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References

  1. Schuster E, Dunn-Coleman N, Frisvad JC, van Dijck PWM. 2002. On the safety of Aspergillus niger - A review. Appl. Microbiol. Biotechnol. 59: 426-435.
    Pubmed CrossRef
  2. Cairns TC, Nai C, Meyer V. 2018. How a fungus shapes biotechnology: 100 years of Aspergillus niger research. Fungal Biol. Biotechnol. 5: 13.
    Pubmed PMC CrossRef
  3. Steiniger C, Hoffmann S, Mainz A, Kaiser M, Voigt K, Meyer V, et al. 2017. Harnessing fungal nonribosomal cyclodepsipeptide synthetases for mechanistic insights and tailored engineering. Chem. Sci. 8: 7834-7843.
    Pubmed PMC CrossRef
  4. Michael W, Cherry L, Victoria DC, Fox BP, Fox JA, Wong DL, et al. 2004. Characterization of humanized antibodies secreted by Aspergillus niger. Appl. Environ. Microbiol. 70: 2567-2576.
    Pubmed PMC CrossRef
  5. Punt PJ. 2002. Filamentous fungi as cell factories for protein production. Trends Biotechnol. 20: 200-206.
    CrossRef
  6. Magaña-Ortíz D, Fernández F, Loske AM, Gómez-Lim MA. 2018. Extracellular expression in Aspergillus niger of an antibody fused to Leishmania sp. antigens.  Curr. Microbiol. 75: 40-48.
    Pubmed CrossRef
  7. Guo Y, Zheng P, Sun J. 2010. Aspergillus niger as a potential cellular factory: prior knowledge and key technology. Sheng Wu Gong Cheng Xue Bao 26: 1410-1418.
    Pubmed
  8. Zoglowek M, Lübeck PS, Ahring BK, Lübeck M. 2015. Heterologous expression of cellobiohydrolases in filamentous fungi - an update on the current challenges, achievements and perspectives. Process Biochem. 50: 211-220.
    CrossRef
  9. Krasevec N, van de Hondel C, Komel R. 2000. Expression of human lymphotoxin alpha in Aspergillus niger. Pflugers Arch. 440: R83-R85.
    Pubmed CrossRef
  10. Svetina M, Krasevec N, Gaberc-Porekar V, Komel R. 2000. Expression of catalytic subunit of bovine enterokinase in the filamentous fungus Aspergillus niger. J. Biotechnol. 76: 245-251.
    Pubmed CrossRef
  11. Roberts IN, Jeenes DJ, Mackenzie DA, Wilkinson AP, Sumner IG, Archer DB. 1992. Heterologous gene expression in Aspergillus niger: a glucoamylase-porcine pancreatic prophospholipase A2 fusion protein is secreted and processed to yield mature enzyme. Gene 122: 155-161.
    Pubmed CrossRef
  12. Zhang H, Yan JN, Zhang H, Qi LT, Xu Y, Zhang YY, et al. 2018. Effect of gpd box copy numbers in the gpdA promoter of Aspergillus nidulans on its transcription efficiency in A. niger. FEMS Microbiol. Lett. 1: 365.
    CrossRef
  13. Liu F, Wang B, Ye Y, Pan L. 2017. High level expression and characterization of tannase tan7 using Aspergillus niger SH-2 with low-background endogenous secretory proteins as the host. Protein Expr. Purif. 144: 71-75.
    Pubmed CrossRef
  14. Zhang H, Wang S, Zhang XX, Ji W, Song FP, Zhao Y, et al. 2016. The amyR-deletion strain of Aspergillus niger CICC2462 is a suitable host strain to express secreted protein with a low background. Microb. Cell Fact. 15: 11.
    Pubmed PMC CrossRef
  15. Kamaruddin N, Storms R, Mahadi NM, Illias RM, Abu Bakar FD, Murad AMA. 2018. Reduction of extracellular proteases increased activity and stability of heterologous protein in Aspergillus niger. Arab. J. Sci. Eng. 43: 3327-3338.
    CrossRef
  16. Zhang XF, Ai YH, Xu Y, Yu XW. 2019. High-level expression of Aspergillus niger lipase in Pichia pastoris: characterization and gastric digestion in vitro. Food Chem. 274: 305-313.
    Pubmed CrossRef
  17. Saxena RK, Davidson WS, Sheoran A, Giri B. 2003. Purification and characterization of an alkaline thermostable lipase from Aspergillus carneus. Process Biochem. 39: 239-247.
    CrossRef
  18. Xia J-l, Huang B, Nie Z-y, Wang W. 2011. Production and characterization of alkaline extracellular lipase from newly isolated strain Aspergillus awamori HB-03. J. Cent. South Univ. 18: 1425.
    CrossRef
  19. Shu ZY, Yan YJ, Yang JK, Xu L. 2007. Aspergillus niger lipase: gene cloning, over-expression in Escherichia coli and in vitro refolding. Biotechnol. Lett. 29: 1875-1879.
    Pubmed CrossRef
  20. Yang J, Yan X, Zhang Z, Jiang X, Yan Y. 2009. Two-step synthesis of the full length Aspergillus niger lipase gene lipA leads to high-level expression in Pichia pastoris. Sheng Wu Gong Cheng Xue Bao 25: 381-387.
    Pubmed
  21. Kozak M. 1986. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44: 283-292.
    Pubmed CrossRef
  22. Ahangarzadeh S, Daneshvar MH, Rajabi-Memari H, Galehdari H, Alamisaied K. 2012. Cloning, transformation and expression of human interferon α2b Gene in tobacco plant (Nicotiana tabacum cv. xanthi). Jundishapur J. Nat. Pharm. Prod. 7: 111-116.
    Pubmed PMC CrossRef
  23. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.
    Pubmed CrossRef
  24. Xu Y, Wang YH, Liu TQ, Zhang H, Zhang H, Li J. 2018. The GlaA signal peptide substantially increases the expression and secretion of α-galactosidase in Aspergillus niger. Biotechnol. Lett. 40: 949-955.
    Pubmed CrossRef
  25. Li M, Zhou L, Liu M, Huang Y, Sun X, Lu F. 2013. Construction of an engineering strain producing high yields of alpha-transglucosidase via Agrobacterium tumefaciens-mediated transformation of Asperillus niger. Biosci. Biotechnol. Biochem. 77: 1860-1866.
    Pubmed CrossRef
  26. Madhavan A, Pandey A, Sukumaran RK. 2017. Expression system for heterologous protein expression in the filamentous fungus Aspergillus unguis. Bioresour. Technol. 245: 1334-1342.
    Pubmed CrossRef
  27. Canseco-Pérez MA, Castillo-Avila GM, Chi-Manzanero B, Islas-Flores I, Apolinar-Hernández MM, Rivera-Muñoz G, et al. 2018. Fungal screening on olive oil for extracellular triacylglycerol lipases: selection of a trichoderma harzianum strain and genome wide search for the genes. Genes 9(2). pii:E62.
    Pubmed PMC CrossRef
  28. Jo BS, Choi SS. 2015. Introns: the functional benefits of introns in genomes. Genomics Inform. 13: 112-118.
    Pubmed PMC CrossRef
  29. Kurachi S, Hitomi Y, Furukawa M, Kurachi K. 1995. Role of intron I in expression of the human factor IX gene. J. Biol. Chem. 270: 5276-5281.
    Pubmed CrossRef
  30. Gniadkowski M, Hemmings-Mieszczak M, Klahre U, Liu HX, Filipowicz W. 1996. Characterization of intronic uridine-rich sequence elements acting as possible targets for nuclear proteins during pre-mRNA splicing in Nicotiana plumbaginifolia. Nucleic Acids Res. 24: 619-627.
    Pubmed PMC CrossRef
  31. Jun X, Zhen GZ. 2003. Intron requirement for AFP gene expression in Trichoderma viride. Microbiology 149: 3093-3097.
    Pubmed CrossRef
  32. Gonzalez-Hilarion S, Paulet D, Lee KT, Hon CC, Lechat P, Mogensen E, et al. 2016. Intron retention-dependent gene regulation in Cryptococcus neoformans. Sci. Rep. 6: 32252.
    Pubmed PMC CrossRef
  33. Kozak M. 2005. Regulation of translation via mRNA structure in prokaryotes and eukaryotes. Gene 361: 13-37.
    Pubmed CrossRef
  34. Du M, Ye L, Liu J, Liu J, Yang L. 2008. Enhancement of GFP expression by Kozak sequence +4G in HEK293 cells. Sheng Wu Gong Cheng Xue Bao 24: 491-494.
    Pubmed
  35. Olafsdóttir G, Svansson V, Ingvarsson S, Marti E, Torsteinsdóttir S. 2008. In vitro analysis of expression vectors for DNA vaccination of horses: the effect of a Kozak sequence. Acta Vet. Scand. 50: 44.
    Pubmed PMC CrossRef
  36. Li J, Liang Q, Song WJ, Marchisio MA. 2017. Nucleotides upstream of the Kozak sequence strongly influence gene expression in the yeast S. cerevisiae. J. Biol. Eng. 11: 25.
    Pubmed PMC CrossRef
  37. Mahadik ND, Puntambekar US, Bastawde KB, Khire JM, Gokhale DV. 2002. Production of acidic lipase by Aspergillus niger in solid state fermentation. Process Biochem. 38: 715-721.
    CrossRef
  38. Guang L. 2015. Purification and characterization of a lipase with high thermostability and polar organic solvent-tolerance from Aspergillus niger AN0512. Lipids 11: 1155-1163.
    Pubmed CrossRef
  39. dos Santos EAL, Lima ÁS, Soares CMF, Santana L. 2017. Lipase from Aspergillus niger obtained from mangaba residue fermentation: biochemical characterization of free and immobilized enzymes on a sol-gel matrix. Acta Sci.Technol. 39: 1-8.
    CrossRef

