L-Lysine production by mutants of Bacillus licheniformis

Summary A bacterium able to grow at 46°C was isolated from soil and identified asBacillus licheniformis. L-Lysine producing mutants were derived from the bacterium by the introduction of resistance to S-(2-amino)-ethylcysteine (thialysine) and auxotrophy.One of the mutants, HBR-2 (Thialysine^r, Leu^–, Homoserine^–), produced L-lysine at a concentration of 30 mg/ml in a molasses medium containing 10% reducing sugar.     

Biocatalytic Knoevenagel reaction using alkaline protease from Bacillus licheniformis

Abstract

BLAP (alkaline protease from Bacillus licheniformis) was used as a biocatalyst in the Knoevenagel condensations of aromatic, hetero-aromatic and α;β-unsaturated aldehydes with less reactive acetylacetone or ethyl acetoacetate. The reactions were performed in organic solvent in the presence of water. The functionalized trisubstituted alkenes and α,β,γ,δ-unsaturated carbonyl compounds could be obtained in moderate to good yields with E/Z selectivities up to >99:1. This biocatalytic reaction provided an alternative pathway for Knoevenagel condensation, which also demonstrates a novel case of unnatural activity of existing enzymes.     

Oxygen-transfer strategy and its regulation effects in serine alkaline protease production by Bacillus licheniformis

The effects of oxygen transfer on the production and product distribution in serine alkaline protease (SAP) fermentation by Bacillus licheniformis and oxygen-transfer strategy in relation to the physiology of the bacilli were investigated on a defined medium with citric acid as sole carbon source in 3.5-dm(3) batch bioreactor systems. By forming a 3 x 3 matrix with the parameters air-inlet rates of Q(O)/V(R) = 0.2, 0.5, 1.0 vvm, and agitation rates of N = 150, 500, 750 min(-1), the effects of oxygen transfer were investigated at nine different conditions. The concentrations of the product SAP and by-products, i.e., neutral protease, alpha-amylase, amino acids, and organic acids, and SAP activities were determined throughout the bioprocess. Among the constant air-flow and agitation-rate fermentations, Q(O)/V(R) = 0.5 vvm, N = 750 min(-1) oxygen-transfer conditions produced maximum SAP activity that was 500 U cm(-3), at t = 37 h. With the increase in Q(O)/V(R) and/or N, Damk?hler number that is the oxygen-transfer limitation decreases; and the process passes from oxygen-transfer limited conditions to biochemical-reaction limited conditions. Further increase in SAP activity, A = 680 U cm(-3) was achieved by applying an oxygen-transfer strategy based on the analysis of the data obtained with the constant oxygen-transfer condition experiments, with a step increase in air-inlet rate, from Q(O)/V(R) = 0.2 to Q(O)/V(R) = 0.5 vvm at N = 750 min(-1) constant agitation rate at t = 24 h. Organic acids and amino acids that were excreted to the fermentation medium varied depending on the oxygen-transfer conditions. With the increase in oxygen-transfer rate acetic acid concentration increased; contrarily, with the decrease in the oxygen-transfer rate the TCA-cycle organic acids alpha-ketoglutaric and succinic acids, and gluconic acid were excreted to the fermentation broth; nevertheless, the application of the oxygen-transfer strategy prevented the increase in acetic acid concentration between t = 35-38 h. Under all the oxygen-transfer conditions, the amino acid having the highest concentration and the amino acid that was not excreted to the fermentation broth were lysine and asparagine, respectively; both of which belong to the aspartic acid-group amino acids. Further, this result indicates the requirement of the genetic regulation directed to the aspartic acid-group enzymes for the progress in SAP production in B. licheniformis.     

