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.     

α-Amylase production with Bacillus subtilis in the presence of PEG and surfactants

Summary -Amylase production with Bacillus subtilis was studied in the presence of PEG 600 and PEG 3350 as well as different surfactants: Trixon X-100, Tween 80, CTAB (cetylammonium-bromide) and SDS (sodiumdodecylsulphate) at concentrations resulting in comparable decreases in surface tension. Only PEG 600, at a concentration of 200 g/kg, was found to increase the enzyme production. Cell growth estimated as optical density at 620 nm and viable counts were not influenced by either PEG or surfactants. The results are discussed in relation to -amylase production in aqueous two-phase systems.     

Reversibility of Acid-Inactivation of Bacillus subtilis’ α-Amylase

The inactivation of Bacillus subtilis’ α-amylase by acid was shown to be reversible. In the experiment, two different Bac. subtilis’ α-amylases, saccharifying and liquefying types, were used and the reversibility was investigated deviding into two processes of inactivation and reactivation. Both amylases showed the reversibility in a similar degree and in general the inactivated enzymes by acid were reactivated only by adjusting the pH to slightly alkaline values followed by incubation under certain conditions. However, the reversibility, especially, the reactivation was greatly influenced by several chemicals, the effect of certain chemicals being different according to the type of the bacterial amylase. Contrary to liquefying amylase, saccharifying amylase was insensitive to metal chelators but, nevertheless, the reactivation of the amylase was prevented by metal chelators. Also the reactivation of saccharifying amylase was inhibited by sulfhydryl reagents, although the native enzyme was quite insensitive to the chemicals. In the acid-inactivation and reactivation process, a reversible change in the ultraviolet absorption spectra of the enzymes was observed, and some discussion of the implication was presented.     

Reversibility of Heat-Inactivation of Bacillus subtilis’ α-Amylase

Inactivation of Bacillus subtilis’ α-amylase by heat was found to be reversible under a certain condition, and the factors affecting there were investigated, distinguishing into two groups: those influencing on the inactivation process by heat and those on the reactivation at the subsequent incubation after heating. Generally, the amylase heated in borate buffer solution was best in the reactivation degree. For reactivation of the heat-inactivated enzyme there was found an optimum in temperature, pH and concentration of enzyme, respectively. The reactivation was temporarily prevented by urea, but irreversibly inhibited by either calcium salts or calcium binding agents. In the reversible heat-inactivation of the enzyme was also found a reversible change in the absorption spectra as well as in the behavior of the enzyme toward proteinase.     

α-Amylase production by Bacillus subtilis and B. amyloliquefaciens in different PEG solutions

-Amylase production by Bacillus subtilis and Bacillus amyloliquefaciens was investigated in polyethyleneglycol (PEG)-containing growth medium. Five different molecular weight PEGs (600, 3000, 4000, 8000 and 20,000) were used. Enzyme production with B. subtilis increased 21% in medium containing 5% PEG 3000, but enzyme production with B. amyloliquefaciens increased 31% in medium containing 5% PEG 600 and 21% in medium containing 2% PEG 8000.     

Structure of Bacillus subtilis Bacteriophage φ25 and φ25 Deoxyribonucleic Acid

The structure of Bacillus subtilis bacteriophage phi25 and phi25 deoxyribonucleic acid (DNA) were studied by electron microscopy. The head of phi25 is a regular polyhedron measuring 75 nm in diameter. The uncontracted tail of phi25 is 130 nm in length and includes a large, complex tail plate. Phage phi25 DNA is double-stranded and has a molecular weight of approximately 100 million as determined by electron microscopic length measurements and analytical band sedimentation in CsCl. The complementary strands of phi25 DNA contain numerous random interruptions. Chemical analysis of phi25 DNA demonstrated that 5-hydroxymethyluracil replaces thymine and that the DNA has a mole per cent (guanine plus cytosine) of 42.     

