P.V. Volkov et al.
Carbohydrate Research 499 (2021) 108211
5
37 host. It is based on a homologous inducible cbhI promoter [17,19].
centrifuged at 4000 rpm for 20 min on a centrifuge Avanti JXN-26
(Beckman Coulter, Atlanta, GA, USA) to remove biomass and insoluble
components of the nutrient medium. The supernatant was freeze-dried
on a VirTisBenchTop 2K ES freeze dryer (SP Scientific, Warminster,
PA, USA).
Productivity of the recipient strain on standard growth medium reaches
up to 50 g of extracellular protein per 1 L of culture fluid.
2
. Experimental
2
.1. Microbial strains and media
2.4. Enzyme purification
The mycelium of the T. harzianum strain was isolated from the sur-
Desalting and fractionation of crude freeze-dried enzyme prepara-
tions were carried out on AKTA Purifier system (GE Healthcare, Swe-
den). Proteins were preliminary precipitated with ammonium sulfate
(80% of saturation) followed by a desalting on a Bio-Gel Р-4 column
(Bio-Rad Laboratories, Hercules, CA, USA) equilibrated by 0.01 М Bis-
Tris/HCl buffer, pH 6.8. The desalted protein solution was fraction-
ated by anion-exchange chromatography (AEC) on a Source 15Q column
(Pharmacia, Uppsala, Sweden) in Bis-Tris/HCl starting buffer (pH 6.8,
0.01 M), the bound proteins were eluted with a gradient of NaCl (0–0.4
M). Protein fractions with pustulanase activity were purified by hydro-
phobic interaction chromatography (HIC) on a Source 15ISO column
(Pharmacia, Uppsala, Sweden). Ammonium sulfate was added to the
face of a crumbling tree and grown on a medium contained (in g/L):
glucose – 5.0, yeast extract – 10.0 and potassium phosphate – 25.0. The
genomic DNA from T. harzianum mycelia was isolated using DNeasy
Plant Mini Kit (QIAGEN, Valencia, CA, USA). Auxotrophic
P. verruculosum B1-537 strain with a knock-out nitrate reductase gene
(
ΔniaD) [19,20] was used as a host strain in transformation. The
TM
Escherichia coli MachI T1R strain (Invitrogen, Carlsbad, CA, USA) was
applied for bacterial transformation and isolation of plasmids containing
target genes.
A medium for cultivation of P. verruculosum transformants contained
(
in g/L): microcrystalline cellulose (MCC) – 40; yeast extract – 10;
КН РО SO – 5; MgSO ⋅7H O – 0.3; СaCl ⋅2H O – 0.3.
– 15; (NH
P. verruculosum B1-537 (ΔniaD) was used as control.
2
4
4
)
2
4
4
2
2
2
sample to give 1.7 M (NH
4
)
SO
2 4
in 50 mM Na-acetate buffer, pH 5.0, the
SO
4
elution of proteins was performed in a reversed gradient of (NH
4
)
2
The composition of the medium for P. verruculosum fermentation was
from 1.7 to 0 M. The fraction with a highest pustulanase activity was
selected for the final purification by gel filtration on a Superose 12
column (GE Healthcare, UK). Elution was performed in the isocratic
mode in 0.01 M Na-acetate buffer, pH 5.0.
the following (in g/L): MCC – 60; glucose – 40; wheat bran – 10; yeast
extract – 10; КН
СaCl
⋅2H
2
РО
4
– 7; (NH
4
)
2
SO
4
– 5; MgSO
4
⋅7H O – 0.3;
2
◦
2
2
O – 0.3 (рН 4.5–5.0, 32 С, fermentation time 144 h).
The enzyme purity was characterized by sodium dodecylsulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectrofocusing
(IEF). SDS-PAGE was carried out in 12% gel using a Mini Protean II
equipment (Bio-Rad Laboratories, Hercules, CA, USA). IEF was per-
formed on a Model 111 Mini IEF Cell (Bio-Rad Laboratories, Hercules,
CA, USA). Staining of protein bands was carried out in Coomassie Blue
R-250 (Ferak, Berlin, Germany).