Cloning and over-expression of an alkaline protease from Bacillus licheniformis

The alkaline protease gene, apr, from Bacillus licheniformis 2709 was cloned into a Bacillus shuttle expression vector, pHL, to yield the recombinant plasmid pHL-apr. The pHL-apr was expressed in Bacillus subtilis WB600, yielding a high expression strain BW-016. The amount of alkaline protease produced in the recombinant increased by 65% relative to the original strain. SDS-PAGE analysis indicated a Mr of 30.5 kDa. The amino acid sequence deduced from the DNA sequence analysis revealed a 98% identity to that of Bacillus licheniformis 6816.     

地衣芽胞杆菌碱性蛋白酶基因克隆与表达特性

目的建立高产量和高活力的地衣芽胞杆菌碱性蛋白酶基因表达体系。方法采用PCR技术克隆获得目的基因,将其连入表达质粒pET-32 a构建原核表达重组质粒,经测序鉴定后,转化BL21大肠埃希菌,不同温度下IPTG诱导表达融合蛋白,测定酶活;进一步对该基因和编码蛋白进行同源性比较和酶学性质分析。结果碱性蛋白酶基因序列全长1 149 bp,编码382个氨基酸,同源性为99%,融合蛋白分子质量为62 kD,蛋白酶酶活为29 000 U/mL,并且在25℃时是以可溶蛋白形式表达,37℃时部分蛋白以包涵体形式存在。结论此种表达体系可以成功表达具有生物活性的碱性蛋白酶,诱导温度对蛋白酶存在形式具有较大影响。     

地衣芽孢杆菌碱性蛋白酶的研究 Ⅰ.碱性蛋白酶高产菌的筛选及产酶条件初探

从成都佳丰食品厂等处采集的样品中平板分离初筛到124株碱性蛋白酶产生菌,进一步复筛出一株高产,且稳定的碱性蛋白酶产生菌株B．L．JF-ld,初步鉴定为地衣穿孢杆菌（Bacilluslicheniformis）。该菌的最适产酶条件为：培养基（％）为麦芽糖7．5,酵母膏3,NaCl0．5,K2HPO4·3H2O0．53,NaHPO4·2H2O0．03,Na2CO30．056,MnSO4l×10－4mol／L,pH8．7,通气量为（1：0．5）～（1：1）（v／v）,37℃发酵40h,酶活力单位高达7180U／ml。     

地衣芽孢杆菌JF-UN122碱性蛋白酶的分离纯化与性质

地衣芽孢杆菌JF—UN122的发酵液,以硫酸铵分段盐析得粗酶,再经DEAE—Sephadex A—50吸附色素、CM—Sephadex C-50离子交换及Sephadex G—75柱层析等步骤获得电泳纯的碱性蛋白酶。SDS-PAGE测得其分子量为31．6KDa。以酪蛋白为底物时,酶的Km为5．26μg／min,Vm为20．8μg／min。酶的最适pH为9．0,最适温度为55℃,pH5～11,55℃以下酶较稳定,对1mol／LH2O2具有一定的耐氧化性。PMSF对酶抑制,二硫苏糖醇(DTT)有保护作用,钙离子、EDTA、SDS、尿素等对酶无明显影响。     

Effect of bioprocess conditions on growth and alkaline protease production by halotolerant Bacillus licheniformis BA17

The effect of bioprocess conditions (pH and temperature) on the growth and alkaline protease production of halotolerant Bacillus licheniformis BA17 bioreactor cultures have been systematically analyzed using response surface methodology in order to assess the importance of these generally disregarded parameters. Two models were proposed differing by the choice of response variable. Under optimized bioprocess conditions, whole alkaline protease activity was about 3 fold higher than the activities obtained in the preliminary studies. Results of this study not only highlight the importance of pH and temperature for further engineering purposes but also serve as basis for understanding the true mechanism lying under the relation between these process parameters and growth and whole alkaline protease production.     

