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1.
凝乳酶在奶酪加工中应用广泛,为获得高活性的凝乳酶制剂,采用乳酸克鲁维酵母为宿主,首次对经密码子优化的牛凝乳酶原基因进行表达。利用DNAWorks3.0软件辅助设计,用两步PCR法合成了小牛凝乳酶原基因(GenBank Accession No.AA30448)。将该基因插入酵母表达载体pKLAC1,构建了重组载体pKLAC1-Prochy,并用电脉冲法将线性化的重组质粒转化到乳酸克鲁维酵母GG799中。通过含1%酪蛋白的YEPD平板活性筛选,PCR鉴定,最后获得了一株多拷贝整合的基因工程菌chy1。该菌株可分泌表达牛凝乳酶原,经SDS-PAGE分析,证明重组牛凝乳酶原的分子量约为41kDa,符合预期大小,酸化处理后为36kDa,证明可以正确自我剪切。液体培养96h后,酶活最高达到99.67SU/mL。分别以半乳糖和葡萄糖为碳源的条件下表达,其酶活性差异不大,说明在发酵期间,可以不经过半乳糖诱导即可产生高水平的牛凝乳酶原产物。该工程菌的获得为进一步优化产酶条件及放大工艺提供了条件,并为凝乳酶的工业化生产奠定了基础。 相似文献
2.
【目的】实现鼠灰链霉菌来源经密码子优化后的腺苷酸脱氨酶基因在乳酸克鲁维酵母(Kluyveromyces lactis GG799)中组成型表达。【方法】以鼠灰链霉菌(Streptomyces murinus)来源的腺苷酸脱氨酶(AMP)基因经密码子优化后作为模板,设计特异性引物,PCR扩增AMP脱氨酶基因opt-AMPD,以p KLAC1为载体构建重组表达质粒p KLAC1-opt-AMPD,经Sac II线性化后电转化法转入K.lactis GG799,筛选得到重组菌株,测定酶活,经His TrapTM HP纯化后得到AMP脱氨酶,并优化重组菌的发酵培养基。【结果】对AMP脱氨酶基因进行了密码子优化后,构建了重组K.lactis GG799/p KLAC1-opt-AMPD,实现组成型表达,密码子优化后AMP脱氨酶酶活提高到586±50 U/m L。SDS-PAGE结果显示,纯化后的AMP脱氨酶为单一条带,蛋白大小约为60 k D。优化的发酵培养基为(g/L):葡萄糖40、蛋白胨20、酵母粉15、Na Cl 8、KCl 10、Mg SO4 2,30°C、200 r/min发酵120 h,酶活达到2 100±60 U/m L。【结论】实现了密码子优化后的腺苷酸脱氨酶基因在乳酸克鲁维酵母GG799内的组成型表达,为实现腺苷酸脱氨酶的重组高效表达和发酵生产进行了有益探索。 相似文献
3.
Chymosin can specifically break down the Phe105–Met106 peptide bond of milk κ-casein to form insoluble para-κ-casein, resulting in milk coagulation, a process that is used in making cheese. In this study, in order to obtain an alternative
milk coagulant which is safe and efficient, and simultaneously can produce cheese with a good taste, bovine prochymosin B
was chosen and constitutively expressed to a high level in Pichia pastoris. The recombinant chymosin was expressed mainly as a secretory form, and it exhibited milk-clotting activity. It was purified
by ammonium sulfate fractionation, anion exchange, followed by cation exchange chromatography. A final yield of 24.2% was
obtained for the purified enzyme, which appeared as a single band in SDS–PAGE having a molecular mass of approximate 36 kDa.
Proteolysis assay showed that it specifically hydrolyzed κ-casein. It was stable at 25–50°C and had optimal activity at 37°C and pH 4.0. The activity of the recombinant chymosin was
activated by cations such as Mn2+, Fe3+, Mg2+ and Na+, but inhibited by K+, Co2+, Zn2+, Ni2+, and to a lesser extent by Cu2+. These results suggested that recombinant bovine chymosin is an acid milk coagulant, and it could be considered as a safe
and efficient enzyme suitable for use in cheese production. 相似文献
4.
