Development of yeast strains for the efficient utilisation of starch: evaluation of constructs that express α-amylase and glucoamylase separately or as bifunctional fusion proteins

Eight constructions involving the Bacillus subtilis -amylase gene (amyE), a mouse pancreatic -amylase cDNA (AMY2) and an Aspergillus awamori glucoamylase cDNA (glaA) were prepared: three fusion genes, involving one -amylase and the glucoamylase, two double-cassette plasmids (expressing one or other -amylase and the glucoamylase) and three single-cassette plasmids, expressing the individual coding sequences. Following transformation of each plasmid into Saccharomyces cerevisiae, a plate test revealed that the largest starch hydrolysis halo was produced by the strain bearing the B. subtilis -amylase/glucoamylase fusion (BsAAase/GAase), and the smallest halo by the one expressing the mouse pancreatic -amylase/glucoamylase fusion (MAAase/GAase). When assayed for enzymatic activity in liquid medium, the strains bearing the fusion and the double-cassette plasmids involving B. subtilis -amylase and the glucoamylase exhibited both enzymic activities. Moreover, the BsAAase/GAase hybrid was able to adsorb and digest raw starch. The MAAse/GAase fusion protein was found to exhibit only -amylase activity. Finally, the capacity to grow on soluble and corn starch was tested in liquid medium for the strains bearing plasmids coding for the fusion proteins and the separate enzymes. The strain carrying the double-cassette BsAAase + GAase, which produced one of the smallest hydrolysis haloes in the place test, showed the best performance, not only in digesting soluble and corn starch but also in using all of the hydrolysis products for growth. The transformant bearing the BsAAase/GAase fusion was able to grow on soluble starch, but not on corn starch.     

Enhancement of α-amylase production by integrating and amplifying the α-amylase gene of Bacillus amyloliquefaciens in the genome of Bacillus subtilis

Summary The -amylase gene of Bacillus amyloliquefaciens was integrated into the genome of Bacillus subtilis by homologous recombination. In the first transformation step, several strains were obtained carrying the -amylase gene as two randomly located copies. These strains produced -amylase in the quantities comparable with that of the multicopy plasmid pKTH10, carrying the same -amylase gene. With the plasmid system, however, the rate of the -amylase synthesis was faster and the production phase shorter than those of the chromosomally encoded -amylase. The two chromosomal gene copies were further multiplied either by amplification using increasing antibiotic concentration as the selective pressure or by performing a second transformation step, identical to the first integration procedure. Both methods resulted in integration strains carrying up to eight -amylase gene copies per one genome and producing up to eightfold higher -amylase activity than the parental strains. Six out of seven transformants, studied in more detail, were stable after growth of 42 h even without antibiotic selection. The number of the DNA and mRNA copies of the -amylase gene was quantitavely determined by sandwich hybridization techniques, directly from culture medium.     

The effect of glucose on the expression of a clonedBacillus amyloliquefaciens α-amylase gene in strains ofBacillus subtilis

Bacillus subtilis strain 1A297 was shown to relieve the glucose repression of a clonedB. amyloliquefaciens -amylase gene carried on the hybrid plasmid pVC102 without affecting its temporal activation. However, glucose repression of -amylase occurred when pVC102, was introduced intoB. subtilis strain 1A289. Glucose repression was relieved by -methyl-d-glucoside, an analog of glucose that blocks its uptake. The relief of glucose repression in 1A297 did not act at the level of plasmid copy number. As 1A297 was capable of exerting glucose repression on a homologous chromosomally encoded gene, it is postulated that the putativetrans-acting product involved in glucose repression inB. subtilis (Nicholson and Chambliss, 1986, J. Bacteriol. 165:663–670) is altered in strain 1A297 and does not recognize theB. amyloliquefaciens -amylase gene.     

Enhanced alpha - amylase production by chromosomal integration of pTVA1 in industrial strain in Bacillus subtilis

Summary Strain Bacillus subtilis MS was constructed with 12–22 fold increase of -amylase production, caused by presence of multiple -amylase gene copies in the chromosome of industrial strain Bacillus subtilis CCM2722, as demonstrated by DNA hybridization. The enhanced production is a result of multiple integration of plasmid pTVA1, carrying a temperature sensitive origin of replication from pE194, and containing the -amylase gene and a modified transposon Tn917.     

