Fermentation
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Polyethylene Glycols
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chemistry
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Polyethylene Terephthalates
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Recombinant Proteins
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biosynthesis
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genetics
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Surface Properties
8.Two-step synthesis of the full length Aspergillus niger lipase gene lipA leads to high-level expression in Pichia pastoris.
Jiangke YANG ; Xiangxiang YAN ; Zhengping ZHANG ; Xueqing JIANG ; Yunjun YAN
Chinese Journal of Biotechnology 2009;25(3):381-387
Aspergillus niger lipases are important biocatalysis widely used in industries for food processing and pharmaceutical preparation. High-level expression recombinants can lead to cost effective lipase large scale production. Full length gene synthesis is an efficient measure to enhance the expression level of the gene. In order to reduce the non-specific binding between oligonucleotides and bases mutation caused by the complicate secondary structure of DNA and excessive PCR amplification, a frequently phenomenon in one-step gene synthesis, we used a two-step method including assembly PCR (A-PCR) and digestion-ligation step to synthesis Aspergillus niger lipase gene lipA. Assisted by DNA2.0 and Gene2Oliga software, we optimized the codon usage and secondary structure of RNA and induced enzyme sites Cla I (237 site) and Pst I (475 site) into the gene. In the first step, fragments F1 (237 bp), F2 (238 bp) and F3 (422 bp) were separately synthesized by assembly PCR. In the second step, fragments F1, F2 and F3 were separately digested by Cla I and Pst I, and then ligated into a full length lipA gene. Two-step method efficiently enhanced successful ratio for full-length gene synthesis and dispersed the risk for gene redesign. The synthesized gene was cloned into pPIC9K vector and transferred into Pichia pastoris. After methanol inducement, the expression level of the codon optimized lipA-syn gene reached 176.0 U/mL, 10.8-fold of the original lipA gene (16.3 U/mL) in Pichia pastoris GS1115. The recombinant offers the possibility for lipase large-scale production.
Base Sequence
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Carboxylic Ester Hydrolases
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biosynthesis
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genetics
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Cloning, Molecular
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Genes, Synthetic
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Genetic Engineering
;
methods
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Genetic Vectors
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genetics
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Molecular Sequence Data
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Pichia
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genetics
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metabolism
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Recombinant Proteins
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biosynthesis
;
genetics
9.Cloning and expression of Fusarium moniliforme CGMCC 0536 D-lactonohydrolase gene in Escherichia coli.
Chinese Journal of Biotechnology 2005;21(3):390-395
The total cDNA obtained through reverse transcription of F. oxysporum CGMCC 0536 mRNA used as template, a fragment about 1.5kb was amplied with oligo(dT)15 primer and a gene specific primer designed on the base of the sequence of both NH2-terminus and the cDNA sequence encoding D-lactonohydrolase of Fusarium oxysporum reported on the NCBI, then the fragment was cloned to the pMD18-T vector and sequenced. The sequence encoding D-lactonohydrolase of F. moniliforme CGMCC 0536 shows a high homology of 90.06% with that of F. oxysporum indicating that the gene encoding D-lactonohydrolase is highly conservative. Two specific primers were designed according to the sequence result, and a fragment, 1146bp, was amplied using hot start PCR with these two specific primers. Subsequently, the resulting products were digested with EcoR I and Sal I and ligated to the pTrc99a vector digested with the same enzymes using T4 DNA ligase. the recombinant plasmid, pTrc99a-LAC, was transformed into Escherichia coli JM109. The two positive clones were induced with IPTG, and enzymes expressed in Escherichia coli JM109, the enzyme activity was about 37U and 41U respectively. The expression products were analyzed by SDS-polyacrylamide gel electrophoresis indicating that about 40kD protein was obtained.
Base Sequence
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Carboxylic Ester Hydrolases
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biosynthesis
;
genetics
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Cloning, Molecular
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Electrophoresis, Polyacrylamide Gel
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Escherichia coli
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genetics
;
metabolism
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Fungal Proteins
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biosynthesis
;
genetics
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Fusarium
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enzymology
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genetics
;
Molecular Sequence Data
10.Synthesis of cefatrizine by recombinant alpha-amino acid ester hydrolase.
Jialin PAN ; Lu WANG ; Duanhua LI ; Lijuan YE
Chinese Journal of Biotechnology 2013;29(4):501-509
To explore the enzymatic route of cefatrizine synthesis, alpha-amino acid ester hydrolase (AEH) gene was cloned from the whole genome of Xanthomonas rubrillineans, and expressed heterologously in Escherichia coli BL21 (DE3). The effects of temperature, pH and substrates' molar ratio upon the transformation yield of cefatrizine by purified recombinant AEH were investigated. The monomer of AEH was determined as 70 kDa by SDS-PAGE. The optimal pH and temperature reaction were (6.0 +/- 0.1) and 36 degrees C for cefatrizine synthesis. The transformation yield was 64.3% under 36 degrees C, pH (6.0 +/- 0.1), when the concentrations of two substrates were about 30 mmol/L (7-ATTC) and 120 mmol/L (HPGM x HCl), respectively, and the enzyme consumption was 22 U/mL. The results pave the way for optimization of the industrial enzymatic synthesis of cefatrizine.
Carboxylic Ester Hydrolases
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biosynthesis
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genetics
;
Catalysis
;
Cefatrizine
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metabolism
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Cloning, Molecular
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Escherichia coli
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genetics
;
metabolism
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Kinetics
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Recombinant Proteins
;
biosynthesis
;
genetics
;
Xanthomonas
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enzymology