Influence of a Cell-Wall-Associated Protease on Production of α-Amylase by Bacillus subtilis

AmyL, an extracellular α-amylase from Bacillus licheniformis, is resistant to extracellular proteases secreted by Bacillus subtilis during growth. Nevertheless, when AmyL is produced and secreted by B. subtilis, it is subject to considerable cell-associated proteolysis. Cell-wall-bound proteins CWBP52 and CWBP23 are the processed products of the B. subtilis wprA gene. Although no activity has been ascribed to CWBP23, CWBP52 exhibits serine protease activity. Using a strain encoding an inducible wprA gene, we show that a product of wprA, most likely CWBP52, is involved in the posttranslocational stability of AmyL. A construct in which wprA is not expressed exhibits an increased yield of α-amylase. The potential role of wprA in protein secretion is discussed, together with implications for the use of B. subtilis and related bacteria as hosts for the secretion of heterologous proteins.The cell envelope of the gram-positive bacterium Bacillus subtilis consists of a single (cytoplasmic) membrane surrounded by a relatively thick cell wall consisting of similar proportions of peptidoglycan and covalently attached anionic polymers. The absence of an outer membrane means that there is no equivalent of the membrane-enclosed periplasm found in gram-negative bacteria. However, by virtue of its thickness and high density of negative charge, the cell wall may perform some of the roles of the periplasm in gram-positive bacteria.The absence of an outer membrane in gram-positive bacteria also simplifies the secretion pathway, and, consequently, B. subtilis and its close relatives have the potential to secrete proteins directly into the growth medium, at concentrations in excess of 5 grams per liter (4). Despite its extensive use in the production of commercially important Bacillus enzymes (e.g., α-amylases and alkaline proteases), attempts to exploit B. subtilis for the production of heterologous proteins at high concentrations have proved disappointing (8). One reason for this failure is the production and release into the culture medium of several extracellular proteases (24, 28, 37). Although native Bacillus proteins are generally resistant to these proteases, heterologous proteins are often rapidly degraded in their presence. As a result, strains of B. subtilis that are multiply deficient in extracellular proteases have been developed (11, 37). The more developed of these strains have less than 1% of the proteolytic activity of the wild type (37). To date, efforts have concentrated mainly on the proteases which reside in a truly extracellular location, while those which remain cell associated have been largely overlooked.Although strains deficient in extracellular proteases have improved the productivity of B. subtilis for the production of heterologous proteins, they have only partially overcome problems of unexpectedly low yields. We and others have recently shown (22, 31) that significant amounts of secretory protein are degraded within minutes of being synthesized. This degradation is observed even for Bacillus proteins that are highly resistant to proteases released into the culture medium, suggesting that a component of this degradation is cell associated.Margot and Karamata recently reported the identification of a cell-wall-associated protease encoded by the wprA gene (21). The primary product of this gene is a 96-kDa polypeptide that is processed into two previously identified cell wall proteins, namely, CWBP52 and CWBP23. The processing of the WprA precursor during secretion accompanies the targeting of CWBP52 and CWBP23 to the cell wall and is analagous to the processing of another B. subtilis cell-wall-bound protein, namely, WapA (5). The amino acid sequence of CWBP52 shows a high degree of similarity with serine proteases of the subtilisin family, and phenylmethylsulfonile fluoride (PMSF)-sensitive protease activity was detected in proteins extracted from the cell wall of a wprA⁺ strain, but not one in which this gene had been insertionally inactivated (21). In the absence of homology to proteins in the databases, the N-terminal CWBP23 moiety was presumed to function as a chaperone-like propeptide that is proteolytically processed on the trans side of the membrane. In this paper, we report on a potential role of products of wprA in the integrity of secretory proteins during late stages in the secretion pathway. We also discuss the potential of wprA mutants to increase the productivity of B. subtilis for secretory proteins.     

Growth of Bacteriophage φ105 and Its Deoxyribonucleic Acid in Radiation-Sensitive Mutants of Bacillus subtilis

Growth of phage phi105 and its deoxyribonucleic acid (DNA) was studied in radiation-sensitive mutants of Bacillus subtilis. The recA gene is required for optimal prophage induction with mitomycin C and for infectivity of prophage DNA. rec B gene is required for marker rescue from mature DNA. The importance of bacterial genes for phage DNA activity seems to depend on phage DNA structure.     