Solvent production from starch: effect of pH on α-amylase and glucoamylase localization and synthesis in synthetic medium

Summary The fermentation of starch by Clostridium acetobutylicum ATCC 824 has been reviewed in an optimised synthetic medium. A progressive increase of pH from 4.4 to 5.2 led to a higher production of extracellular -amylase whereas glucoamylase was poorly affected. A portion of these enzymes was cell-associated and on increasing the pH from 4.4 to 5.8 a decrease was noted in cell-bound enzymes. The association was higher for the glucoamylase than for the -amylase. The highest rate of starch consumption was at pH 5.2 whereas due to the earlier shift to solvent production at low pH, the highest solvent production was at pH 4.4. This study suggested that the level of -amylase and then the rate of starch hydrolysis was the limiting step of sugar catabolism in C. acetobutylicum ATCC 824. Correspondence to: P. Soucaille     

Effect of polyethylene glycol on Bacillus subtilis α-amylase purification with raw and modified starch

Polyethylene glycol was found to enhance adsorption of Bacillus subtilis -amylase on starch in optimum concentration 10 % (w/w). Degree of adsorption at 12°C was increased from 83 to 98 % and from 30 to 81 % for cross-linked and raw starch, resp. Higher sorption capacity and easy desorption of -amylase without temperature or pH change was reached at 22 °C. Yield of -amylase 95 % and purification factor 8.3 were achieved on the cross-linked starch column. The method is suitable for -amylase isolation from PEG phase after its microbial production in aqueous two-phase systems.     

Selective growth of a mutant in continuous culture of Bacillus caldolyticus for production of α-amylase

Summary In a continuous culture of Bacillus caldolyticus strain SP, which requires maltose as an inducer for production of -amylase in batch culture, a predominant mutant strain M1 which produced high amounts of -amylase in the absence of maltose in batch culture, developed. The change of cell population from strain SP to strain M1 in maltose-casitone medium was linear with time in the transient state after the change from batch to continuous culture at a dilution rate of 0.17 h^-1, and was completed in about 11 generations of bacterial growth. The dilution rate effect of continuous culture on -amylase activity was almost the same with both strains SP and M1. The maximum -amylase activity of 380 units/ml was observed at an intermediate dilution rate that was 11.5 times higher than -amylase activity at the end of a batch culture using the same medium. It was deduced that the enhancement of -amylase production in continuous culture was attributed partly to the predominant growth of a mutant strain with higher -amylase productivity.     

Colony switching in an alpha-amylase-producing strain ofBacillus subtilis

Four colony variants (two stable and two unstable phenotypes) were observed inBacillus subtilis -10, an -amylase-hyperproducing strain. The stable variants lost the ability to produce -amylase, while the unstable ones reverted to the typical morphology after restreaking. Unstable expanding sectors appeared in typical colonies, and their appearance was influenced by the culture origin and age.     

Characterization of a new Bacillus stearothermophilus isolate: a highly thermostable α-amylase-producing strain

A novel strain of Bacillus stearothermophilus was isolated from samples of a potato-processing industry. Compared to known -amylases from other B. stearothermophilus strains, the isolate was found to produce a highly thermostable -amylase. The half-time of inactivation of this -amylase was 5.1 h at 80°C and 2.4 h at 90°C. The temperature optimum for activity of the -amylase was 70°C; the pH optimum for activity was relatively low, in the range 5.5–6.0. -Amylase synthesis was regulated by induction and repression mechanisms. An inverse relationship was found between growth rate and -amylase production. Low starch concentrations and low growth temperatures were favourable for enzyme production by the organism. At the optimal temperature for growth, 65°C, the -amylase was a growth-associated enzyme. The optimal temperature for -amylase production, however, was 40°C, with -amylase increasing from 3.9 units (U)/ml to 143 U/ml when lowering the growth temperature from 65°C to 40°C. Maximal -amylase production in a batch fermentor run at 65°C was 102 U/ml, which was 26-fold higher than in erlenmeyer flasks at 65°C. The dissolved O₂ concentration was found to be a critical factor in production of the -amylase.     