Juan J. Jiménez Juan Borrero Loreto Gútiez Sara Arbulu Carmen Herranz Luis M. Cintas Pablo E. Hernández 《Molecular biotechnology》2014,56(6):571-583
The use of synthetic genes may constitute a successful approach for the heterologous production and functional expression of bacterial antimicrobial peptides (bacteriocins) by recombinant yeasts. In this work, synthetic genes with adapted codon usage designed from the mature amino acid sequence of the bacteriocin enterocin A (EntA), produced by Enterococcus faecium T136, and the mature bacteriocin E 50-52 (BacE50-52), produced by E. faecium NRRL B-32746, were synthesized. The synthetic entA and bacE50-52 were cloned into the protein expression vectors pPICZαA and pKLAC2 for transformation of derived vectors into Pichia pastoris X-33 and Kluyveromyces lactis GG799, respectively. The recombinant vectors were linearized and transformed into competent cells selecting for P. pastoris X-33EAS (entA), P. pastoris X-33BE50-52S (bacE50-52), K. lactis GG799EAS (entA), and K. lactis GG799BE50-52S (bacE50-52). P. pastoris X-33EAS and K. lactis GG799EAS, but not P. pastoris X-33BE50-52S and K. lactis GG799BE50-52S, showed antimicrobial activity in their supernatants. However, purification of the supernatants of the producer yeasts permitted recovery of the bacteriocins EntA and BacE50-52. Both purified bacteriocins were active against Gram-positive bacteria such as Listeria monocytogenes but not against Gram-negative bacteria, including Campylobacter jejuni. 相似文献
5.
Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning,
expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame
(ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant
plasmid was transformed into E. coli
Rosetta. SDS–PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis
activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A
gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant
plasmid containing Attacin A, which includes 570 bp in all. SDS–PAGE analysis demonstrated that the fusion protein expressed
was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity
to several G− bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was
cloned and expressed successfully. It was the basis for further study of Attacin. 相似文献
6.
Zhao Jin-Fang Song Wen-Lu Cheng Jun Zhang Chuan-Xi 《World journal of microbiology & biotechnology》2010,26(1):177-181
The hydrogenase gene from Enterobacter cloacae (IIT-BT 08) was amplified and inserted into a prokaryotic expression vector to create a recombinant plasmid (pGEX-4T-2-Cat/hydA).
The recombinant plasmid was transformed into a hydrogen-producing strain of Enterobacter aerogenes (ATCC13408). SDS–PAGE and western blot analysis confirmed the successful expression of the GST-tagged hydA protein. Anaerobic
fermentation for the production of hydrogen from glucose was investigated using E. aerogenes ATCC13408 and the recombinant strain. The results showed that the hydrogen yield markedly increased, from 442.82 ± 22.61 ml/g
glucose in the ATCC13408 strain to 864.02 ± 36.8 ml/g glucose in the recombinant. The maximum rate of hydrogen production
was found to be 53.49 ± 3.34 ml l−1 h−1 using 1% (w/v) glucose as the substrate at pH 6.0 and a reaction temperature of 37°C. 相似文献
7.
Jing Yu Jiaxi Jiang Zian Fang Yuyang Li Hong Lv Jianping Liu 《Biotechnology letters》2010,32(4):507-512
Inulinase gene (Kcinu) derived from Kluyveromyces cicerisporus was expressed extracellularly in Kluyveromyces lactis using an episomal vector directed by Kcinu promoter. The influence of hap1 gene disruption on the expression of inulinase was studied. Inulinase activity in the supernatant of the recombinant Klhap1Δ strain was 391 U ml−1 after cultured 120 h, which was 2.2-fold that of the wild type host. The relative inulinase mRNA level of the Klhap1Δ strain was 11.3-fold that of the wild type strain, and the expression plasmid was more stable in the mutant host. Based on
these results, the disruption of hap1 facilitated the high and stable expression of inulinase controlled by Kcinu promoter in K. lactis. 相似文献
8.
To obtain the protein expression of a hybrid xylanase in yeast, the gene encoding it was modified according to the codon bias
of Pichia pastoris and expressed extracellularly in this yeast as an active xylanase, MBtx, exhibited a molecular mass of approximately 35 kDa
on SDS–PAGE. The pH behavior of MBtx in terms of both activity and stability was similar to that of Btx, original gene product
in Escherichia coli, while a certain difference was observed in optimal temperature for activity and in thermal stability. HPLC analysis revealed
the xylan in wheat could be hydrolyzed by MBtx and the major hydrolysis product was xylotriose. These results showed codon
usage played a key role in regulating the expression of the hybrid xylanase in P. pastoris and the recombinant hybrid xylanase, MBtx, produced by P. pastoris could be potentially useful in feed industry. 相似文献
9.