Production of alkaline protease by Bacillus licheniformis in an aqueous two-phase system

Alkaline protease production by Bacillus licheniformis was studied in an aqueous two-phase system composed of 5% (w/w) polyethylene glycol 6000 (PEG 6000) and 5% (w/w) dextran T500. The top phase was continuous and rich in PEG while the bottom phase was dispersed and rich in dextran. The cells were retained in the bottom phase and at the interface. The two-phase system produced less enzyme in total amount than the control in the early phase, but after 50 h the enzyme produced in the control system decreased while the aqueous two-phase system continued its production and finally the total enzyme activity reached 1.3 times that of the control culture. In order to improve the productivity of protease, repeated batch cultivation were successfully carried out four times by optimizing the top phas composition of freshly added media, which resulted in 13.8, 35.9, 27.8 and 34.7 units ml⁻¹ h⁻¹ of protease based on the amounts of replaced top phases, respectively.     

Analysis of nucleotide pools during protease production with Bacillus licheniformis

Summary During development of a fed-batch procedure for protease production with Bacillus licheniformis the nucleotide pools of the culture were assayed. Transitions between different growth phases or different nutrient limitations could easily be discerned by alterations in the nucleotide pool. As already described for B. subtilis, induction of sporulation was marked by a drop in the guanosine triphosphate (GTP) pool of the cells, which always preceded protease production. This was also valid for closed-loop controlled fed-batch processes in which the concentration of ammonium, which repressed protease biosynthesis, was kept constant at low levels. A marked decrease in the GTP content of the cells, e.g. after addition of mycophenolic acid during the exponential phase, increased protease formation during the stationary phase. During protease production the energy charge was lower (0.6–0.8) than during the exponential and early stationary phases (0.8–0.95) although very low energy charges (<0.5) did not support protease formation. Offprint requests to: G. Bierbaum     

Production of alkaline protease with Bacillus licheniformis in a controlled fed-batch process

Summary A production method for alkaline serine protease with Bacillus licheniformis in a synthetic medium was developed. Employing closed-loop control of oxygen, nitrogen and carbon source the pO₂ was held at 5%, the ammonium concentration kept below 1 mM and the glycerol concentration was maintained between 20 and 100 mM. Protease production was monitored by flow injection analysis. Thus, in a fed-batch procedure production could be increased 4.6-fold in comparison to an uncontrolled batch process. Offprint requests to: G. Bierbaum     

Isolation and identification of alkaline protease producer halotolerantBacillus licheniformis strain BA17

An alkaline protease producerBacillus licheniformis strain was isolated from Van Lake in Turkey. The strain is Gram positive, aerobic, motile, sporulating rod-shaped bacterium. Spores were ellipsoidal and positioned central in nonswollen sporangium. The cells were able to grow well at a pH range of 5.7–10. The optimal growth temperature was found to be 37 °C. Growth at a wide range of NaCl concentration (from 0 to 20%) showed that BA17 is halotolerant. Main fatty acid composition of BA17 was anteiso-C15:0 and iso-C15∶0. The strain was presumptively identified asB. licheniformis according to 16S rDNA gene sequence analysis. The most appropriate medium for the growth and protease production is composed of 0.5% yeast extract, 0.5% NaNO₃, 0.02% MgSO₄\7H₂O, 0.1% K₂HPO₄ and 0.5% maltose. The optimum temperature and pH of the alkaline protease of strain BA17 were found to be 60 °C and pH 11, respectively. The activity was completely lost in the presence of PMSF, suggesting that the preparation contains serine-alkaline protease(s).     

Production of protease with Bacillus licheniformis mutants insensitive to repression of exoenzyme biosynthesis