α-Amylase production by immobilized whole cells of Bacillus subtilis

Summary Whole cells of Bacillus subtilis were immobilized in polyacrylamide gel prepared from 5% total acrylamide (85% acrylamide and 15% N,N-methylenebisacrylamide). Production of -amylase by the immobilized whole cells was attempted in a batch system. -Amylase produced by the immobilized whole cells was about three times larger than that produced by washed cells at optimum conditions. The reusability of the immobilized whole cells and washed cells was examined. The activity of -amylase production by washed cells decreased with increasing use cycles. On the other hand, the activity of the immobilized cells increased gradually, and it reched a steady state after seven cycles. -Amylase was produced from a simple reaction medium containing 1% meat extract and 0.05% yeast extract by the immobilized whole cells. The rate of -amylase production by the immobilized whole cells was the same as in submerged cultivation using starch bouillon medium. Growth of B. subtilis in polyacrylamide gel was observed by electron microscopy.     

Secretion of the sweet-tasting plant protein thaumatin byBacillus subtilis

Summary With the -amylase promoter and ribosome binding site,Bacillis subtilis was used to express the sweet plant protein thaumatin II cDNA fused in the correct reading frame to the -amylase leader peptide. The r-thaumatin was purified from the medium on a S-Sepharose column and detected with western blots by sheep -thaumatin antibodies. The r-thaumatin and authentic thaumatin were the same size when reduced by 2-ME and the same size when not reduced.     

Differential expression of α-amylase genes in germinating rice and barley seeds

Steady-state levels of mRNA from individual -amylase genes were measured in the embryo and aleurone tissues of rice (Oryza sativa) and two varieties of barley (Hordeum vulgare L. cv. Himalaya and cv. Klages) during germination. Each member of the -amylase multigene families of rice and barley was differentially expressed in each tissue. In rice, -amylase genes displayed tissue-specific expression in which genes RAmy3B, RAmy3C, and RAmy3E were preferentially expressed in the aleurone layer, genes RAmy1A, RAmy1B and RAmy3D were expressed in both the embryo and aleurone, and genes RAmy3A and RAmy2A were not expressed in either tissue. Whenver two or more genes were expressed in any tissue, the rate of mRNA accumulation from each gene was unique. In contrast to rice, barley -amylase gene expression was not tissue-specific. Messenger RNAs encoding low- and high-pI -amylase isozymes were detectable in both the embryo and aleurone and accumulated at different rates in each tissue. In particular, peak levels of mRNA encoding high-pI -amylases always preceded those encoding low-pI -amylases. Two distinct differences in -amylase gene expression were observed between the two barley varieties. levels of high-pI -amylase mRNA peaked two days earlier in Klages embryos than in Himalaya embryos. Throughout six days of germination, Klages produced three times as much high-pI -amylase mRNA and nearly four times as much low-pI -amylase mRNA than the slower-germinating Himalaya variety.     

Production of amylolytic enzymes by Bacillus amyloliquefaciens in pure culture and in co-culture with Zymomonas mobilis

Mixed cultures of Bacillus amyloliquefaciens MIR-41 and Zymomonas mobilis Flo-B3 showed a 2.5 fold increase in -amylase production, and a 20 times fold decrease in ethanol production compared with pure cultures. Enhanced -amylase production by B. amyloliquefaciens in mixed cultures after 24 h could be attributed to the lack of repression in the synthesis of -amylase by ethanol and protease inhibition by the pH of the culture medium.     

Selection of microorganisms which produce raw-starch degrading enzymes

Summary Microorganisms which produce strong raw-starch degrading enzymes were isolated from soil using a medium containing a unique carbon source, -amylase resistant starch (-RS), which is insoluble in water and hardly digested with Bacillus amyloliquefaciens -amylase. Among the isolates, three strains showing high activities were characterized. Two of them, K-27 (fungus) and K-28 (yeast), produced -amylase and glucoamylase, and the final product from starch was only glucose. The third strain, K-2, was a bacterium and produced -amylase, which produced glucose and malto-oligosaccharides from starch. The enzyme preparation of these strains degraded raw corn starch rapidly.     

