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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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Glucoamylase and α-amylase production of recombinantP. pastoris. (a) Time course of saccharogenic activity of KM71/9KGla (■) and KM71/9KGla-ZαAmy (●); (b) Time course of dextrinogenic activity of KM71/ZαAmy (■) and KM71/9KGla-ZαAmy (●). The induction were performed in 500 ml shake flask with 50 ml BMMY media incubating in 30°C, 230 rpm. The induction period was 5 days with the addition of 0.5% (v/v) methanol per day.
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Fig2: Glucoamylase and α-amylase production of recombinantP. pastoris. (a) Time course of saccharogenic activity of KM71/9KGla (■) and KM71/9KGla-ZαAmy (●); (b) Time course of dextrinogenic activity of KM71/ZαAmy (■) and KM71/9KGla-ZαAmy (●). The induction were performed in 500 ml shake flask with 50 ml BMMY media incubating in 30°C, 230 rpm. The induction period was 5 days with the addition of 0.5% (v/v) methanol per day.

Mentions: After induction at 30°C, 230 rpm for 96 h, the supernatants of KM71/9KGla, KM71/ZαAmy and KM71/9KGla-ZαAmy all had a clear band between 45.0 kDa and 66.2 kDa (Figure 1a), while the supernatant of negative controls showed no band. The highest protein concentration of supernatant of KM71/9KGla-ZαAmy was 0.94 ± 0.13 mg ml−1, which was increased 23% compared to that of KM71/9KGla (0.76 ± 0.08 mg ml−1), and was greatly increased 298% compared to that of KM71/ZαAmy (0.24 ± 0.03 mg ml−1). The protein concentration of supernatant of negative controls were zero. Figure 2 illustrates the marked increase in activity. KM71/9KGla-ZαAmy achieved the highest saccharogenic activity (defined as the same with glucoamylase activity) of 2218 U ml−1, which was improved by 79% compared to that of KM71/9KGla (glucoamylase activity, 1237 U ml−1). The dextrinogenic activity (defined as the same with α-amylase activity) of KM71/9KGla-ZαAmy has a maximum activity of 8285 U ml−1 and was improved by 183% compared to that of KM71/ZαAmy (α-amylase activity, 2927 U ml−1). No amylase activity was detected in the original P. pastoris host.Figure 1


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)

Glucoamylase and α-amylase production of recombinantP. pastoris. (a) Time course of saccharogenic activity of KM71/9KGla (■) and KM71/9KGla-ZαAmy (●); (b) Time course of dextrinogenic activity of KM71/ZαAmy (■) and KM71/9KGla-ZαAmy (●). The induction were performed in 500 ml shake flask with 50 ml BMMY media incubating in 30°C, 230 rpm. The induction period was 5 days with the addition of 0.5% (v/v) methanol per day.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4362842&req=5

Fig2: Glucoamylase and α-amylase production of recombinantP. pastoris. (a) Time course of saccharogenic activity of KM71/9KGla (■) and KM71/9KGla-ZαAmy (●); (b) Time course of dextrinogenic activity of KM71/ZαAmy (■) and KM71/9KGla-ZαAmy (●). The induction were performed in 500 ml shake flask with 50 ml BMMY media incubating in 30°C, 230 rpm. The induction period was 5 days with the addition of 0.5% (v/v) methanol per day.
Mentions: After induction at 30°C, 230 rpm for 96 h, the supernatants of KM71/9KGla, KM71/ZαAmy and KM71/9KGla-ZαAmy all had a clear band between 45.0 kDa and 66.2 kDa (Figure 1a), while the supernatant of negative controls showed no band. The highest protein concentration of supernatant of KM71/9KGla-ZαAmy was 0.94 ± 0.13 mg ml−1, which was increased 23% compared to that of KM71/9KGla (0.76 ± 0.08 mg ml−1), and was greatly increased 298% compared to that of KM71/ZαAmy (0.24 ± 0.03 mg ml−1). The protein concentration of supernatant of negative controls were zero. Figure 2 illustrates the marked increase in activity. KM71/9KGla-ZαAmy achieved the highest saccharogenic activity (defined as the same with glucoamylase activity) of 2218 U ml−1, which was improved by 79% compared to that of KM71/9KGla (glucoamylase activity, 1237 U ml−1). The dextrinogenic activity (defined as the same with α-amylase activity) of KM71/9KGla-ZαAmy has a maximum activity of 8285 U ml−1 and was improved by 183% compared to that of KM71/ZαAmy (α-amylase activity, 2927 U ml−1). No amylase activity was detected in the original P. pastoris host.Figure 1

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