Bacillus Stearothermophilus Maltogenic Amylase Research Articles

Effect of codon-optimized E. coli signal peptides on recombinant Bacillus stearothermophilus maltogenic amylase periplasmic localization, yield and activity

Recombinant proteins can be targeted to the Escherichia coli periplasm by fusing them to signal peptides. The popular pET vectors facilitate fusion of target proteins to the PelB signal. A systematic comparison of the PelB signal with native E. coli signal peptides for recombinant protein expression and periplasmic localization is not reported. We chose the Bacillus stearothermophilus maltogenic amylase (MA), an industrial enzyme widely used in the baking and brewing industry, as a model protein and analyzed the competence of seven, codon-optimized, E. coli signal sequences to translocate MA to the E. coli periplasm compared to PelB. MA fusions to three of the signals facilitated enhanced periplasmic localization of MA compared to the PelB fusion. Interestingly, these three fusions showed greatly improved MA yields and between 18- and 50-fold improved amylase activities compared to the PelB fusion. Previously, non-optimal codon usage in native E. coli signal peptide sequences has been reported to be important for protein stability and activity. Our results suggest that E. coli signal peptides with optimal codon usage could also be beneficial for heterologous protein secretion to the periplasm. Moreover, such fusions could even enhance activity rather than diminish it. This effect, to our knowledge has not been previously documented. In addition, the seven vector platform reported here could also be used as a screen to identify the best signal peptide partner for other recombinant targets of interest.

Hydrolysis of amylopectin by amylolytic enzymes: level of inner chain attack as an important analytical differentiation criterion

Differences in amylase action pattern on amylopectin were demonstrated by the relation between the decrease in potassium iodide–iodine binding of waxy maize starch and the increase in reducing value during hydrolysis, as expressed by the RV 80 value (i.e., the reducing value for a potassium iodide–iodine binding value of 80% of that of the starting material). In the initial stages of the hydrolysis, the ratio of the increase in the level of reducing polysaccharides to the increase in the total level of reducing sugars formed during amylolysis of amylopectin can be considered as a measure of the level of inner chain attack (LICA) in the overall hydrolysis of the amylopectin structure and correlated with the respective RV 80 value. Bacillus amyloliquefaciens α-amylase and Aspergillus oryzae α-amylase, with the lowest RV 80 and the highest LICA values, hydrolysed the inner chains of amylopectin to a greater extent than did porcine pancreatic α-amylase. In the initial stages of hydrolysis, Bacillus stearothermophilus maltogenic amylase, like the Bacillus cereus β-amylase, did not display any significant degree of internal hydrolysis of amylopectin, in line with the high RV 80 and very low LICA values for these enzymes. However, at the later stages of hydrolysis, the maltogenic amylase probably exhibited a significant degree of internal hydrolysis of amylopectin, which itself seems to depend on temperature. The temperature dependence of the hydrolysis pattern of this enzyme is relevant for interpretation of its action as antifirming enzyme in bread-making applications.

Amylolytically-resistant tapioca starch modified by combined treatment of branching enzyme and maltogenic amylase

Tapioca starch was modified using branching enzyme (BE) isolated from Bacillus subtilis 168 and Bacillus stearothermophilus maltogenic amylase (BSMA), and their molecular fine structure and susceptibility to amylolytic enzymes were investigated. By BE treatment, the molecular weight decreased from 3.1 × 10 8 to 1.7 × 10 6, the number of shorter branch chains (DP 6–12) increased, the number of longer branch chains (DP >25) decreased, and amylose content decreased from 18.9% to 0.75%. This indicated that α–1,4 linkages of amylose and amylopectin were cleaved, and moiety of glycosyl residues were transferred to another amylose and amylopectin to produce branched glucan and BE-treated tapioca starch by forming α–1,6 branch linkages. The product was further modified with BSMA to produce highly-branched tapioca starch with 9.7% of extra branch points. When subject to digestion with human pancreatic α-amylase (HPA), porcine pancreatic α-amylase (PPA) and glucoamylase, highly-branched tapioca starch gave significantly lowered α-amylase susceptibility (7.5 times, 14.4 times and 3.9 times, respectively), compared to native tapioca starch.