Construction of Transducing Phage ρ 11 Containing α-Amylase Structural Gene of Bacillus subtilis

We constructed a reporter system to detect a superoxide-generating methyl viologen using SoxRS of Escherichia coli and GFP of Aequorea victoria. E. coli carrying this plasmid exhibited strong fluorescence when grown in the presence of a superoxide-generating reagent methyl viologen. The fluorescence intensity observed in the stationary phase culture of the transformant increased in response to the methyl viologen concentration in a range of 0.01 μM to 10 μM.     

Glucosyl Glycerophosphoric Acid and its Polymer Excreted by α-Amylase-forming Bacillus subtilis

α-Amylase formation by washed cell suspensions of Bac. subtilis was found to be accompanied by the excretion of a compound consisting of glucose, glycerol and phosphoric acid. It was excreted as a polymer and a monomer. The former, a kind of teichoic acid, was significantly dominant in quantity when the cells were incubated under the conditions suitable for α-amylase formation. On the other hand, the monomer prevailed when the bacterial cells were under the unfavorable conditions for the enzyme formation.

Both compounds were purified by ion exchange column chromatography. Chemical and enzymatic investigations revealed the following structures: 2-O-α-d-glucopyranosyl-glycerol-3-monophosphoric acid for the monomer, and a polymerized form of the monomer through phosphodiester linkages involving the hydroxyl groups on C3 of the glycerol, for the polymer.     

Conversion of Purine Bases and Their Ribosides to 5′-Inosinic Acid by Bacillus subtilis

Among various nutritional mutants with weak 5′-nucleotidase derived from Bacillus subtilis IAM 1145, the adenine-requiring mutants could convert exogenously added hypo- xanthine, guanine, xanthine and their ribosides to 5′-inosinic acid (IMP) and accumulate it in the medium. Synthesis of IMP from purine derivatives was observed predominantly in an early stage of the cultivation. The conversion was stimulated by Fe²⁺ or Mn²⁺, and markedly depressed by an excess amount of adenine in the production-medium.     

Characterization of Infectious Deoxyribonucleic Acid from Temperate Bacillus subtilis Bacteriophage φ 105

Phenol-extracted, infectious deoxyribonucleic acid (DNA) species from phi105 phage particles, from phi105 lysogenic bacteria, and from induced phi105 lysogenic bacteria were sedimented in sucrose gradients. Infectious DNA from phi105 particles sedimented like the bulk of mature phage DNA in neutral sucrose. Infectivity of prophage DNA was associated with fast-sedimenting material of heterogenous size. Infectious vegetative phage DNA sedimented somewhat faster than mature phage DNA; it was rapidly converted to a poorly infectious form during the infection.     

α-LNA (α-D-Configured Locked Nucleic Acid)

Abstract

Two pyrimidine α-LNA nucleoside monomers have been synthesised and incorporated into α-configured oligonucleotides. A fully modified mixed α-LNA sequence displays unprecedented parallel stranded hybridisation with complementary RNA and a remarkable selectivity for RNA over DNA. Modelling shows α-LNA : RNA to form an extended duplex with a very broad major groove.     

Stepwise Introduction of Regulatory Genes Stimulating Production of α-Amylase into Bacillus subtilis : Construction of an α-Amylase Extrahyper Producing Strain

The production of extracellular α-amylase in Bacillus subtilis is probably regulated by many genetic elements, such as amyR, tmrA7, pap, amyB and sacU. Additional genetic elements, C-108 and A-2 for production of the α-amylase were found in D-cycloserine and ampicillin resistant mutants (C108 and A2) of B. subtilis 6160, respectively. Strain C108 increased the production of α-amylase about 5 times and protease about 80 times compared to parental 6160 strain. Strain A2 showed a nearly 6-fold increased α-amylase production.

These genetic elements displayed a synergistic effect with other genetic factors in production of extracellular α-amylase when these elements were transferred by DNA mediated transformation. By stepwise introduction of these and other genetic elements into B. subtilis 6160 by transformation and mutation, strains with higher α-amylase producing activity were obtained. The finally obtained strain, T2N26, produced about 1,500-2,000 times more α-amylase than parental 6160 strain.     