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Article

Research article

J. Microbiol. Biotechnol. 2020; 30(2): 196-205

Published online February 28, 2020 https://doi.org/10.4014/jmb.1906.06028

Copyright © The Korean Society for Microbiology and Biotechnology.

Improved Homologous Expression of the Acidic Lipase from Aspergillus niger

Zhu Si-Yuan , Xu Yan and Yu Xiao-Wei *

Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, P.R. China

Received: June 14, 2019; Accepted: November 19, 2019

Abstract

In this study, the acidic lipase from Aspergillus niger (ANL) was homologously expressed in A. niger. The expression of ANL was significantly improved by the expression of the native ANL with the introns, the addition of the Kozak sequence and the optimization of the signal sequences. When the cDNA sequence of ANL fused with the glaA signal was expressed under the gpdA promoter in A. niger, no lipase activity could be detected. We then tried to improve the expression by using the full-length ANL gene containing three introns, and the lipase activity in the supernatant reached 75.80 U/ml, probably as a result of a more stable mRNA structure. The expression was further improved to 100.60 U/ml by introducing a Kozak sequence around the start codon due to a higher translation efficiency. Finally, the effects of three signal sequences including the cbhI signal, the ANL signal and the glaA signal on the lipase expression were evaluated. The transformant with the cbhI signal showed the highest lipase activity (314.67 U/ml), which was 1.90-fold and 3.13-fold higher than those with the ANL signal and the glaA signal, respectively. The acidic lipase was characterized and its highest activity was detected at pH 3.0 and a temperature of 45ºC. These results provided promising strategies for the production of the acidic lipase from A. niger.

Keywords: Aspergillus niger, acidic lipase, expression, characterization, Agrobacterium-mediated transformation

1. Introduction

Aspergillus niger is one of the most important microorganisms in the production of organic acids and a wide range of enzymes [1]. Many substances produced in A. niger are considered as Generally Recognized as Safe (GRAS) by the United States Food and Drug Administration [1]. A variety of industrially important enzymes such as oxidases, cellulases, dehydrogenases, hydrolases and pectinases have been produced in A. niger [2]. In addition, other structurally complex antibody proteins or small peptides, such as nucleoside hydrolase, sterol 24-c-methyltransferase, humanized immunoglobulin G1 antibodies, cyclodepsi-peptides (octa-enniatin and octa-beauvericin), were also successfully expressed in A. niger [3-6]. In recent years, A. niger has been used for the production of high-value recombinant antibodies with a lower cost since the protein produced in A. niger is processed more similarly to mammals [6, 7]. The products from A. niger have been widely applied in food, feed, bioenergy and pharmaceutical processes, amongst others [2].

Many attempts have been made to increase the expression of proteins in filamentous fungi [8], for example, by optimi-zation of promoters and copy numbers, or expression as fusion protein [9-11]. The expression of xylanase was improved by 2.27-fold through modification of the gpdA promoter [12]. The A. niger glucoamylase gene is commonly used as a fusion tag for improvement of foreign gene expression in A. niger, including the porcine pancreatic prophospholipase A2 (proPLA2) gene [11], the human lymphotoxin alpha (LTα) gene [9] and the catalytic subunit of the bovine enterokinase (EKL) gene [10].