Saul Nitsche Rocha José Abrahão-Neto María Esperanza Cerdán Andreas Karoly Gombert María Isabel González-Siso 《Applied microbiology and biotechnology》2011,89(2):375-385
In the present work, a thermophilic esterase from Thermus thermophilus HB27 was cloned into Kluyveromyces marxianus and into Kluyveromyces lactis using two different expression systems, yielding four recombinant strains. K. lactis showed the highest esterase expression levels (294 units per gram dry cell weight, with 65% of cell-bound enzyme) using an
episomal system with the PGK promoter and terminator from Saccharomyces cerevisiae combined with the K. lactis k1 secretion signal. K. marxianus showed higher secretion efficiency of the heterologous esterase (56.9 units per gram dry cell weight, with 34% of cell-bound
enzyme) than K. lactis. Hydrolytic activities for the heterologous esterases were maximum at pH values between 8.0 and 9.0 for both yeast species
and at temperatures of 50 °C and 45 °C for K. marxianus and K. lactis, respectively. When compared to previously published data on this same esterase produced in the original host or in S. cerevisiae, our results indicate that Kluyveromyces yeasts can be considered good hosts for the heterologous secretion of thermophilic esterases, which have a potential application
in biodiesel production or in resolving racemates. 相似文献
10.
José D. Antonino de SouzaJr. Sona Jain Claudia Maria Fontes de Oliveira Constância F. Ayres Wagner Alexandre Lucena 《BioControl》2009,54(3):467-473
Bacillus thuringiensis (Bt) Berliner is a promising agent for microbial control of agriculturally and medically important insects. This study aimed
at searching for Bt strains encoding Cry proteins that act more efficiently against fall armyworm. Thirty Bt strains were
isolated from soil samples in Pernambuco State and evaluated through bioassays. Among these, strain I4A7 was the most efficient
against the fall armyworm, Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae), and thus it was characterized by biochemical sodium dodecyl sulfate–polyacrylamide
gel electrophoresis (SDS–PAGE) and molecular (polymerase chain reaction (PCR) and sequencing reaction) methods. The protein
pattern of this strain on a SDS–PAGE was similar to that of B. thuringiensis
israelensis (Bti). Moreover, I4A7 cry DNA sequence showed high identity (99–100%) to genes cry4Aa, 4Ba, 10Aa, 11Aa, cyt1Aa and cyt2B from Bti. The toxicity of the newly isolated Bti-like strain upon S. frugiperda should be considered as this strain might be used in combination with other Bt strains, such as B. thuringiensis var. kurstaki (Btk).
Handling Editor: Helen Roy. 相似文献
11.
Zhongbiao Tan Jianfang Li Minchen Wu Cunduo Tang Huimin Zhang Junqing Wang 《World journal of microbiology & biotechnology》2011,27(12):2767-2774
A 777-bp cDNA fragment encoding a mature alkaline lipase (LipI) from Penicillium cyclopium PG37 was amplified by RT–PCR, and inserted into the expression plasmid pPIC9 K. The recombinant plasmid, designated as pPIC9 K-lipI,
was linearized with SalI and transformed into Pichia pastoris GS115 (his4, Mut+) by electroporation. MD plate and YPD plates containing G418 were used for screening of the multi-copy P. pastoris transformants (His+, Mut+). One transformant resistant to 4.0 mg/ml of G418, numbered as P. pastoris GSL4-7, expressing the highest recombinant LipI (rLipI) activity was chosen for optimizing expression conditions. The integration
of the gene LipI into the P. pastoris GS115 genome was confirmed by PCR analysis using 5′- and 3′-AOX1 primers. SDS–PAGE and lipase activity assays demonstrated that the rLipI, a glycosylated protein with an apparent molecular
weight of about 31.5 kDa, was extracellularly expressed in P. pastoris. When the P. pastoris GSL4-7 was cultured under the optimized conditions, the expressed rLipI activity was up to 407 U/ml, much higher than that
(10.5 U/ml) expressed with standard protocol. The rLipI showed the highest activity at pH 10.5 and 25°C, and was stable at
a broad pH range of 7.0–10.5 and at a temperature of 30°C or below. 相似文献
12.
Si-Xiu Liu Zhong-Ping Fu Rui-Min Mu Zhi-Bi Hu Fu-Jun Wang Xiang-Rong Wang 《Molecular biology reports》2010,37(4):1781-1786
A nucleic acid sequence MC, encoding Momordica Chanrantia anti-hyperglycaemic peptide MC6 (accession: AAX06814) synthesized according to Escherichia coli preferred codons, was cloned and expressed in E. coli. Recombinant protein pQE8-MC (about 3.5 kDa) was purified and analyzed by 20% SDS–PAGE and western blot. It revealed that
the expressed pQE8-MC had good solubility in aqueous media. An HPLC assay was used to confirm the expression of pQE8-MC. Subsequent
pharmacological activity assay revealed a significant hypoglycemic effect of low dose treatments of pQE8-MC on male kunming
mice. Four hours after an intravenous tail injection, the blood sugar levels of mice treated with pQE8-MC saline solution
A3 (1 mg/kg BW) decreased greatly (P < 0.01) relative to the levels of a control group. This suggests that pQE8-MC, expressed in bioengineered E. coli, has a similar hypoglycemic function to the natural protein MC6 from M. Chanrantia. These results reveal the possibility of using bio-engineered bacteria as an anti-diabetic agent. 相似文献
13.