Enzymatic Synthesis and Properties of Highly Branched Rice Starch Amylose and Amylopectin Cluster

We enzymatically modified rice starch to produce highly branched amylopectin and amylose and analyzed the resulting structural changes. To prepare the highly branched amylopectin cluster (HBAPC), we first treated waxy rice starch with Thermus scotoductus alpha-glucanotransferase (TSalphaGT), followed by treatment with Bacillus stearothermophilus maltogenic amylase (BSMA). Highly branched amylose (HBA) was prepared by incubating amylose with Bacillus subtilis 168 branching enzyme (BBE) and subsequently treating it with BSMA. The molecular weight of TSalphaGT-treated waxy rice starch was reduced from 8.9 x 10(8) to 1.2 x 10(5) Da, indicating that the alpha-1,4 glucosidic linkage of the segment between amylopectin clusters was hydrolyzed. Analysis of the amylopectin cluster side chains revealed that a rearrangement in the side-chain length distribution occurred. Furthermore, HBAPC and HBA were found to contain significant numbers of branched maltooligosaccharide side chains. In short, amylopectin molecules of waxy rice starch were hydrolyzed into amylopectin clusters by TSalphaGT in the enzymatic modification process, and then further branched by transglycosylation using BSMA. HBAPC and HBA showed higher water solubility and stability against retrogradation than amylopectin clusters or branched amylose. The hydrolysis rates of HBAPC and HBA by glucoamylase and alpha-amylase greatly decreased. The k cat/ K m value of glucoamylase acting on the amylopectin cluster was 45.94 s(-1)(mg/mL)(-1) and that for glucoamylase acting on HBAPC was 11.10 s(-1)(mg/mL)(-1), indicating that HBAPC was 4-fold less susceptible to glucoamylase. The k cat/ K m value for HBA was 15.90 s(-1)(mg/mL)(-1), or about three times less than that for branched amylose. The k cat/ K m values of porcine pancreatic alpha-amylase for HBAPC and HBA were 496 and 588 s(-1)(mg/mL)(-1), respectively, indicating that HBA and HBAPC are less susceptible to hydrolysis by glucoamylase and alpha-amylase. HBAPC and HBA show potential as novel glucan polymers with low digestibility and high water solubility.

Influence of amylases on the rheological and molecular properties of partially damaged wheat starch

AbstractThe effects of Bacillus subtilis, porcine pancreatic and Aspergillus oryzae α‐amylases, sweet potato β‐amylase and Bacillus stearothermophilus maltogenic amylase (BStA) on the rheological properties (measured with a Rapid Visco Analyser) of partially damaged wheat starch were studied and the accompanying changes in starch molecular properties were analysed by high‐performance size exclusion chromatography. Pasting and gelation of starch slurries (with an increased level of damaged starch) were significantly affected by the supplemented amylases and greatly depended on the mode of action and properties of the enzymes added. In general, at low endo‐amylase concentrations, peak, hot paste and cold paste viscosities were more reduced for enzyme‐supplemented partially damaged starch than for enzyme‐supplemented native wheat starch, demonstrating the significance of damaged starch levels in determining amylase functionality. Higher dosages of thermostable amylases ruled out most of the differences between amylase‐supplemented native starch and partially damaged starches, except for BStA. Furthermore, the (limited) endo‐action of BStA determines to a great extent the rheological properties of the starch paste. These results contribute to a better understanding of (maltogenic) amylase functionality in processing (damaged) starch‐containing foods. Copyright © 2006 Society of Chemical Industry

Enzymatic synthesis of a new inhibitor of α-amylases: acarviosinyl-isomaltosyl-spiro-thiohydantoin

Synthesis of acarviosinyl-isomaltosyl-spiro-thiohydantoin in yields up to 20%, has been achieved by Bacillus stearothermophilus maltogenic amylase (BSMA). BSMA is capable of transferring the acarviosine–glucose residue from an acarbose donor onto glucopyranosylidene-spiro-thiohydantoin. Reactions were followed using HPLC and MALDI-TOF MS. 1H and 13C NMR studies revealed that the enzyme reserved its stereoselectivity. Glycosylation took place mainly at C-6 resulting in α-acarviosinyl-(1→4)-α- d-glucopyranosyl-(1→6)- d-glucopyranosylidene-spiro-thiohydantoin. This compound was found to be a much more efficient salivary amylase inhibitor than glucopyranosylidene-spiro-thiohydantoin with kinetic constants of K EI = 0.19 μM and K ESI = 0.24 μM.