In addition, a superior host strain is very important to in achieving target protein expression at a high level. A host with a lower protein background could be obtained by disrupting the gene of the original high-yield protein in the strain. Without the interference of endogenous proteins, a higher yield could be obtained for recombinant proteins in the host. At present, tannase, xylanase, mannase and asparaginase have been successfully expressed at a high level in strains with a lower endogenous protein background [13, 14]. In another example, Kamaruddin et al. [15] increased the yield of cutinase by 36-fold in A. niger by down-regulating the protease expression.

Acidic lipases are in a category of extreme enzymes that are stable and exhibit high catalytic activity in extreme acidic conditions. Due to the acidogenic properties of A. niger, most of the proteins or metabolites in A. niger are acid-tolerant. The A. niger lipase (ANL) is an acidic lipase that maintains high activity under acidic conditions and has a wide range of applications in food and chemical industries [16]. ANL has exhibited better resistance to acidic conditions compared to the other Aspergillus sp. lipases [17, 18]. For example, the lipase from A. carneus was not stable at pH 6 as its optimum pH was 9.0 [17], and the highest stability of the lipase from A. awamori was at pH 8.0-9.0 [18]. However, the high cost for the production of the acidic A. niger lipase remains a barrier for its industrial applications. At present, ANL has been heterologously expressed in Escherichia coli and Pichia pastoris. The specific activity of the ANL was 110 U/(mg protein) in E. coli and could be improved two folds by addition of Ca2+ [19]. When expressed in P. patoris the activity of ANL fused with a small ubiquitin-related modifier (SUMO) reached 173 U/(ml supernatant) and 432 U/(mg protein) in the shake flasks [20].

In this study, the ANL gene was homologously expressed in A. niger. Improvement of the A. niger lipase (ANL) expression was achieved through three strategies, including the addition of Kozak sequences for enhancing the translation efficiency, the expression of the native ANL with the introns for improving the stability of the mRNA, and optimization of signal sequences for enhancing the efficiency of extracellular secretion.

Materials and Methods

Strains, Plasmids, Media and Culture Conditions

E. coli TOP10 was used to maintain and amplify the plasmids. A. niger 89 was preserved in our lab and used as the expression host, as it has a low-background of endogenous secretory proteins and is hygromycin B-sensitive. Agrobacterium tumefaciens AGL1 was used for A. tumefaciens-mediated transformation (ATMT).

LB medium (1% w/v peptone, 0.5% w/v yeast extract, 1% w/v NaCl with 50 μg/ml kanamycin) was used for cultivation of E. coli. YEB medium (0.1% w/v yeast extract, 0.5% w/v peptone, 0.5% w/v beef extract, 0.5% w/v sucrose, 0.0493% w/v MgSO4·7H2O, pH 7.0 with 50 μg/ml Rif ) was used for the A. tumefaciens growth at 28°C 200 rpm for 12-16 h. IM medium (0.145% w/v KH2PO4, 0.205% w/v K2HPO4, 0.05% w/v (NH4)2SO4, 0.05% w/v MgSO4·7H2O, 0.015% w/v NaCl, 0.0066% w/v CaCl2, 0.000248% w/v FeSO4·7H2O, 0.18% w/v glucose, 0.5% v/v glycerin with 40 mM MES (pH5.3) and 200 μM Acetosyringone (AS)) was used for pre-incubation of A. tumefaciens at 28ºC 200 rpm and IM medium supplemented with 2% agar (IM plate) was used for co-cultivation of A. tumefaciens and A. niger at 23ºC. The potato dextrose agar (PDA plate, OKA Co. Ltd., China) was used for sporulation of A. niger. Fermentation medium (0.2% w/v NaNO3, 0.05% w/v KCl, 0.05% w/v MgSO4·7H2O, 0.1% w/v K2HPO4·3H2O, 0.001% w/v FeSO4·7H2O, 1% w/v (NH4)2SO4, 4% w/v corn starch, 2% v/v olive oil, 10% v/v 10 × 100 mmol/l pH 6.0 phosphate buffer) was used for the protein expression in A. niger. The rhodamine-olive oil plate (0.2% w/v NaNO3, 0.05% w/v KCl, 0.05% w/v MgSO4·7H2O, 0.1% w/v K2HPO4·3H2O, 0.25% w/v glucose, 0.25% w/v corn starch, 0.25% w/v maltose, 10% v/v 10 × 100 mmol/l pH 6.0 phosphate buffer, 1*10-3% w/v Rhodamine B (Biosharp Co. Ltd., China), 1% v/v olive oil emulsion (olive oil was emulsified with 4% w/v polyvinyl alcohol (PVA) in a ratio of 1:3 (v/v) by a high-speed homogenizer), 2% w/v agar) was used for screening.

Construction of Recombinant Strains

The full-length gene of ANL (ANL1000) (GenBank: DQ647700.1) contains three introns. ANL1000 was amplified from the A. niger genome and the intron-free ANL gene (ANL) was amplified by overlap extension PCR. In the plasmid pCAMglaS-ANL, the intron-free ANL gene was fused with the glaA signal sequence and expressed under the gpdA promoter. The ANL gene in pCAMglaSANL was replaced with ANL1000 by restriction enzyme digestion (ApaI and BamHI) to construct the plasmid pCAMglaS-ANL1000 (Fig. 1). Three kinds of signal sequences were used, those of glucoamylase, ANL and exoglucanase cbhI. The signal sequences were introduced through PCR. Six vectors were constructed with different combinations of the three signal sequences and two lengths of A. niger lipase gene (ANL, ANL1000). According to the design principle of the kozak sequence [21], the kozak sequence (GCCA-3CCA+1TGG+4) was added around the start codon A+1TG, and two other nucleotides were added downstream of the Kozak sequence to avoid frameshift. As indicated in Table S3, the other two added nucleotides are AT for the glaA signal and AG for the ANL signal and the cbhI signal right after the G+4. All plasmids contained the Hyg marker gene for selection on hygromycin B. 6×his-tag was added at the C-terminus of the lipase for purification of the expressed enzyme by affinity chromatography. The work flow for construction of the plasmids is shown in Fig. 2. All primers used are listed in Table S1. All vectors proved to be correctly assembled by restriction endonuclease digestion and sequencing of the assembly joint.

Figure 1. Construction of pCAMglaS-ANL and pCAMglaS-ANL1000 expression vectors.
Figure 2. Construction of 6 kinds of expression vectors.

