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Cloning of a novel thermostable glucoamylase from thermophilic fungus Rhizomucor pusillus and high-level co-expression with α-amylase in Pichia pastoris.

He Z, Zhang L, Mao Y, Gu J, Pan Q, Zhou S, Gao B, Wei D - BMC Biotechnol. (2014)

Bottom Line: Although various amylases have been found in fungi, the amylases from Aspergillus dominate the commercial application.The maximum saccharogenic activity of KM71/9KGla-ZαAmy was 2218 U ml(-1), which improved 79% compared to KM71/9KGla.Besides, fungal glucoamylase and α-amylase from R. pusillus were shown as promising candidates for further application in starch hydrolysis.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Fungal amylase, mainly constitute of fungal α-amylase and glucoamylase, are utilized in a broad range of industries, such as starch hydrolysis, food and brewing. Although various amylases have been found in fungi, the amylases from Aspergillus dominate the commercial application. One of main problems exist with regard to these commercial use of amylases is relatively low thermal and acid stability. In order to maximize the efficiency of starch process, developing fungal amylases with increased thermostability and acid stability has been attracting researchers' interest continually. Besides, synergetic action of glucoamylase and α-amylase could facilitate the degradation of starch. And co-expressing glucoamylase with α-amylase in one host could avoid the need to ferment repeatedly and improves cost-effectiveness of the process.

Results: A novel fungal glucoamylase (RpGla) gene encoding a putative protein of 512 amino acid residues was cloned from Rhizomucor pusillus. BLAST analysis revealed that RpGla shared highest identity of 51% with the Rhizopus oryzae glucoamylase (ABB77799.1). The fungal glucoamylase RpGla was expressed in Pichia pastoris (KM71/9KGla) with maximum activity of 1237 U ml(-1). The optimum pH and temperature of RpGla were pH 4.0 and 70 °C, respectively. Fungal α-amylase (RpAmy) gene was also cloned from R. pusillus and transformed into KM71/9KGla, resulted in recombinant yeast KM71/9KGla-ZαAmy harboring the RpGla and RpAmy genes simultaneously. The maximum saccharogenic activity of KM71/9KGla-ZαAmy was 2218 U ml(-1), which improved 79% compared to KM71/9KGla. Soluble starch hydrolyzed by purified RpGla achieved 43% glucose and 34% maltose. Higher productivity was achieved with a final yield of 48% glucose and 47% maltose catalyzed by purified enzyme preparation produced by KM71/9KGla-ZαAmy.

Conclusions: A novel fungal glucoamylase and fungal α-amylase genes were cloned from Rhizomucor pusillus. The two enzymes showed good thermostability and acid stability, and similar biochemical properties facilitated synergetic action of the two enzymes. A dramatic improvement was seen in amylase activity through co-expressing RpGla with RpAmy in Pichia pastoris. This is the first report of improving activity through co-expression glucoamylase with α-amylase in P. pastoris. Besides, fungal glucoamylase and α-amylase from R. pusillus were shown as promising candidates for further application in starch hydrolysis.

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HPLC analysis of catalyzed products of starch and glucose by recombinant enzymes. (a) Hydrolysis products of starch catalyzed by purified RpGla; (b) Hydrolysis products of starch catalyzed by purified RpAmy; (c) Hydrolysis products of starch catalyzed by co-expressed enzyme preparation; (d) Blank control: soluble starch processed under the same procedure with deactivated enzyme preparation. The loading amount was all 10 μl. Substrate solution was prepared by mixing 1 g of soluble starch in 100 ml of citrate-sodium citrate buffer (50 mM, pH 5.0). 200 μl of appropriate diluted purified enzyme solution was added into 1 ml of substrate solution. After incubation at 50°C for 48 h, the mixture was boiled to stop the reaction and centrifuged. The retention time of glucose and maltose were about 5.0 min and 8.4 min respectively; (e) Reaction product of 40% glucose catalyzed by recombinant glucoamylase after 24 h of incubation at 50°C.
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Fig4: HPLC analysis of catalyzed products of starch and glucose by recombinant enzymes. (a) Hydrolysis products of starch catalyzed by purified RpGla; (b) Hydrolysis products of starch catalyzed by purified RpAmy; (c) Hydrolysis products of starch catalyzed by co-expressed enzyme preparation; (d) Blank control: soluble starch processed under the same procedure with deactivated enzyme preparation. The loading amount was all 10 μl. Substrate solution was prepared by mixing 1 g of soluble starch in 100 ml of citrate-sodium citrate buffer (50 mM, pH 5.0). 200 μl of appropriate diluted purified enzyme solution was added into 1 ml of substrate solution. After incubation at 50°C for 48 h, the mixture was boiled to stop the reaction and centrifuged. The retention time of glucose and maltose were about 5.0 min and 8.4 min respectively; (e) Reaction product of 40% glucose catalyzed by recombinant glucoamylase after 24 h of incubation at 50°C.

Mentions: According to HPLC analysis result (Figure 4a), the main products of soluble starch hydrolyzed by purified RpGla were glucose (43%) and maltose (34%) with no detection of other oligosaccharides. Utilizing purified RpAmy as catalyst, the main products were maltose (69%) and glucose (22%) (Figure 4b) with no detectable maltotriose and maltotetraose. However, the main products hydrolyzed by purified mixed enzyme preparation of KM71/9KGla-ZαAmy were glucose (48%) and maltose (47%) with no detectable of maltotriose and maltotetraose (Figure 4c).Figure 4


Cloning of a novel thermostable glucoamylase from thermophilic fungus Rhizomucor pusillus and high-level co-expression with α-amylase in Pichia pastoris.

