Cloning Of Thermostable Amylases From Rhizomucor Pusillus And Expression In Pichia Pastoris

Amylase is a class of enzymes which could hydrolyze the α-1,4or a-1,6glycosidic bond of starch and its related compounds to generate oligosaccharide or monosaccharide. Amylase is widely used in starch sugar production, bread baking and food industry, alcohol brewing and fermentation industry, as well as detergent, textile, pharmaceutical and other industries. The amylases which most widely used in current industry include a-amylase, glucoamylase and pullulanase.In this project, we establish a systematic methodology to screen filamentous fungi which could produce amylase. We obtained12strains of filamentous fungi which showed higher amylase activity from soil samples, Chinese traditional distiller’s yeast, etc. from6provinces and cities across China:Henan, Yunnan, Shanghai, Hunan, Gansu and Guizhou. Among them, filamentous fungus GX-3grew well even under the condition of50℃. Determining enzymatic properties of the crude enzyme solution obtained by shake flask liquid fermentation of GX-3, it showed that its a-amylase activity optimum pH was4.0, the optimum temperature was80℃. According to analysis results of18S rDNA and ITS sequences of fungus GX-3in NCBI, the filamentous fungus GX-3is belonging to Rhizomucor pusillus.The α-amylase gene of Rhizomucor pusillus GX-3was cloned by reverse transcription PCR. The DNA sequence of the gene consists of1572nucleotides, cDNA sequence consists of1416nucleotides, and the reading frame encodes471amino acids. Compared with the amino acid sequence of Rhizomucor pusillus a-amylase published in patent of Novozymes(WO2004/055178A1), six amino acid residues are changed:Gly31â†'Ala, Ala127â†'Thr, Asp135â†'Gly, Ser263â†'Thr, Thr365â†'Ala, Ala391â†'Thr.The glucoamylase gene of Rhizomucor pusillus GX-3was cloned by chromosome walking techniques and reverse transcription PCR. The total length of DNA sequence of the gene is1971bp and cDNA sequence is1539bp. The open reading frame encodes512amino acids. According to the results of analysis by Blast, the Rhizomucor pusillus glucoamylase showed low similarity with the glucoamylase sequence which had been reported. The highest similarity of amino acids sequence was only51%. This is the first glucoamylase gene cloned from the Rhizomucor pusillus.The recombinant vectors pPIC9KAmy which carries R.pusillus a-amylase gene and pPIC9KGLA which carries R.pusillus glucoamylase gene were constructed and transformed into Pichia pastoris KM71respectively. Recombinants KM71/9KAmy which carries high copy of a-amylase gene and KM71/9KGLA which carries high copy of R.pusillus glucoamylase gene were obtained in the YPDS plates containing4.0mg/ml G418. By inducing expression, the protein expression levels of Recombinants KM71/9KAmy achieved 1.5mg/mL, the a-amylase activity was31279U/ml; the protein expression levels of Recombinants KM71/9KGLA achieved0.762mg/mL, the glucoamylase activity was1237U/ml. This paper was the first project reporting the heterologous expression of R.pusillus a-amylase and glucoamylase in Pichia pastoris.The result of determination of enzymatic properties of R.pusillus a-amylase and glucoamylase showed that the optimum pH of a-amylase was5.0with high stability in the condition of pH4.0-9.0. The residual enzyme activity was83%after incubation for30minutes in the condition of pH4.0, and90%in the condition of pH9.0. The optimum temperature was70℃, and showed high thermostability. The residual enzyme activity was73%after incubation for30minutes at60℃. Comparing with the enzyme preparation of a-amylase of Novozymes and domestic company obtained from current market, the optimum pH of R.pusillus a-amylase is lower and the optimum temperatures is higher. The pH stability and temperature stability are also improved.The optimum pH of glucoamylase is4.0with high stability in the condition of pH4.0～9.0. The residual enzyme activity was71%after incubation for30minutes in the condition of pH4.0, and89%in the condition of pH9.0. The optimum temperature was70℃, and showed high thermostability below60℃. The residual enzyme activity was85%after incubation for30minutes at60℃.By thin layer chromatography and HPLC analysis, the results showed that the major product of soluble starch(1%, w/v) hydrolyzed by R.pusillus a-amylase was maltose (69%), and glucose (22%); the main product hydrolyzed by R.pusillus glucoamylase were glucose (43%) and maltose (34%).In order to study the synergistic effect of the the the R. pusillus a-amylase and glucoamylase hydrolysis of starch, the recombinant expression vector pPICZaAmy was constructed and transformed into KM71, then obtained recombinant KM71/ZaAmy which could secrete R. pusillus a-amylase. The expression vector pPICZaAmy then transferred into Pichia pastoris KM71/9KGLA containing glucoamylase gene. The recombinant KM71/9KGLA-ZaAmy which containing both a-amylase and glucoamylase gene was screened in YPDS plats containing200ug/ml Zeocin, and verified by PCR amlification. By inducible expression of recombinant Pichia pastoris KM71/9KGLA-ZaAmy, compared with KM71/9KGLA, the protein expression level increased by23%, glucoamylase activity increased by79%under the same conditions. Thus, a-amylase and glucoamylase showed a synergistic effect in the process of the hydrolysis of starch.