α-Amylase production in batch and continuous cultures by Bacillus caldolyticus

Summary The concentration and productivity of -amylase increased remarkably, 15- and 11-fold respectively, in a continuous culture of Bacillus caldolyticus DSM 405 compared with batch culture, provided starch was used as the sugar source in a casitone medium. In the casitone medium with or without glucose hardly any improvement of enzyme production was observed in continuous culture. The addition of a small amount of starch to the glucose-casitone medium had a marked effect in stimulating amylase formation in continuous culture but no effect in batch culture.It was suggested that the higher production of -amylase in the continuous culture using starch as the inducer was partly related to the predominance of some conditional non-sporulating variants with a higher amylase forming activity and to derepression of the enzyme at a low glucose concentration.     

Propeptide of Bacillus subtilis amylase enhances extracellular production of human interferon-α in Bacillus subtilis

The Gram-positive bacterium, Bacillus subtilis and related species are widely used industrially as hosts for producing enzymes. These species possess a high potential to produce secreted proteins into the culture medium. Nevertheless, the secretion of heterologous proteins by these species is frequently inefficient. In this study, the human interferon-α2b (hIFN-α2b) was used as a heterologous model protein, to investigate the effect of B. subtilis AmyE propeptide in enhancing the secretion of heterologous proteins in B. subtilis. We found that the secretion production and activity of hIFN-α2b with AmyE propeptide increased by more than threefold, compared to that without AmyE propeptide. The maximum amount of secreted hIFN-α2b with propeptide was 14.8 ± 0.6 μg ml⁻¹. In addition, the pro-hIFN-α2b bioactivity reached 5.4 ± 0.5 × 10⁷ U mg⁻¹, which is roughly the same level as that of the non-propeptide hIFN-α2b. These results indicated that AmyE propeptide enhanced the secretion of the hIFN-α2b protein from B. subtilis. This study provides a useful method to enhance the extracellular production of heterologous proteins in B. subtilis.     

Cloning of Thermostable α-Amylase Gene Using Bacillus subtilis Phage ρ11 as a Vector

Using a method consisting of two repetitions of “prophage transformation,” the thermostable α-amylase gene in Bacillus subtilis has been cloned in temperate phage ρ11.     

Formation of Phage-Induced γ-Polyglutamic Acid Depolymerase in Lysogenic Strain of Bacillus natto

The influence of tea catechins on the absorption of starch or sucrose was investigated in vivo. Tea catechins were administered orally to rats before soluble starch or sucrose administration. Saccharide-dosed rats were killed and the blood and the contents of the intestine were collected at intervals over two hours. Catechins of certain concentrations suppressed the increase of plasma glucose levels, thus concurrently suppressing insulin activity. Increased activity of intestinal α-amylase by starch dosing was inhibited markedly in the catechin-administered rats. Sucrase on the brush border membrane was also inhibited by prior catechin administration. From these results it was assumed that orally administered catechins will inhibit intestinal α-amylase or sucrase, thereby deterring the digestion of certain amounts of starch or sucrose and eventually reducing the plasma glucose levels.     

Hybrid On-Line Optimal Control Strategy for Producing α-Amylase by Bacillus subtilis

The combined effect of macronutrients in the extraction medium on α-amylase produced by Bacillus subtilis were studied by using response surface methodology in shaken flask cultures. The production of amylase was significantly affected by the interaction between wheat bran and the cotton seed extract in the extraction medium and by the interaction between the cotton seed extract and starch. The optimal combination in the extraction medium for maximum α-amylase production was determined as 10.80 g·L^?1 of wheat bran, 9.90 g·L^?1 of cotton seed extract, 0.5 g·L^?1 of starch, 2.0 g·L^?1 of yeast extract, 5.00 g·L^?1 of NaCl and 2.00 g·L^?1 of CaCl₂. A 12.55-fold increase of enzyme activity was recorded in the optimized medium compared to the result acquired in a minimum essential medium. The optimized medium was used to compare different cultivation strategies in fermenters. The pH-stat strategy for reducing cellular stress response and the substrate concentration-stat strategy for reducing substrate inhibition were independently investigated. The temperature-limited strategy has been proposed to solve the proteolytic digestion problem, although the high-pressure strategy resulted in high productivity. A hybrid strategy simultaneously controlling pH, temperature, substrate concentration and pO₂ was finally investigated to enhance the efficiency of the process. This hybrid strategy resulted in high activity of α-amylase, increasing the productivity almost three-fold as compared to an ordinary fed-batch culture.