Agrobacterium-Mediated Transformation (ATMT)

In the recombinant plasmid the fragment including the target gene between two elements, RB (right boundary) and LB (left boundary), was randomly inserted into the genome of A. niger by ATMT. The recombinant plasmids were transformed into A. tumefaciens AGL1 by the freeze-thaw method [22]. Positive transformants of AGL1 were cultivated in 5 mL of YEB at 28ºC for 16 h, and then collected and incubated in IM liquid medium at 28ºC to an OD600 of 0.8-1.0. A. niger spore suspension was generated from strains cultivated for 3-5 days at 30ºC. A mixture of 100 μl of the A. niger spore suspension (107 spores/ml) and 100 μl of the positive transformant of AGL1 was spread on the cellophane covering the IM plates. The co-cultivation was carried out in the dark at 23ºC for 48 h. Afterwards, the cellophane was transferred to the PDA plates with hygromycin B (200 μg/ml) and cefotaxime sodium (200 μg/ml). The plates were incubated at 30ºC for 2-3 d. Then, the transformants were rescreened on the PDA plates with hygromycin B (200 μg/ml). The transformants were purified by spore isolation for at least three successive generations on the PDA plates with hygromycin B (200 μg/ml). The positive transformants were confirmed by PCR with the identification primers (Table S2).

Expression of ANL in A. niger

The wild-type strain and the positive transformants of A. niger were grown on the PDA plates for 3-5 d, and then the spores were washed with saline. The spore suspension was inoculated into 50 ml fermentation medium with a final concentration of 2 × 105 spores/ml in 250 ml flasks shaken (200 rpm) at 28ºC for 168 h. Protein concentrations of the supernatant were determined by the Bradford method [23].

Purification and Identification of ANL

ANL fused with a 6×his-tag at the C-terminus was purified by Ni-NTA column affinity chromatography using the ÄKTA protein purification system. The fermentation supernatants were collected by filtering through a 200-mesh nylon membrane, then filtered through 0.22 μm aqueous microfiltration membrane. The system was equilibrated with buffer A (150 mM NaCl, 20 mM Tris-HCl, pH 8.0) and the target protein was eluted with buffer B (150 mM NaCl, 20 mM Tris-HCl, 0.5 M imidazole, pH 8.0). The purified protein was examined by SDS-PAGE and stored in buffer A at -80ºC for further analysis. For preparation of the SDS-PAGE samples, the purified protein solution or the fermentation supernatant was mixed with the 2X SDS loading buffer in equal volume. The samples were boiled for 10 min, centrifuged for 5 min, and then 10 μl of each sample was loaded into the SDS-PAGE gel.

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) was used for protein identification. The target protein on SDS-PAGE was cut out and digested. The peptide mass fingerprinting of the target protein was analyzed using Mascot software (http://www.matrixscience.com).

Assay of Enzyme Activity

The enzyme activity of ANL was determined by alkali titration using olive oil as the substrate. Olive oil was emulsified with 4%w/v polyvinyl alcohol (PVA) at a ratio of 1:3 (v/v) by a high-speed homogenizer and used as the substrate. Each reaction contained 4 ml of substrate and 5 ml of 50 mM citrate-phosphate buffer (pH 3.0) and 1 ml enzyme solution. For measurement of the purified enzyme, the activity assays were done using 1 ml of 0.25 mg /ml enzyme solution. For measurement of the activity of the supernatant, the activity assays were done using 1 ml of the supernatant. The reaction was incubated at 45ºC for 15 min, and then terminated by adding 15 ml of 95% ethanol solution. The heat-inactivated enzyme solution was used as the blank control. The reaction solution was titrated with 0.1 mol/l NaOH standard solution. The amount of enzyme that produces 1 μmol of fatty acid in one minute is defined as one unit of enzyme activity. The enzyme activity was calculated using the following equation.

X=(B-A)×C×nt

X: enzyme activity, U/L; B: volume of NaOH consumed by titrating the sample, ml; A: volume of NaOH consumed by titrating the blank; C: NaOH concentration, mol/L; n: dilution factor; t: reaction time.

Characterization of the Purified Enzyme ANL

The optimum pH of the purified enzyme ANL was determined by measuring the lipase activity at 45ºC in various 50 mM citrate-phosphate buffers (pH 2.0-9.0). The pH stability was studied by incubating the purified enzyme ANL in different 50 mM citrate-phosphate buffers with pH ranging from 2.0 to 10.0 for 24 h at 45ºC. The samples were taken at 2-h intervals for the first 12 h and for the last timepoint at 24 h. The residual activity was then assayed under the standard assay conditions.

The optimum temperature of the purified enzyme ANL was determined by measuring the enzyme activity at various temperatures (25ºC-60ºC) in 50 mM of citrate-phosphate buffer, pH 3. The thermal stability was determined by incubating the purified enzyme ANL for 24 h at the desired temperatures (30ºC-60ºC) followed by measuring the residual activity. The samples were taken at 2-h intervals for the first 12 h and for the last timepoint at 24 h.

The kinetic parameters of ANL were determined using the emulsified triglyceride olive oil (0-120 g/l) as the substrate and assayed using the alkali titration method under optimum assay conditions. The values of the kinetic parameters Km and kcat were determined by Lineweaver-Burk plot and Michaelis-Menten equation.

Results and Discussion

Design of the Expression Vectors

The expression cassettes of the plasmids are shown in Table 1. ANL is the cDNA gene without intron, and ANL1000 represents the native full-length ANL gene containing three introns. The length of the native ANL gene is about 1,000 bp. Thus, we named the native ANL ‘ANL1000.’ The Kozak sequence (GCCA-3CCA+1TGG+4) was added around the start codon of the ANL gene in the plasmid pCAMkoglaS-ANL, and two other nucleotides were added downstream of the kozak sequence G+4 to form a codon to avoid frameshift. Three signal sequences were selected including the glucoamylase (glaA) signal sequence, the ANL signal sequence and the cellobiohydrolase I (cbhI) signal sequence for the expression of ANL in A. niger. The glaA signal sequence is used widely in A. niger, because glucoamylase is expressed in high amounts in A. niger [24], and the signal sequence from a highly expressed gene usually helps the extracellular expression of the target gene [25]. The ANL signal sequence is its own signal sequence. Zoglowek et al.[8] suggest that natural signal sequence may be more suitable for gene expression than other signal sequences. The cbhI signal sequence was also used frequently in A. niger. In the study of Madhavan et al. [26], the cbhI signal sequence exhibited higher secretion than the glaA signal sequence. The signal sequences are listed in Table S3.