Helicobacter pylori is the principal cause of chronic active gastritis, peptic ulcer, and gastric cancer. To develop an oral vaccine against
H. pylori infection, we had expressed the H. pylori
ureB gene (Genbank accession no. FJ436980) in nisin-controlled expression vectors using Lactococcus lactis NZ3900 as host. The ureB gene was amplified by PCR from a H.pylori strain MEL-Hp27. Then the ureB gene was fused translationally downstream of the nisin-inducible promoter nisA in a L. lactis plasmid pNZ8149. Lactose utilization based on the complementation of the lacF gene was used as a dominant selection marker for the food-grade expression system employing L. lactis NZ3900. The conditions of UreB expression in this system were optimized by orthogonal experiment. The optimized conditions
have been determined as follows: induction of expression was carried out at the cells density of OD600 ≈ 0.4 with 25 ng/ml nisin, and harvest after 5 h. The maximum percentage of recombinant UreB was estimated to be 7% of total
soluble cellular proteins and the yield was 12.9 μg/ml. Western blot demonstrated that the UreB protein was expressed in the
L. lactis transformant and had favorable immunoreactivity. These results indicated that the lactococci-derived vaccines could be promising
candidates as alternative vaccine strategies for preventing H. pylori infection. 相似文献
14.
Chun Xia Hu Zi Rong Xu Wei Fen Li Niu Dong Ping Lu Ling Lin Fu 《Biotechnology letters》2009,31(7):991-997
K88 (F4) fimbrial adhesin, FaeG, was expressed extracellularly in Lactococcus lactis using a nisin-controlled gene expression system. The antibody response and protective efficacy of the recombinant bacteria
(L. lactis [spNZ8048-faeG]) against live enterotoxigenic E. coli (ETEC) C83549 challenge were evaluated in ICR mice. Mice vaccinated with L. lactis [spNZ8048-faeG] had a significantly increased antigen-specific IgG level in the serum and decreased mortality rate (P < 0.05) compared with the control. This indicates that oral immunization of L. lactis [spNZ8048-faeG] can induce an immune-response protection upon challenge with live ETEC in ICR mice.
An erratum to this article can be found at 相似文献
15.
Shigemori S Yonekura S Sato T Nakanishi M Otani H Shimosato T 《Current microbiology》2012,64(6):569-575
In this study, we successfully developed a recombinant strain of Lactococcus lactis NZ9000 (NZ9000) that produced green fluorescent protein fused to αS1-casein (GFP-αS1Cas). A modified lactic acid bacterial vector (pNZ8148#2) was constructed by inserting genes for GFP and αS1-casein, a major cow’s milk allergen, and the resulting vector, pNZ8148#2-GFP-αS1Cas, was applied to the expression of recombinant GFP-αS1Cas protein (rGFP-αS1Cas) in NZ9000. After inducing expression with nisin, the production of rGFP-αS1Cas was confirmed by confocal laser microscopic analysis, and the expression conditions were optimized based on fluorescent
analysis and western blotting results. Moreover, the in vitro treatment of splenocytes isolated from α-casein (≥70 % αS-casein)-immunized mice with rGFP-αS1Cas resulted in increased IL-13 mRNA expression. The observed allergic activity is indicative of the Th2-cell mediated immune
response and is similar to the effects induced by exposure to α-casein. Our results suggest that the expression of rGFP-αS1Cas in NZ9000 may facilitate in vivo applications of this system aimed at improving the specificity of immunological responses
to specific milk allergen. 相似文献
16.
Xianming Wu Songfeng Wu Dong Li Jiyang Zhang Lin Hou Jie Ma Wanlin Liu Daming Ren Yunping Zhu Fuchu He 《BMC bioinformatics》2010,11(1):61
Background
Codon bias is believed to play an important role in the control of gene expression. In Escherichia coli, some rare codons, which can limit the expression level of exogenous protein, have been defined by gene engineering operations. Previous studies have confirmed the existence of codon pair's preference in many genomes, but the underlying cause of this bias has not been well established. Here we focus on the patterns of rarely-used synonymous codons. A novel method was introduced to identify the rare codons merely by codon pair bias in Escherichia coli. 相似文献17.