Transglycosylation of tagatose with maltotriose by Bacillus stearothermophilus maltogenic amylase (BSMA)

d-Tagatose was transglycosylated by Bacillus stearothermophilus maltogenic amylase (BSMA), and the physicochemical properties of the transfer products were analyzed. Maltosyl-tagatose was the main product of the transglycosylation reaction using maltotriose as the donor and d-tagatose as the acceptor. Glucosyl-tagatose was produced from maltosyl-tagatose by removal of a glucosyl moiety by glucoamylase. The 13C NMR analysis of glucosyl-tagatose suggested that a linkage was formed between the C-1 carbon of the glucose unit and the C-1 carbon of the tagatose unit, thereby producing 1,1-α-glucosyl-tagatose from the transglycosylation reaction. Hygroscopicity measurements showed that glucosyl-tagatose had greater water sorption than did tagatose or sucrose. The glass transition temperature of glucosyl-tagatose was −29 °C, which is considerably higher than that of tagatose, −45 °C. Its structure and physicochemical properties suggest that glucosyl-tagatose has potential as a low-calorie sweetener and cryostabilizer.

In vitro enzymatic modification of puerarin to puerarin glycosides by maltogenic amylase

Puerarin (daidzein 8- C-glucoside), the most abundant isoflavone in Puerariae radix, is prescribed to treat coronary heart disease, cardiac infarction, problems in ocular blood flow, sudden deafness, and alcoholism. However, puerarin cannot be given by injection due to its low solubility in water. To increase its solubility, puerarin was transglycosylated using various enzymes. Bacillus stearothermophilus maltogenic amylase (BSMA) was the most effective transferase used compared with Thermotoga maritima maltosyl transferase (TMMT), Thermus scotoductus 4-α-glucanotransferase (TS4αGTase), and Bacillus sp. I-5 cyclodextrin glucanotransferase (BSCGTase). TMMT and TS4αGTase lacked acceptor specificity for puerarin, which lacks an O-glucoside linkage between d-glucose and 7-OH-daidzein. The yield exceeded 70% when reacting 1% puerarin (acceptor), 3.0% soluble starch (donor), and 5 U/100 μL BSMA at 55 °C for 45 min. The two major transfer products of the BSMA reaction were purified using C 18 and GPC chromatography. Their structures were identified as α- d-glucosyl-(1→6)-puerarin and α- d-maltosyl-(1→6)-puerarin using ESI + TOF MS–MS and 13C NMR spectroscopy. The solubility of the transfer products was 14 and 168 times higher than that of puerarin, respectively.

Cooperative action of alpha-glucanotransferase and maltogenic amylase for an improved process of isomaltooligosaccharide (IMO) production.

Maltogenic amylase and alpha-glucanotransferase (alpha-GTase) were employed in an effort to develop an efficient process for the production of isomaltooligosaccharides (IMOs). Bacillus stearothermophilus maltogenic amylase (BSMA) and alpha-GTase from Thermotoga maritima were overexpressed in Escherichia coli using overexpression vectors. An IMO mixture containing 58% of various IMOs was produced from liquefied corn syrup by the hydrolyzing and transglycosylation activities of BSMA alone. When BSMA and alpha-GTase were reacted simultaneously, the IMO content increased to 68% and contained relatively larger IMOs compared with the products obtained by the reaction without alpha-GTase. Time course analysis of the IMO production suggested that BSMA hydrolyzed maltopentaose and maltohexaose most favorably into maltose and maltotriose and transferred the resulting molecules simultaneously to acceptor molecules to form IMOs. alpha-GTase transferred donor sugar molecules to the hydrolysis products such as maltose and maltotriose to form maltopentaose, which was then rehydrolyzed by BSMA as a favorable substrate.

Inhibition of β-glycosidases by acarbose analogues containing cellobiose and lactose structures

Acarbose analogues, containing cellobiose and lactose structures, were prepared by reaction of the two disaccharides with acarbose and Bacillus stearothermophilus maltogenic amylase. The kinetics for the inhibition by the two analogues was studied for β-glucosidase, β-galactosidase, cyclomaltodextrin glucanosyltransferase (CGTase), and α-glucosidase. Both analogues were potent competitive inhibitors for β-glucosidase, with K I values in the range of 0.04–2.44 μM, and the lactose analogues were good uncompetitive inhibitors for β-galactosidase, with K I values in the range of 159–415 μM, while acarbose was not an inhibitor for either enzyme at 10 and 5 mM, respectively. Both analogues were also potent mixed inhibitors for CGTase, with K I values in the range of 0.1–9.3 μM. The lactose analogue was a 6.4-fold better competitive inhibitor for α-glucosidase than was acarbose.