Table 1 . Plasmids used for the acidic lipase expression..

PlasmidTransformantsExpression cassette
1pCAMglaS-ANLglaS-ANLPgpdA- glaA signal-ANL-his tag-Tcgr
2pCAMglaS-ANL1000glaS-ANL1000PgpdA- glaA signal-ANL1000- his tag-Tcgr
3pCAMkoglaS-ANLkoglaS-ANLPgpdA-kozak seq-glaA signal-ANL- his tag-Tcgr
4pCAMkoANLS-ANLkoANLS-ANLPgpdA-kozak seq-ANL signal-ANL- his tag-Tcgr
5pCAMkocbhS-ANLkocbhS-ANLPgpdA-kozak seq-cbhⅠ signal-ANL- his tag-Tcgr
6pCAMkoglaS-ANL1000koglaS-ANL1000PgpdA-kozak seq-glaA signal-ANL1000- his tag-Tcgr
7pCAMkoANLS-ANL1000koANLS-ANL1000PgpdA-kozak seq-ANL signal-ANL1000- his tag-Tcgr
8pCAMkocbhS-ANL1000kocbhS-ANL1000PgpdA-kozak seq-cbhⅠ signal-ANL1000- his tag-Tcgr



Expression of ANL in A. niger

Identification of positive transformants. We isolated several A. niger strains from the environment. The endogenous secretory proteins in the supernatant were detected on SDS-PAGE and we found that the secretory proteins in A. niger 89 were much lower than those in other strains (data not shown). Thus, we chose A. niger 89 as the expression host in our study. The plasmids indicated in Table 1 were transformed into A. niger 89 by ATMT for the expression of ANL. The positive transformants verified by PCR were plated on the rhodamine-olive oil plate as shown in Fig. 3. The lipase produced by the transformant can hydrolyze olive oil to generate fatty acids. The fatty acids then react with rhodamine B to develop a red color under natural light and to emit fluorescence under ultraviolet light [27]. The larger the color halo around the colony, the higher the lipase activity. Thus, the positive transformants with higher lipase activity were selected according to the size of the color halo. The wild-type A. niger 89 didn’t show an obvious color halo. The other 8 kinds of transformants showed different sizes of color halos on the rhodamine-olive oil plates. Although transformed with the same plasmid, the expression level of lipase varied among different transformants due to the random insertion of the expression cassette in the genome [26].

Figure 3. A. niger 89 transformed with eight different plasmids grown on the rhodamine-olive oil plates for 2 days (the wild-type A. niger 89 as the control). The letters represent 8 different constructs. The bigger the red halo, the higher the lipase activity. To determine the lipase activity shown in Fig. 4 we selected three transformants for each construct with relatively bigger halos indicated with a square.

The effects of the introns, the Kozak sequence and the signal sequences on the ANL expression. The wild-type strain A. niger 89 and the transformants were cultivated using corn starch and olive oil as the main carbon sources. Olive oil was used both for the carbon source and the inducer of the lipase. We followed the lipase production during fermentation. The lipase began to secrete into the medium between 48 h and 72 h and the highest lipase production was reached at around 168 h during the fermentation. The wild-type strain A. niger 89, the transformants of pCAMglaS-ANL and pCAMglaS-ANL1000 were characterized for the effect of the introns. As shown in Fig. 4A, there was no obvious target band for either the wild type (A. niger 89) or the transformants of pCAMglaS-ANL (glaS-ANL). The target band for the transformant of pCAMglaS-ANL1000 with the introns (glaS-ANL1000) could be detected and the lipase activity in the supernatant reached 75.80 U/ml. Although introns do not participate in protein-coding sequences, they do play significant functional roles in eukaryotes, including regulation of alternative splicing, positive regulation of gene expression, regulation of nonsense-mediated decay, and provision of a new gene source as well as impact on natural selection [28]. In our case, the presence of the introns may enable a more stable secondary structure, which protects pre-mRNA from degradation in the nucleus [29, 30]. Moreover, the introns can assist in the transport of pre-mRNA [31] and regulation of mRNA maturation [32]. In this study, the expression of the ANL gene with introns was significantly higher compared to the cDNA.

Figure 4. SDS-PAGE and the lipase activities of the transformants. (A) SDS-PAGE of the fermentation supernatants after cultivation for 168 h. (B) Enzyme production in the supernatants after cultivation for 168 h. The lipase activity was measured on emulsified olive oil at pH 3.0 and 45ºC. Error bars indicated the standard deviation of the enzyme activities of three transformants for each construct in biological triplicates.

The effect of the Kozak sequence on the lipase expression was also evaluated using two sets of comparison (pCAMglaS-ANL vs pCAMkoglaS-ANL, pCAMglaS-ANL1000 vs pCAMkoglaS-ANL1000). After addition of the kozak sequence in pCAMkoglaS-ANL, the lipase activity increased from undetectable to 61.30 U/ml. When the Kozak sequence was added in pCAMkoglaS-ANL1000 with the introns, the lipase activity was improved by 1.33-fold compared with that of pCAMglaS-ANL1000.

Kozak et al. [21] found that the base pair near the initiation codon has a certain effect in translation, and the certain sequence, GCCACCATGG, is known as the ‘Kozak sequence’ and shows a higher translation level. The Kozak sequence enhances the translation efficiency by optimization of the ATG environment to avoid the leaky ribosomal-scanning [33]. The Kozak sequence has been applied to increase the expression level of foreign genes in mammalian cells [34, 35] and Saccharomyces cerevisiae [36], but there is no report on the effect of the Kozak sequence on protein expression in A. niger.

The effects of three signal sequences (glaA signal sequence, ANL signal sequence and cbhI signal sequence) were analyzed and tested with both ANL1000 and ANL. As shown in Fig. 4 and Table 2, the enzyme production and protein concentration in the supernatant of the transformants were evaluated. The enzyme production in the supernatant has a positive correlation with the protein concentration. Higher enzyme activity per milliliter (U/ml) indicates higher enzyme production in the supernatant. The enzyme production of the pCAMkocbhS-ANL1000 transformant (kocbhS-ANL1000) was the highest (314.67 U/ml), followed by koANLS-ANL1000, koglaS-ANL1000, kocbhS-ANL, koANLS-ANL, glaS-ANL1000, koglaS-ANL, glaS-ANL. The enzyme production of all ANL1000 proteins was higher compared to ANL. Regarding the signal sequence, the transformants with the cbhI signal sequence showed the highest expression, followed by those with the ANL and glaA signal sequence. It is worth noting that the thickness of the protein bands on SDS-PAGE was not correlated very well with the average protein concentration in Table 2. The reason was that the loading sample for each construct was not a mixture of three transformants, but one randomly selected from these replicates.