The effects of alpha amylase on collagen-proteoglycans and collagen-glycoprotein complexes in connective tissue matrices

Summary Different connective tissues were digested with Bacillus subtilis -amylase in an effort to determine the effects of this enzyme on the glycoprotein-collagen complexes. It was found that in some tissues insoluble collagen could not be attacked by Clostridium histolyticum collagenase unless a glycoprotein fraction, strongly associated with the fibers, was removed. This removal was achieved by -amylase, thus supporting earlier observations that sugar-containing compounds are strongly bound to collagen. Using alpha amylase an unexpected and interesting result demonstrated that this enzyme(s) releases acid glycosaminoglycans from the various connective tissues. Chemical studies have shown that the -amylase preparation was not contaminated to any significant extent by hyaluronidase or by a protease.Supported by Grant DE-02110-05 of the National Institutes for Public Health, Bethesda, Maryland.     

Protein secretion by minicells of Bacillus subtilis

Summary The minicell producing strain Bacillus subtilis IA292 was transformed with plasmids encoding the Bacillus enzymes -glucanase, -amylase and neutral protease. Purified minicells were shown to be free of detectable proteolytic activity. Minicells containing plasmids were found to synthesise all three enzymes internally, but evidence of secretion was only observed in the unique case of neutral protease secretion by minicells prepared from cultures grown in BHI medium.     

Production of heterologous proteins in Bacillus subtilis: the effect of the joint between signal sequence and mature protein on yield

Summary We have previously made a set of DNA constructs by fusing the mature part of Bacillus licheniformis -amylase with the signal sequence of B. amyloliquefaciens -amylase at various distances from the signal sequence cleavage site. We observed that the level of -amylase production in B. subtilis depended strongly on the distance of the junction from the signal sequence cleavage site, with quite a sharp optimum distance. To test whether the effect is limited to the pair of -amylase signal sequence and mature protein, we analysed the protein production in a set of constructs in which an Escherichia coli \-lactamase was similarly joined at different distances from the -amylase signal sequence. Also in this case the distance seemed to be an important factor in affecting the level of production in B. subtilis. The observed effect might depend on the modulation of pre-protein folding, which in turn could affect the secretion level. Offsprint requests to: M. Sibakov     

α-Amylase and glucoamylase production by Schwanniomyces castellii

A chromogenic substrate (Cibachron blue-amylose), and soluble starch and maltose were used to characterize the amylolytic system from Schwanniomyces castellii 3754. The strain was able to produce inducible -amylase (EC 3.2.1.1) and glucoamylase (EC 3.2.1.3) when grown on different C sources. The effect of the C source was slightly different for -amylase and glucoamylase production. Melezitose, maltose and soluble starch enhanced both -amylase and glucoamylase synthesis to nearly the same extent; amylose, trehalose and cellobiose particularly induced -amylase synthesis. The optimal pH for the release of both amylases was 5.5–7.0; maximal -amylase synthesis, on the other hand, was observed in the medium buffered at pH 6.0. The optimal pH for -amylase and glucoamylase activity was in the range of 4.5–7.2 and 4.2–5.5, respectively. Temperatures allowing maximal activity were 45°C for -amylase and 45–52°C for glucoamylase; a rapid decline of both activities was observed just above these temperatures.The species Schwanniomyces castellii (together with Schw. alluvius) is now considered to be synonymous with Schw. occidentalis var. occidentalis (Kreger-Van Rij 1984).     

Regio- and stereo-selective synthesis of vinyl glucose ester catalyzed by an alkaline protease of Bacillus subtilis

The transesterification of -d-glucose with divinylsuccinate, divinyladipate and divinylsebacate in pyridine at 55 °C for 3 days was catalyzed by an alkaline protease from Bacillus subtilis to give corresponding 6-O-vinyl glucose esters at 30%, 53% and 35% yield, respectively. The stereo-selectivity of the alkaline protease toward the -anomer was affected by the acyl donor chain length. 6-O-Vinylsuccinyl-d-glucose was mixture of - and -anomers (/=44/56), the other two products were the pure -d-glucose derivatives.