2
.2. Construction of the expression plasmids and transformation of P.
verruculosum
Plasmid construction and production of recombinant strains was
carried out as described previously [19–21]. Primers were designed
based on the homology of genes encoding similar β-glucanases from
NCBI database: CAA55789.1, XP_024773174.1 and OPB46546.1.
Briefly, using the freshly isolated genomic DNA from T. harzianum and
two pairs of primers shown below, the 1352 bp PCR-product was
amplified by polymerase chain reaction (PCR) using a MyCycler
equipment (Bio-Rad Laboratories, Hercules, CA, USA):
Protein concentration in samples was determined by the modified
method of Lowry et al. [25], using bovine serum albumin as the
standard.
2.5. Enzyme identification by MALDI-TOF mass spectrometry
′
Pust-LIC5 5 - caa-aca-gaa-gca-acc-gac-aca-atg-aag-tac-tcc-atc-gtt-
gct-ccg-g - 3’.
Identification of the pustulanase was carried out by MALDI-TOF
mass spectrometry (MS) peptide fingerprinting of trypsin-digested pro-
teins from the bands after IEF as described earlier [23,26]. MALDI-TOF
MS of peptides was carried out on an ultrafleXtreme TOF/TOF mass
spectrometer (Bruker Daltonik GmbH, Bremen, Germany). Peptides in
tides obtained by theoretical trypsinolysis of the bgl 1.6 gene amino acid
sequence of T. harzianum pustulanase. Glycopeptides and structures of
′
Pust-LIC3 5 - gag-gag-aag-cc-ggt-tac-ctg-aat-cca-gcg-cag-aca-tcc-tgg-
t - 3’.
Then, using the LIC-cloning method [22], the bgl1.6 gene was cloned
into the pCBHI vector under control of cbhI promoter encoding homol-
ogous cellobiohydrolase
Fig. S1). Protoplasts of P. verruculosum B1-537 host strain were trans-
formed by pCBHI-Pust plasmid together with the pSTA10
co-transforming plasmid at the ratio of 6:1 g using the transformation
I [23]. Thus, pCBHI-Pust was obtained
(
μ
protocol [24]. The pSTA10 plasmid contained a homologous nitrate
reductase (niaD) gene, allowing the selection of the resulting trans-
formants on medium supplemented with NaNO
formation frequency was 20–40 clones per 1 g of pCBHI-Pust plasmid,
which corresponds to the literature data for Penicillium strains [24].
3
(10 mM). The trans-
μ
2.6. Enzyme activity assays
Enzyme activity against polymeric substrates was assayed using the
modified Nelson-Somogyi method [27] of determination of the reducing
sugars (RS) released from pustulan (InvivoGen, France), laminarin
(Sigma, St. Louis, MO, USA), β-glucan (Megazyme), Na-salt carbox-
imetylcellulose (CMC, Sigma, St. Louis, MO, USA) and gentiobiose
(Megazyme). A solution of the substrate (10 g/L) in 0.1 M Na acetate
2
.3. Screening and cultivation of transformants
Screening of transformants was carried out in glass tubes (total
volume 50 mL, fermentation medium volume 10 mL). As a result of
primary screening, several clones of P. verruculosum (PV4, PV15, PV35
and PV44) with a high activity of the culture filtrates against pustulan
were selected.
◦
buffer, pH 5.0, was incubated with the enzyme at 50 C for 10 min
(reaction volume 0.2 mL), and the reaction was stopped by adding 0.2
ml of the Somogyi reagent. After incubation of the obtained solution for
40 min in a boiling water bath, 0.2 ml of the Nelson reagent was added.
The resulting solution was cooled in cold water for 10 min and 0.4 mL of
acetone and 1 mL of distilled water were added. After centrifugation of
the sample for 2 min at 13,000 rpm, the absorbance of the supernatant at
The best selected clones were cultivated in 3-L glass bioreactors KF-
1
04/3 (Prointex, Moscow, Russia). Fermentation was carried out in fed-
batch mode with glucose addition (every 12 h after 2 days), three ad-
ditions of MCC and one addition of salts.
After completion of cultivation in fermenters, the culture broth was
2