Table 2 . Protein concentration in the supernatant of the transformants after cultivation for 168 h..

TransformantsProtein concentration (mg/mL)*
A. niger 890.08±0.01
glaS-ANL0.09±0.01
glaS-ANL10000.14±0.02
koglaS-ANL0.12±0.01
koANLS-ANL0.18±0.02
kocbhS-ANL0.19±0.02
koglaS-ANL10000.20±0.01
koANLS-ANL10000.23±0.03
kocbhS-ANL10000.32±0.02

*The standard deviation indicates three biological replicates of three transformants for each construct..



Purification and Identification of ANL

As shown in Fig. 5 the fermentation supernatant and the purified enzyme were detected by SDS-PAGE. About 0.15 g of ANL enzyme was purified from 1 L fermentation supernatant. The specific activity of the purified ANL is 680.34 ± 4.75 U/mg. Two bands were detected for both the fermentation supernatant samples and the purified protein. The four bands of proteins were identified by MALDI-TOF-MS. The results showed that band-A and band-C contained four unique fragments of VTHLNDIVPR, VGNYALAEHITSQGSGANFR, MLLEFDLTNNFGGTAGF LAADNTNKR and NDGYSVELYTYGCPR, the band-B and the band-D contained two unique fragments of VGNYALAEHITSQGSGANFR and VTHLNDIVPR. By comparison, all these unique fragments belong to ANL. The difference in size may be due to post-translational modifications such as glycosylation.

Figure 5. SDS-PAGE of the fermentation supernatant and the purified protein. 1. The fermentation supernatant of kocbhS-ANL1000 after cultivation for 168 h. 2. The purified protein loaded with 3 μg. Band A, B, C, and D were cut from gel and analyzed using MALDI-TOF-MS.

Enzymatic Characterization

Effects of pH on the lipase activity and stability. As shown in Fig. 6A the optimum pH of ANL is pH 3. ANL exhibits a high stability in a broad range of pH from 3 to 10. After being retained under pH 3 for 24 h, the relative activity of ANL is still more than 60% (Fig. 6B). In other reports the optimum pH of ANL was around 2.5-5 [37-39]. During fermentation A. niger produces a high amount of acid, which might be the reason for a very acid-resistant ANL.

Figure 6. Effect of pH on the activity and stability of ANL. (A) The optimal pH. The activity was determined at pH 2.0-9.0 and 45ºC using olive oil as the substrate. (B) The pH stability of ANL. The pH stability of ANL was determined at 45ºC and pH 3.0 after being incubated in pH 2.0-10.0 50 mM citrate-phosphate buffer for up to 24 h. Error bars indicate the standard deviation of three biological replicates.

Effects of temperature on the lipase activity and stability. As shown in Fig. 7 the optimum temperature of ANL is 45ºC. In the analysis of thermostability, the relative activity of the purified ANL was still more than 70% after incubation at 30ºC and 40ºC for 24 h. However, the relative activity decreased significantly when incubated at 50ºC for 12 h. At 60ºC the relative activity dropped to 30% within 5 h and was completely inactivated within 12 h.

Figure 7. Effect of temperature on the activity and stability of ANL. (A) The optimal temperature. The activity was determined at 25-60ºC and pH 3.0. (B) The temperature stability of ANL. The temperature stability of ANL was determined at 45ºC and pH 3.0 after incubation for up to 24 h at temperatures of 30-60ºC. Error bars indicate the standard deviation of three biological replicates.

Determination of kinetic parameters. The olive oil emulsion was used as the substrate to determine the kinetic parameters. As shown in Table 3, the Km and kcat values are 9.30 ± 1.04 g/l and 391.66 ± 8.69 s-1, respectively. Km reflects the affinity of the enzyme to the substrate. The lower the Km, the higher the affinity with the substrate. In this study, ANL has a better affinity towards olive oil compared with other studies in which the Km values of the lipases from A. niger were 77 mM (22 g/l) [39] and 108 g/l [16] using olive oil as the substrates.

Table 3 . The kinetic parameters of ANL..

LipaseKm (g/L)kcat(1/s)kcat/Km(L/(s g))
ANL9.30±1.04391.66±8.6942.25±3.35

Supplemental Materials

Acknowledgements

Financial support from the National Natural Science Foundation of China (31671799), Six Talent Peaks Project in Jiangsu Province (NY-010), the National First-Class Discipline Program of Light Industry Technology and Engineering (LITE2018-09), and the National High Technology Research and Development Program of China (863 Program) (2012AA022207) is greatly appreciated.

Conflict of Interest

The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.Construction of pCAMglaS-ANL and pCAMglaS-ANL1000 expression vectors.
Journal of Microbiology and Biotechnology 2020; 30: 196-205https://doi.org/10.4014/jmb.1906.06028

Fig 2.

Figure 2.Construction of 6 kinds of expression vectors.
Journal of Microbiology and Biotechnology 2020; 30: 196-205https://doi.org/10.4014/jmb.1906.06028

Fig 3.

Figure 3.A. niger 89 transformed with eight different plasmids grown on the rhodamine-olive oil plates for 2 days (the wild-type A. niger 89 as the control). The letters represent 8 different constructs. The bigger the red halo, the higher the lipase activity. To determine the lipase activity shown in Fig. 4 we selected three transformants for each construct with relatively bigger halos indicated with a square.
Journal of Microbiology and Biotechnology 2020; 30: 196-205https://doi.org/10.4014/jmb.1906.06028

Fig 4.

Figure 4.SDS-PAGE and the lipase activities of the transformants. (A) SDS-PAGE of the fermentation supernatants after cultivation for 168 h. (B) Enzyme production in the supernatants after cultivation for 168 h. The lipase activity was measured on emulsified olive oil at pH 3.0 and 45ºC. Error bars indicated the standard deviation of the enzyme activities of three transformants for each construct in biological triplicates.
Journal of Microbiology and Biotechnology 2020; 30: 196-205https://doi.org/10.4014/jmb.1906.06028

Fig 5.

Figure 5.SDS-PAGE of the fermentation supernatant and the purified protein. 1. The fermentation supernatant of kocbhS-ANL1000 after cultivation for 168 h. 2. The purified protein loaded with 3 μg. Band A, B, C, and D were cut from gel and analyzed using MALDI-TOF-MS.
Journal of Microbiology and Biotechnology 2020; 30: 196-205https://doi.org/10.4014/jmb.1906.06028

Fig 6.