Two mutant strains of Bacillus licheniformis insensitive to catabolite repression were selected by classical mutagenesis in connection with the development of a fed-batch procedure for protease production. B. licheniformis 4a produced up to 20 U (Anson-Units) subtilisin Carlsberg/ml in fed-batch experiments in the presence of up to 1.5 m glycerol, but was inhibited by excess ammonium. Formation of spores, excretion of -amylase and the biosynthesis of citrate synthase and isocitrate dehydrogenase were likewise not repressed by glycerol. The strain was characterized by unusually low activity of the -oxoglutarate dehydrogenase complex and increased biosynthesis of polyglutamic acid in the presence and excretion of -oxoglutarate in the absence of ammonium, respectively. The results are discussed in view of a possible connection between the defect in the -oxoglutarate dehydrogenase complex and insensitivity to catabolite repression. The second strain B. licheniformis 114 was able to synthesize 11.5 U protease/ml independently of the glycerol and ammonium concentration in the medium. Correspondence to: G. Bierbaum     

Production of alkaline protease by entrapped Bacillus licheniformis cells in repeated batch process

In this study, Bacillus licheniformis cells were immobilized by entrapment in calcium alginate beads and were used for production of alkaline protease by repeated batch process. In order to increase the stability of the beads, the immobilization procedure was optimized by statistical full factorial method, by which three factors including alginate type, calcium chloride concentration, and agitation speed were studied. Optimization of the enzyme production medium, by the Taguchi method, was also studied. The obtained results showed that optimization of the cell immobilization procedure and medium constituents significantly enhanced the production of alkaline protease. In comparison with the free-cell culture in pre-optimized medium, about 7.3-fold higher productivity was resulted after optimization of the overall procedure. Repeated batch mode of operation, using optimized conditions, resulted in continuous production of the alkaline protease for 13 batches in 19 days.     

Evaluation of various parameters of calcium-alginate immobilization method for enhanced alkaline protease production by Bacillus licheniformis NCIM-2042 using statistical methods

Calcium-alginate immobilization method for the production of alkaline protease by Bacillus licheniformis NCIM-2042 was optimized statistically. Four variables, such as sodium-alginate concentration, calcium chloride concentration, inoculum size and agitation speed were optimized by 2(4) full factorial central composite design and subsequent analysis and model validation by a second-order regression equation. Eleven carbon, 11 organic nitrogen and seven inorganic nitrogen sources were screened by two-level Plackett-Burman design for maximum alkaline protease production by using optimized immobilized conditions. The levels of four variables, such as Na-alginate 2.78%; CaCl(2), 2.15%; inoculum size, 8.10% and agitation, 139 rpm were found to be optimum for maximal production of protease. Glucose, soybean meal and ammonium sulfate were resulted in maximum protease production at 644 U/ml, 720 U/ml, and 806 U/ml when screened for carbon, organic nitrogen and inorganic nitrogen sources, respectively, using optimized immobilization conditions. Repeated fed batch mode of operation, using optimized immobilized conditions, resulted in continuous operation for 12 cycles without disintegration of beads. Cross-sectional scanning electron microscope images have shown the growth pattern of B. licheniformis in Ca-alginate immobilized beads.     

Purification and characterisation of an alkaline protease used in tannery industry from Bacillus licheniformis

An extracellular alkaline protease produced by Bacillus licheniformis AP-1 was purified 76-fold, yielding a single 28 kDa band on SDS-PAGE. It was optimally active at pH 11 and at 60 degrees C (assayed over 10 min). The protease was completely inhibited by phenylmethylsulfonyl fluoride and diodopropyl fluorophosphate, with little increase upon Ca2+ and Mg2+ addition.     

Isolation and characterization of Tn917-generated bacitracin deficient mutants of Bacillus licheniformis

Abstract Two Tn917-generated bacitracin deficient mutants of Bacillus licheniformis were isolated. Southern blot analysis of chromosomal DNA extracted from both insertional mutants showed that Tn917 inserted in the vicinity of the gene coding for the enzyme BA2 of the bacitracin synthetase enzyme complex. Measurements of bacitracin synthetase activity in cell-free extracts and positive hybridization signals in the vicinity of the BA2 gene indicate that in both bacitracin deficient mutants Tn917 could be inserted in the BA1 gene or in segments involved in regulation. Thus, it could be possible that the genes for bacitracin synthetase are clustered in the B. licheniformis genome.