Synthesis of acarbose transfer products by Bacillus stearothermophilus maltogenic amylase with simmondsin

Simmondsin, a material related to food intake inhibition from jojoba ( Simmondsia chinensis), was transglycosylated by Bacillus stearothermophilus maltogenic amylase (BSMA) reaction with acarbose to synthesize an antiobese compound with hypoglycemic activity. Ten percent each of acarbose and simmondsin were mixed and incubated with BSMA at 55°C. Glycosylation products of simmondsin were observed by thin layer chromatography (TLC) and high performance ion chromatography (HPIC). The major transfer product was purified by using Biogel P-2 column. The structure was determined by matrix-assisted laser desorption ionization with time of flight (MALDI-TOF)/mass spectrometry (MS) and 13C-NMR. The major transglycosylation product was pseudotrisaccharide (PTS)-simmondsin, in which PTS was attached by an α-(1→6) glycosidic linkage to simmondsin. The administration of transglycosylated simmondsin with acarbose (200 mg/kg per day for 6 days) significantly reduced the food intake by 74%, comparable to 62% of simmondsin versus control in ob/ob mice. The transfer product (10 mg/kg) significantly suppressed the postprandial blood glucose response to starch (2 g/kg) by 68%, comparable to 60% of acarbose in Zucker fa/fa rats. The results indicated that the transfer products would be effective agents in lowering both food intake and blood glucose.

Transglycosylation of neohesperidin dihydrochalcone by Bacillus stearothermophilus maltogenic amylase.

Neohesperidin dihydrochalcone (NHDC), a sweet compound derived from citrus fruits, was modified to a series of its oligosaccharides by transglycosylation activity of Bacillus stearothermophilus maltogenic amylase (BSMA). Maltotriose as a donor was reacted with NHDC as an acceptor to glycosylate for the purpose of increasing the solubility of NHDC. Maltosyl-NHDC was a major transglycosylation product among the several transfer products by TLC analysis. The structure of the major transglycosylation product was determined to be maltosyl-alpha-(1,6)-neohesperidin dihydrochalcone by MALDI-TOF/MS and (1)H and (13)C NMR. Maltosyl-NHDC was 700 times more soluble in water and 7 times less sweet than NHDC.

Comparative Study of the Inhibition of α-Glucosidase, α-Amylase, and Cyclomaltodextrin Glucanosyltransferase by Acarbose, Isoacarbose, and Acarviosine–Glucose

Bacillus stearothermophilus maltogenic amylase hydrolyzes the first glycosidic linkage of acarbose to give acarviosine–glucose. In the presence of carbohydrate acceptors, acarviosine–glucose is primarily transferred to the C-6 position of the acceptor. When d-glucose is the acceptor, isoacarbose is formed. Acarbose, acarviosine–glucose, and isoacarbose were compared as inhibitors of α-glucosidase, α-amylase, and cyclomaltodextrin glucanosyltransferase. The three inhibitors were found to be competitive inhibitors for α-glucosidase and mixed noncompetitive inhibitors for α-amylase and cyclomaltodextrin glucanosyltransferase. The Ki values were dependent on the type of enzyme and their source. Acarviosine–glucose was a potent inhibitor for baker's yeast α-glucosidase, inhibiting 430 times more than acarbose, and was an excellent inhibitor for cyclomaltodextrin glucanosyltransferase, inhibiting 6 times more than acarbose. Isoacarbose was the most effective inhibitor of α-amylase and cyclomaltodextrin glucanosyltransferase, inhibiting 15.2 and 2.0 times more than acarbose, respectively.

Transglycosylation of naringin by Bacillus stearothermophilusMaltogenic amylase to give glycosylated naringin.

Naringin, a bitter compound in citrus fruits, was transglycosylated by Bacillus stearothermophilus maltogenic amylase reaction with maltotriose to give a series of mono-, di-, and triglycosylnaringins. Glycosylation products of naringin were observed by TLC and HPLC. The major glycosylation product was purified by using a Sephadex LH-20 column. The sturcture was determined by using MALDI-TOF MS, methylation analysis, and (1)H and (13)C NMR. The major transglycosylation product was maltosylnaringin, in which the maltose unit was attached by an alpha-1-->6 glycosidic linkage to the D-glucose moiety of naringin. This product was 250 times more soluble in water and 10 times less bitter than naringin.