Figure 6.Effect of pH on the activity and stability of ANL. (A) The optimal pH. The activity was determined at pH 2.0-9.0 and 45ºC using olive oil as the substrate. (B) The pH stability of ANL. The pH stability of ANL was determined at 45ºC and pH 3.0 after being incubated in pH 2.0-10.0 50 mM citrate-phosphate buffer for up to 24 h. Error bars indicate the standard deviation of three biological replicates.
Journal of Microbiology and Biotechnology 2020; 30: 196-205https://doi.org/10.4014/jmb.1906.06028

Fig 7.

Figure 7.Effect of temperature on the activity and stability of ANL. (A) The optimal temperature. The activity was determined at 25-60ºC and pH 3.0. (B) The temperature stability of ANL. The temperature stability of ANL was determined at 45ºC and pH 3.0 after incubation for up to 24 h at temperatures of 30-60ºC. Error bars indicate the standard deviation of three biological replicates.
Journal of Microbiology and Biotechnology 2020; 30: 196-205https://doi.org/10.4014/jmb.1906.06028

Table 1 . Plasmids used for the acidic lipase expression..

PlasmidTransformantsExpression cassette
1pCAMglaS-ANLglaS-ANLPgpdA- glaA signal-ANL-his tag-Tcgr
2pCAMglaS-ANL1000glaS-ANL1000PgpdA- glaA signal-ANL1000- his tag-Tcgr
3pCAMkoglaS-ANLkoglaS-ANLPgpdA-kozak seq-glaA signal-ANL- his tag-Tcgr
4pCAMkoANLS-ANLkoANLS-ANLPgpdA-kozak seq-ANL signal-ANL- his tag-Tcgr
5pCAMkocbhS-ANLkocbhS-ANLPgpdA-kozak seq-cbhⅠ signal-ANL- his tag-Tcgr
6pCAMkoglaS-ANL1000koglaS-ANL1000PgpdA-kozak seq-glaA signal-ANL1000- his tag-Tcgr
7pCAMkoANLS-ANL1000koANLS-ANL1000PgpdA-kozak seq-ANL signal-ANL1000- his tag-Tcgr
8pCAMkocbhS-ANL1000kocbhS-ANL1000PgpdA-kozak seq-cbhⅠ signal-ANL1000- his tag-Tcgr


Table 2 . Protein concentration in the supernatant of the transformants after cultivation for 168 h..

TransformantsProtein concentration (mg/mL)*
A. niger 890.08±0.01
glaS-ANL0.09±0.01
glaS-ANL10000.14±0.02
koglaS-ANL0.12±0.01
koANLS-ANL0.18±0.02
kocbhS-ANL0.19±0.02
koglaS-ANL10000.20±0.01
koANLS-ANL10000.23±0.03
kocbhS-ANL10000.32±0.02

*The standard deviation indicates three biological replicates of three transformants for each construct..


Table 3 . The kinetic parameters of ANL..

