Secretory Expression And Characterization Of Multifunctional Amylase OPMA In Bacillus Subtitlis

Multifunctional amylases, including neopullulanase, maltogenic amylase and cyclomaltodextrinase have been reported to be a new type ofα-amylase family. They have the four highly conserved regions inα-amylase family in their primary sequence which is known the common structural property of the members ofα-amylase family. Meanwhile they have dual activity of glycoside hydrolase and glycosyltransferase.Usually, there are lots of glucose, maltose and limit dextrin in the end products from starch catalysed by the traditional amylase. The starch-degrading enzymes were secreted by ZW2531-1, a strain newly isolated from Chinese soil. They had multi-catalytic activities like neopullulanase compared with the traditional amylase. They only catalyzed the degradation of starch to produce maltooligosaccharide and isomaltooligosaccharide, including maltose, maltotriose, isomaltotriose and isomaltotetraose, but not to produce glucose. This fact will greatly enhance the value of these enzymes. Cloning and expression of the OPMA-N and OPMA-G gene obtained from ZW2531-1 have been succeeded in E.coli. However, the expressed OPMA-N and OPMA-G mainly existed in the intracellular with a considerable portion of the form of inclusion bodies. This will increase the cost for its industrial applications. With the development of genetic engineering, Bacillus subtilis is becoming a more attractive host to molecular biologists. The advantages of B. subtilis such as fast and easy growth, high secretion capacity, and non-pathogenic GRAS (Generally Regarded as Safe) status have made it an attractive host for the production of heterologous enzymes.In this work,OPMA-N and OPMA-G genes obtained from pET28a-OPMA-N, pET28a-OPMA-G by PCR were cloned into expression plasmid pMA5, and transformed into Bacillus subtilis 168, respectively. The engineering strain with OPMA-N or OPMA-G was cultured in LB medium and the expressed products were identifid by SDS-PAGE. The results showed that both OPMA-N and OPMA-G were expressed and secreted to the culture medium, while both also intracellularly expressed. In addition, analysis of gray showed that secretory OPMA-N or OPMA-G was about 30% or 52% of total extracellular proteins. Therefore, the engineering strain for secretory expression of OPMA-N or OPMA-G was obtained.In this work, the enzymatic characterization of expressed OPMA-N or OPMA-G was analyzed. The results showed that the specific activity of OPMA-N or OPMA-G was 4.01U/mg, 4.20U/mg in the fermentation supernatant, respectively. Product components generated from starch catalyzed by OPMA-N or OPMA-G were analyzed by TLC and HPLC. The results indicated that the two enzymes only catalyzed the degradation of starch to produce maltooligosaccharide and isomaltooligosaccharide including maltose, maltotriose, isomaltotriose and isomaltotetraose, but not to produce glucose. Therefore, compared with other multifunctional amylases, both OPMA-N and OPMA-G have unique specificities of substrate and product, which made them more suitable for the production of functional oligosaccharide. Kinetic analysis showed that the substrate binding and catalytic efficiency of OPMA-N with N domain were better than OPMA-G without N domain.The fermentation conditions of the engineering strain with OPMA-N or OPMA-G were optimized in LB medium to further enhance the activity and yield of secreted enzyme. The results showed that the maximum activity of OPMA-N was obtained in medium containing 0.5% yeast extract, 0.3% urea, 1% MgSO4, 16% liquid volume of medium, pH6.5, after 48h of fermentation, the maximum activity of OPMA-G was obtained in medium containing 1% starch, 0.8% yeast extract, 1% NaCl, pH7.0, 32% liquid volume of medium, after 36h of fermentation. The expressed products under optimum conditions were identified by SDS-PAGE and enzyme activities were detected. The results showed that secretory OPMA-N or OPMA-G was about 30% or 52% of total extracellular proteins. The specific activities of secretory OPMA-N and OPMA-G were 4.57U/mg and 4.65U/mg, respectively.However, the expression levels of foreign gene in Bacillus subtilis considered as the ideal host for heterologous protein expression and secretion are generally lower. Studies on the process of protein secretion reveal that the action of the signal peptide is very important. To explore the impact of signal peptide on the target enzyme secretion, the DNA sequence encoded AmyE signal peptide had been obtained from the genome of Bacillus subtilis 168 by PCR. The constructed expression vectors pMA5-OPMA-SN and pMA5-OPMA-SG (with signal peptide gene) were transformed into Bacillus subtilis 168, respectively. Compared to OPMA-N or OPMA-G, the secretory levels of OPMA-SN and OPMA-SG were improved, but not very significant. In addition, molecular weight analysis showed that the signal sequences in OPMA-SN and OPMA-SG were not cut finally. Compared with OPMA-N, the specific activity of OPMA-SN was slightly increased, but the specific activity of OPMA-SG decreased compared with OPMA-G. It was speculated that the signal peptide might affect the spatial structures of OPMA-N and OPMA-G, and then affecting their catalytic activities. However, the impact on the OPMA-N was less than OPMA-G. Further analysis showed that OPMA-SN, OPMA-SG, OPMA-N and OPMA-G had the same substrate and product specificities. They only catalyzed the degradation of starch to produce maltooligosaccharide and isomalto- oligosaccharide including maltose, maltotriose, isomaltotriose and isomaltotetraose, but not to produce glucose. These results suggested that the introduction of the signal peptide only increased the secretory level of OPMA-N and OPMA-G, but not affected their catalytic properties.With the rapid development of food industry, isomaltooligosaccharide is favored to make up the shortage of the traditional sweetener. Using starch as raw materials to produce isomaltooligosaccharide has been considered a good way. Our country is very rich in starch resources which are widely distributed, large output, low price and no seasonal restrictions on its production. Thus, isomaltooligosaccharide produced from starch will become the main functional oligosaccharide in our country. Obviously, the absence of glucose in the products generated from starch catalyzed by OPMA-N or OPMA-G advanced the value of the products. In addition, OPMA-N or OPMA-G had been achieved successfully in the form of extracellular secretion. The fermentation supernatant can be made directly to applications as enzyme sources to further reduce costs industrial preparation. Moreover, the optimal fermentation conditions of OPMA-N or OPMA-G were determined. Therefore, Extracellular enzyme OPMA-N and OPMA-G will be expected as more valuable candidate enzymes for the industrial production of isomaltooligosaccharides from starch than the traditional ones.