BackgroundFungal amylase, mainly constitute of fungal ¿-amylase and fungal glucoamylase, are utilized in a broad range of industries, such as starch hydrolysis, food and brewing. Although various ¿-amylase and glucoamylase have been found in various fungi, the amylases from Aspergillus dominate the commercial application. One of the main problems exist with regard to these commercial use of amylases is the relatively low thermal and acid stability. In order to maximize the efficiency of starch processing operations, the operating range of glucoamylase and ¿-amylase, in terms of pH, temperature and compatibility with other enzymes should be improved. So developing fungal amylases with increased thermostability and acid stability which could be used in commercial starch process has been attracting researchers¿ interest continually. Besides, synergetic action of glucoamylase and ¿-amylase could facilitate the degradation of starch. And co-expressing glucoamylase with ¿-amylase in one host could avoid the need to ferment repeatedly and improves cost-effectiveness of the process.ResultsA 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 the highest identity of 51% with the Rhizopus oryzae glucoamylase ( ABB77799.1 ). The fungal glucoamylase RpGla was expressed in Pichia pastoris (resulting in 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 ¿-amylase (RpAmy) gene was also cloned from R. pusillus and transformed into KM71/9KGla, resulted in recombinant yeast KM71/9KGla-Z¿Amy, which harboring the RpGla and RpAmy genes simultaneously. The maximum saccharogenic activity of KM71/9KGla-Z¿Amy 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-Z¿Amy.ConclusionsA novel fungal glucoamylase and fungal ¿-amylase genes were cloned from Rhizomucor pusillus. The two enzymes showed good thermostability and acid stability, and the 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 ¿-amylase in P. pastoris. Besides, fungal glucoamylase and ¿-amylase from R. pusillus were shown as promising candidates for further application in starch hydrolysis.