He Z, Zhang L, Mao Y, Gu J, Pan Q, Zhou S, Gao B, Wei D - BMC Biotechnol. (2014)

HPLC analysis of catalyzed products of starch and glucose by recombinant enzymes. (a) Hydrolysis products of starch catalyzed by purified RpGla; (b) Hydrolysis products of starch catalyzed by purified RpAmy; (c) Hydrolysis products of starch catalyzed by co-expressed enzyme preparation; (d) Blank control: soluble starch processed under the same procedure with deactivated enzyme preparation. The loading amount was all 10 μl. Substrate solution was prepared by mixing 1 g of soluble starch in 100 ml of citrate-sodium citrate buffer (50 mM, pH 5.0). 200 μl of appropriate diluted purified enzyme solution was added into 1 ml of substrate solution. After incubation at 50°C for 48 h, the mixture was boiled to stop the reaction and centrifuged. The retention time of glucose and maltose were about 5.0 min and 8.4 min respectively; (e) Reaction product of 40% glucose catalyzed by recombinant glucoamylase after 24 h of incubation at 50°C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4362842&req=5

Fig4: HPLC analysis of catalyzed products of starch and glucose by recombinant enzymes. (a) Hydrolysis products of starch catalyzed by purified RpGla; (b) Hydrolysis products of starch catalyzed by purified RpAmy; (c) Hydrolysis products of starch catalyzed by co-expressed enzyme preparation; (d) Blank control: soluble starch processed under the same procedure with deactivated enzyme preparation. The loading amount was all 10 μl. Substrate solution was prepared by mixing 1 g of soluble starch in 100 ml of citrate-sodium citrate buffer (50 mM, pH 5.0). 200 μl of appropriate diluted purified enzyme solution was added into 1 ml of substrate solution. After incubation at 50°C for 48 h, the mixture was boiled to stop the reaction and centrifuged. The retention time of glucose and maltose were about 5.0 min and 8.4 min respectively; (e) Reaction product of 40% glucose catalyzed by recombinant glucoamylase after 24 h of incubation at 50°C.
Mentions: According to HPLC analysis result (Figure 4a), the main products of soluble starch hydrolyzed by purified RpGla were glucose (43%) and maltose (34%) with no detection of other oligosaccharides. Utilizing purified RpAmy as catalyst, the main products were maltose (69%) and glucose (22%) (Figure 4b) with no detectable maltotriose and maltotetraose. However, the main products hydrolyzed by purified mixed enzyme preparation of KM71/9KGla-ZαAmy were glucose (48%) and maltose (47%) with no detectable of maltotriose and maltotetraose (Figure 4c).Figure 4

Bottom Line: Although various amylases have been found in fungi, the amylases from Aspergillus dominate the commercial application.The maximum saccharogenic activity of KM71/9KGla-ZαAmy was 2218 U ml(-1), which improved 79% compared to KM71/9KGla.Besides, fungal glucoamylase and α-amylase from R. pusillus were shown as promising candidates for further application in starch hydrolysis.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Fungal amylase, mainly constitute of fungal α-amylase and glucoamylase, are utilized in a broad range of industries, such as starch hydrolysis, food and brewing. Although various amylases have been found in fungi, the amylases from Aspergillus dominate the commercial application. One of main problems exist with regard to these commercial use of amylases is relatively low thermal and acid stability. In order to maximize the efficiency of starch process, developing fungal amylases with increased thermostability and acid stability has been attracting researchers' interest continually. Besides, synergetic action of glucoamylase and α-amylase could facilitate the degradation of starch. And co-expressing glucoamylase with α-amylase in one host could avoid the need to ferment repeatedly and improves cost-effectiveness of the process.

Results: A novel fungal glucoamylase (RpGla) gene encoding a putative protein of 512 amino acid residues was cloned from Rhizomucor pusillus. BLAST analysis revealed that RpGla shared highest identity of 51% with the Rhizopus oryzae glucoamylase (ABB77799.1). The fungal glucoamylase RpGla was expressed in Pichia pastoris (KM71/9KGla) with maximum activity of 1237 U ml(-1). The optimum pH and temperature of RpGla were pH 4.0 and 70 °C, respectively. Fungal α-amylase (RpAmy) gene was also cloned from R. pusillus and transformed into KM71/9KGla, resulted in recombinant yeast KM71/9KGla-ZαAmy harboring the RpGla and RpAmy genes simultaneously. The maximum saccharogenic activity of KM71/9KGla-ZαAmy was 2218 U ml(-1), which improved 79% compared to KM71/9KGla. Soluble starch hydrolyzed by purified RpGla achieved 43% glucose and 34% maltose. Higher productivity was achieved with a final yield of 48% glucose and 47% maltose catalyzed by purified enzyme preparation produced by KM71/9KGla-ZαAmy.

Conclusions: A novel fungal glucoamylase and fungal α-amylase genes were cloned from Rhizomucor pusillus. The two enzymes showed good thermostability and acid stability, and similar biochemical properties facilitated synergetic action of the two enzymes. A dramatic improvement was seen in amylase activity through co-expressing RpGla with RpAmy in Pichia pastoris. This is the first report of improving activity through co-expression glucoamylase with α-amylase in P. pastoris. Besides, fungal glucoamylase and α-amylase from R. pusillus were shown as promising candidates for further application in starch hydrolysis.

Show MeSH
Related in: MedlinePlus