LipaseKm (g/L)kcat(1/s)kcat/Km(L/(s g))
ANL9.30±1.04391.66±8.6942.25±3.35

References

  1. Schuster E, Dunn-Coleman N, Frisvad JC, van Dijck PWM. 2002. On the safety of Aspergillus niger - A review. Appl. Microbiol. Biotechnol. 59: 426-435.
    Pubmed CrossRef
  2. Cairns TC, Nai C, Meyer V. 2018. How a fungus shapes biotechnology: 100 years of Aspergillus niger research. Fungal Biol. Biotechnol. 5: 13.
    Pubmed KoreaMed CrossRef
  3. Steiniger C, Hoffmann S, Mainz A, Kaiser M, Voigt K, Meyer V, et al. 2017. Harnessing fungal nonribosomal cyclodepsipeptide synthetases for mechanistic insights and tailored engineering. Chem. Sci. 8: 7834-7843.
    Pubmed KoreaMed CrossRef
  4. Michael W, Cherry L, Victoria DC, Fox BP, Fox JA, Wong DL, et al. 2004. Characterization of humanized antibodies secreted by Aspergillus niger. Appl. Environ. Microbiol. 70: 2567-2576.
    Pubmed KoreaMed CrossRef
  5. Punt PJ. 2002. Filamentous fungi as cell factories for protein production. Trends Biotechnol. 20: 200-206.
    CrossRef
  6. Magaña-Ortíz D, Fernández F, Loske AM, Gómez-Lim MA. 2018. Extracellular expression in Aspergillus niger of an antibody fused to Leishmania sp. antigens.  Curr. Microbiol. 75: 40-48.
    Pubmed CrossRef
  7. Guo Y, Zheng P, Sun J. 2010. Aspergillus niger as a potential cellular factory: prior knowledge and key technology. Sheng Wu Gong Cheng Xue Bao 26: 1410-1418.
    Pubmed
  8. Zoglowek M, Lübeck PS, Ahring BK, Lübeck M. 2015. Heterologous expression of cellobiohydrolases in filamentous fungi - an update on the current challenges, achievements and perspectives. Process Biochem. 50: 211-220.
    CrossRef
  9. Krasevec N, van de Hondel C, Komel R. 2000. Expression of human lymphotoxin alpha in Aspergillus niger. Pflugers Arch. 440: R83-R85.
    Pubmed CrossRef
  10. Svetina M, Krasevec N, Gaberc-Porekar V, Komel R. 2000. Expression of catalytic subunit of bovine enterokinase in the filamentous fungus Aspergillus niger. J. Biotechnol. 76: 245-251.
    Pubmed CrossRef
  11. Roberts IN, Jeenes DJ, Mackenzie DA, Wilkinson AP, Sumner IG, Archer DB. 1992. Heterologous gene expression in Aspergillus niger: a glucoamylase-porcine pancreatic prophospholipase A2 fusion protein is secreted and processed to yield mature enzyme. Gene 122: 155-161.
    Pubmed CrossRef
  12. Zhang H, Yan JN, Zhang H, Qi LT, Xu Y, Zhang YY, et al. 2018. Effect of gpd box copy numbers in the gpdA promoter of Aspergillus nidulans on its transcription efficiency in A. niger. FEMS Microbiol. Lett. 1: 365.
    CrossRef
  13. Liu F, Wang B, Ye Y, Pan L. 2017. High level expression and characterization of tannase tan7 using Aspergillus niger SH-2 with low-background endogenous secretory proteins as the host. Protein Expr. Purif. 144: 71-75.
    Pubmed CrossRef
  14. Zhang H, Wang S, Zhang XX, Ji W, Song FP, Zhao Y, et al. 2016. The amyR-deletion strain of Aspergillus niger CICC2462 is a suitable host strain to express secreted protein with a low background. Microb. Cell Fact. 15: 11.
    Pubmed KoreaMed CrossRef
  15. Kamaruddin N, Storms R, Mahadi NM, Illias RM, Abu Bakar FD, Murad AMA. 2018. Reduction of extracellular proteases increased activity and stability of heterologous protein in Aspergillus niger. Arab. J. Sci. Eng. 43: 3327-3338.
    CrossRef
  16. Zhang XF, Ai YH, Xu Y, Yu XW. 2019. High-level expression of Aspergillus niger lipase in Pichia pastoris: characterization and gastric digestion in vitro. Food Chem. 274: 305-313.
    Pubmed CrossRef
  17. Saxena RK, Davidson WS, Sheoran A, Giri B. 2003. Purification and characterization of an alkaline thermostable lipase from Aspergillus carneus. Process Biochem. 39: 239-247.
    CrossRef
  18. Xia J-l, Huang B, Nie Z-y, Wang W. 2011. Production and characterization of alkaline extracellular lipase from newly isolated strain Aspergillus awamori HB-03. J. Cent. South Univ. 18: 1425.
    CrossRef
  19. Shu ZY, Yan YJ, Yang JK, Xu L. 2007. Aspergillus niger lipase: gene cloning, over-expression in Escherichia coli and in vitro refolding. Biotechnol. Lett. 29: 1875-1879.
    Pubmed CrossRef
  20. Yang J, Yan X, Zhang Z, Jiang X, Yan Y. 2009. Two-step synthesis of the full length Aspergillus niger lipase gene lipA leads to high-level expression in Pichia pastoris. Sheng Wu Gong Cheng Xue Bao 25: 381-387.
    Pubmed
  21. Kozak M. 1986. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44: 283-292.
    Pubmed CrossRef
  22. Ahangarzadeh S, Daneshvar MH, Rajabi-Memari H, Galehdari H, Alamisaied K. 2012. Cloning, transformation and expression of human interferon α2b Gene in tobacco plant (Nicotiana tabacum cv. xanthi). Jundishapur J. Nat. Pharm. Prod. 7: 111-116.
    Pubmed KoreaMed CrossRef
  23. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.
    Pubmed CrossRef
  24. Xu Y, Wang YH, Liu TQ, Zhang H, Zhang H, Li J. 2018. The GlaA signal peptide substantially increases the expression and secretion of α-galactosidase in Aspergillus niger. Biotechnol. Lett. 40: 949-955.
    Pubmed CrossRef
  25. Li M, Zhou L, Liu M, Huang Y, Sun X, Lu F. 2013. Construction of an engineering strain producing high yields of alpha-transglucosidase via Agrobacterium tumefaciens-mediated transformation of Asperillus niger. Biosci. Biotechnol. Biochem. 77: 1860-1866.
    Pubmed CrossRef
  26. Madhavan A, Pandey A, Sukumaran RK. 2017. Expression system for heterologous protein expression in the filamentous fungus Aspergillus unguis. Bioresour. Technol. 245: 1334-1342.
    Pubmed CrossRef
  27. Canseco-Pérez MA, Castillo-Avila GM, Chi-Manzanero B, Islas-Flores I, Apolinar-Hernández MM, Rivera-Muñoz G, et al. 2018. Fungal screening on olive oil for extracellular triacylglycerol lipases: selection of a trichoderma harzianum strain and genome wide search for the genes. Genes 9(2). pii:E62.
    Pubmed KoreaMed CrossRef
  28. Jo BS, Choi SS. 2015. Introns: the functional benefits of introns in genomes. Genomics Inform. 13: 112-118.
    Pubmed KoreaMed CrossRef
  29. Kurachi S, Hitomi Y, Furukawa M, Kurachi K. 1995. Role of intron I in expression of the human factor IX gene. J. Biol. Chem. 270: 5276-5281.
    Pubmed CrossRef
  30. Gniadkowski M, Hemmings-Mieszczak M, Klahre U, Liu HX, Filipowicz W. 1996. Characterization of intronic uridine-rich sequence elements acting as possible targets for nuclear proteins during pre-mRNA splicing in Nicotiana plumbaginifolia. Nucleic Acids Res. 24: 619-627.
    Pubmed KoreaMed CrossRef
  31. Jun X, Zhen GZ. 2003. Intron requirement for AFP gene expression in Trichoderma viride. Microbiology 149: 3093-3097.
    Pubmed CrossRef
  32. Gonzalez-Hilarion S, Paulet D, Lee KT, Hon CC, Lechat P, Mogensen E, et al. 2016. Intron retention-dependent gene regulation in Cryptococcus neoformans. Sci. Rep. 6: 32252.
    Pubmed KoreaMed CrossRef
  33. Kozak M. 2005. Regulation of translation via mRNA structure in prokaryotes and eukaryotes. Gene 361: 13-37.
    Pubmed CrossRef
  34. Du M, Ye L, Liu J, Liu J, Yang L. 2008. Enhancement of GFP expression by Kozak sequence +4G in HEK293 cells. Sheng Wu Gong Cheng Xue Bao 24: 491-494.
    Pubmed
  35. Olafsdóttir G, Svansson V, Ingvarsson S, Marti E, Torsteinsdóttir S. 2008. In vitro analysis of expression vectors for DNA vaccination of horses: the effect of a Kozak sequence. Acta Vet. Scand. 50: 44.
    Pubmed KoreaMed CrossRef
  36. Li J, Liang Q, Song WJ, Marchisio MA. 2017. Nucleotides upstream of the Kozak sequence strongly influence gene expression in the yeast S. cerevisiae. J. Biol. Eng. 11: 25.
    Pubmed KoreaMed CrossRef
  37. Mahadik ND, Puntambekar US, Bastawde KB, Khire JM, Gokhale DV. 2002. Production of acidic lipase by Aspergillus niger in solid state fermentation. Process Biochem. 38: 715-721.
    CrossRef
  38. Guang L. 2015. Purification and characterization of a lipase with high thermostability and polar organic solvent-tolerance from Aspergillus niger AN0512. Lipids 11: 1155-1163.
    Pubmed CrossRef
  39. dos Santos EAL, Lima ÁS, Soares CMF, Santana L. 2017. Lipase from Aspergillus niger obtained from mangaba residue fermentation: biochemical characterization of free and immobilized enzymes on a sol-gel matrix. Acta Sci.Technol. 39: 1-8.
    CrossRef