A novel gene, EG encoding enzymes involved in carboxymethyl cellulose (CMC) degradation was isolated, sequenced from the filamentous fungus Rhizopus stolonifer var. reflexus TP-02, and expressed in Escherichia coli BL21. The results showed that the gene amplified from the cDNA of the strain could be classified as the family of endoglucanase. During the fermentation process, the maximum endoglucanase activity (i.e. 0.715 IU/ml) of the recombinant bacteria was obtained at 36 h. The SDS–PAGE analysis on purified samples showed that a band with apparent molecular weight of about 40 kDa was detected after staining with Coomassie brilliant blue. 相似文献
18.
Qi Wang Cui Min Tingting Yan Hefang Pu Yinqiang Xin Shuangquan Zhang Lan Luo Zhimin Yin 《World journal of microbiology & biotechnology》2011,27(11):2603-2610
l-glutamine (Gln) is an important conditionally necessary amino acid in human body and potential demand in food or medicine
industry is expected. High efficiency of l-Gln production by coupling genetic engineered bacterial glutamine synthetase (GS) with yeast alcoholic fermentation system
has been developed. We report here first the application of small ubiquitin-related modifier (SUMO) fusion technology to the
expression and purification of recombinant Bacillus subtilis GS. In order to obtain GS with high Gln-forming activity, safety and low cost for food and pharmaceutics industry, 0.1% (w/v)
lactose was selected as inducer. The fusion protein was expressed in totally soluble form in E. coli, and expression was verified by SDS–PAGE and western blot analysis. The fusion protein was purified to 90% purity by nickel
nitrilo-triacetic acid (Ni–NTA) resin chromatography with a yield of 625 mg per liter fermentation culture. After the SUMO/GS
fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni–NTA column. Finally, about 121 mg
recombinant GS was obtained from 1 l fermentation culture with no less than 96% purity. The recombinant purified GS showed
great transferase activity (23 U/mg), with 25 U recombinant GS in a 50 ml reaction system, a biosynthesis yield of 27.5 g/l l-Gln was detected by high pressure liquid chromatography (HPLC) or thin-layer chromatography. Thus, the application of SUMO
technology to the expression and purification of GS potentially could be employed for the industrial production of l-Gln. 相似文献
19.
Kong Wentao Kong Jian Hu Shumin Lu Wenwei Wang Ke Ji Mingjie 《World journal of microbiology & biotechnology》2011,27(3):651-657
Capsid protein (Cap) of porcine circovirus type 2 (PCV2) encoded by orf2 is a main structural protein with strong immunoreactivity. However, capsid protein is expressed poorly in prokaryotic organisms
because of differences in codon usage. In this study, we introduce 24 synonymous mutations into orf2 by mutagenic primers and overlap extension polymerase chain reaction (OE-PCR) technique. Fourteen rare codons of orf2 were replaced with preferable codons used in Escherichia coli cells. Moreover, the nuclear localization signal (NLS) region rich in rare codon clusters at the 5′ end was deleted. The
codon-optimized genes demonstrated higher levels of expression compared with wild-type genes. The influence of rare codons
on the gene expression was eliminated by mutation. Western blot analysis confirmed the immunoreactivity of the proteins expressed
by mutated genes. Further testing demonstrated that the mutated genes were also expressed successfully in Lactococcus lactis NZ9000. The immunologically active Cap proteins produced by recombinant strains have the potential applications for serological
diagnostic assays and vaccine development against PCV2-associated diseases. 相似文献
20.
Jia Ouyang Shen Wang Yan Wang Xin Li Mu Chen Qiang Yong Shiyuan Yu 《World journal of microbiology & biotechnology》2011,27(4):751-758
The purpose of this study was to produce a Trichoderma reesei xylanase (XYN2) in Pichia pastoris and to test its potential application for pulp bleaching. The recombinant xylanase was purified by a two-step process of
ultrafiltration and gel filtration chromatography. The molecular mass of the recombinant enzyme was 21 and 25 kDa by SDS–PAGE
analysis, due to different glycosylation of the native protein. The optimum pH and temperature of the recombinant XYN2 was
5.0 and 50 °C. Enzyme activity was stable at 50 °C and at pH 5.0–7.0. The bleaching ability of the recombinant xylanase was
also studied at 50 °C and pH 6.0, using wheat straw pulp. Biobleaching of the xylanase produced chlorine dioxide savings of
up to 60%, while retaining brightness at the control level and led to a lower kappa number and small enhancements in tensile,
burst and tear strength of pulp fibers. 相似文献