99
6.3.5 Recombinant Pectinase Production by Fungi
The recombinant DNA technology has some of the efficient tools like gene cloning
and mutation which attracted significant interest in carrying out research studies in
the modern era of biotechnology for the production of useful enzymes. By means of
these incredible tools, scientists not only can study the specific gene of interest but
also can achieve targeted or overexpression of a specific protein for some particular
interest (Alimardani et al. 2011). Pectinase has been studied for a very long time
aiming to retrieve a large-scale expression due to its potential for various industrial
applications. Recombinant technology has provided the different types of techniques to produce this particular enzyme of interest on a commercial scale in costefficient manner. Various pectinolytic genes have been cloned and expressed in the
vast range of host organisms in order to obtain their homologous as well as heterologous expression with very minimum energy, cost, and time (Barbosa et al. 2015).
According to the study conducted by Gonçalves et al. (2012), the genetically
modified version of P. griseoroseum was used to gain a high production of pectinases. This recombinant was constructed and coupled with additional copies of the
pectinase genes which resulted in 13 folds and 424 folds of hyperproduction of
pectin lyase and PGase. In recent times, the expression of pectinase from Aspergillus
in P. pastoris results in high activity in culture broth using high-cell-density batch
fermentation, and the yield was 8 times greater than the corresponding shake flask
cultivation. Some studies have been conducted for the expression of acid-tolerant
endopolygalacturonase gene from P. oxalicum to the host of P. pastoris. By doing
this, the yield of pectinase enzyme was maximized up to 1828.7 U/mL. When constitutive promoter such as the promoter of gpd gene is used to substitute the indigenous promoter, the production level of pectinase significantly increases.
Besides that, the UV-induced spontaneous catabolite repression-resistant mutant
of P. griseoroseum led to the elevated production level up to 7–8 folds of pectinase
titer when compared to its native strain (Li et al. 2017). The expression of the pelB
gene in E. coli is noteworthy as further optimization on this recombinant strain in 7
liters bioreactor can enhance the pectinase titer up to 1816.2 U/mL. Most of the
industrial enzymes are being produced at a large scale from the source of the fungus. Most of the fungal strains secrete enzymes similar to other similar proteins
(Asgher et al. 2016). According to the study conducted by Hadj-Taieb et al. (2006),
when the strain P. occitanis genetically modified with the single round of nitrous
acid mutagenesis, the mutant strain secretes 50 times more endo- and exo-pectinase
enzyme, unlike the wild-type strain which relatively produces lesser amount of pectinase enzyme. A cloned pectate lyase gene from Klebsiella sp. was expressed in
E. coli BL21 strain which later was grown in LB medium. The culture supernatant
and the cell lysate showed around 4.5 U/mL and 1.2 U/mL of pectinase enzyme
level (Yuan et al. 2011). Yang et al. (2011) also reported that the clone of endopolygalacturonase gene (pga1) from the acidophilic fungus of Bispora sp. was expressed
in P. pastoris GS115 by using the pPIC9 as the expression vector. They transformed
P. pastoris then only cultivated in yeast extract peptone dextrose medium at
6 Fungal Pectinases: Production and Applications in Food Industries
6.3.5 Recombinant Pectinase Production by Fungi
The recombinant DNA technology has some of the efficient tools like gene cloning
and mutation which attracted significant interest in carrying out research studies in
the modern era of biotechnology for the production of useful enzymes. By means of
these incredible tools, scientists not only can study the specific gene of interest but
also can achieve targeted or overexpression of a specific protein for some particular
interest (Alimardani et al. 2011). Pectinase has been studied for a very long time
aiming to retrieve a large-scale expression due to its potential for various industrial
applications. Recombinant technology has provided the different types of techniques to produce this particular enzyme of interest on a commercial scale in costefficient manner. Various pectinolytic genes have been cloned and expressed in the
vast range of host organisms in order to obtain their homologous as well as heterologous expression with very minimum energy, cost, and time (Barbosa et al. 2015).
According to the study conducted by Gonçalves et al. (2012), the genetically
modified version of P. griseoroseum was used to gain a high production of pectinases. This recombinant was constructed and coupled with additional copies of the
pectinase genes which resulted in 13 folds and 424 folds of hyperproduction of
pectin lyase and PGase. In recent times, the expression of pectinase from Aspergillus
in P. pastoris results in high activity in culture broth using high-cell-density batch
fermentation, and the yield was 8 times greater than the corresponding shake flask
cultivation. Some studies have been conducted for the expression of acid-tolerant
endopolygalacturonase gene from P. oxalicum to the host of P. pastoris. By doing
this, the yield of pectinase enzyme was maximized up to 1828.7 U/mL. When constitutive promoter such as the promoter of gpd gene is used to substitute the indigenous promoter, the production level of pectinase significantly increases.
Besides that, the UV-induced spontaneous catabolite repression-resistant mutant
of P. griseoroseum led to the elevated production level up to 7–8 folds of pectinase
titer when compared to its native strain (Li et al. 2017). The expression of the pelB
gene in E. coli is noteworthy as further optimization on this recombinant strain in 7
liters bioreactor can enhance the pectinase titer up to 1816.2 U/mL. Most of the
industrial enzymes are being produced at a large scale from the source of the fungus. Most of the fungal strains secrete enzymes similar to other similar proteins
(Asgher et al. 2016). According to the study conducted by Hadj-Taieb et al. (2006),
when the strain P. occitanis genetically modified with the single round of nitrous
acid mutagenesis, the mutant strain secretes 50 times more endo- and exo-pectinase
enzyme, unlike the wild-type strain which relatively produces lesser amount of pectinase enzyme. A cloned pectate lyase gene from Klebsiella sp. was expressed in
E. coli BL21 strain which later was grown in LB medium. The culture supernatant
and the cell lysate showed around 4.5 U/mL and 1.2 U/mL of pectinase enzyme
level (Yuan et al. 2011). Yang et al. (2011) also reported that the clone of endopolygalacturonase gene (pga1) from the acidophilic fungus of Bispora sp. was expressed
in P. pastoris GS115 by using the pPIC9 as the expression vector. They transformed
P. pastoris then only cultivated in yeast extract peptone dextrose medium at
6 Fungal Pectinases: Production and Applications in Food Industries