Facile syntheses of <Emphasis Type="SmallCaps">l</Emphasis>-β-haloalanine derivatives from <Emphasis Type="SmallCaps">l</Emphasis>-cysteine or <Emphasis Type="SmallCaps">l</Emphasis>-cystine

l-β-Haloalanines are physiologically active unnatural amino acids and they are useful intermediates for the synthesis of natural and unnatural amino acids, S-linked glycopeptides, and lanthionines. In general l-β-haloalanines were prepared predominantly from l-serine via functional group transformation. Here we reported an alternative approach for the preparation of l-β-haloalanines via halogenation of protected l-cysteine esters which was obtained from l-cysteine or l-cystine, respectively. The mercapto group of protected l-cysteine esters was efficiently transformed to halo groups by triphenylphosphine/N-halosuccinimides. It has been proved to be a versatile desulfurization strategy via this functional group transformation.     

The α-<Emphasis Type="SmallCaps">l</Emphasis>-fucosidase from <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis>

Glycoside hydrolases form hyperthermophilic archaea are interesting model systems for the study of catalysis at high temperatures and, at the moment, their detailed enzymological characterization is the only approach to define their role in vivo. Family 29 of glycoside hydrolases classification groups α-l-fucosidases involved in a variety of biological events in Bacteria and Eukarya. In Archaea the first α-l-fucosidase was identified in Sulfolobus solfataricus as interrupted gene expressed by programmed −1 frameshifting. In this review, we describe the identification of the catalytic residues of the archaeal enzyme, by means of the chemical rescue strategy. The intrinsic stability of the hyperthermophilic enzyme allowed the use of this method, which resulted of general applicability for β and α glycoside hydrolases. In addition, the presence in the active site of the archaeal enzyme of a triad of catalytic residues is a rather uncommon feature among the glycoside hydrolases and suggested that in family 29 slightly different catalytic machineries coexist.     

Efficient 40°C fermentation of <Emphasis Type="SmallCaps">l</Emphasis>-lysine by a new<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis> mutant developed by genome breeding

We have recently developed a new L-lysine-producing mutant of Corynebacterium glutamicum by "genome breeding" consisting of characterization and reconstitution of a mutation set essential for high-level production. The strain AHP-3 was examined for L-lysine fermentation on glucose at temperatures above 35 degrees C, at which no examples of efficient L-lysine production have been reported for this organism. We found that the strain had inherited the thermotolerance that the original coryneform bacteria was endowed with, and thereby grew and produced L-lysine efficiently up to 41 degrees C. A final titer of 85 g/l after only 28 h was achieved at temperatures around 40 degrees C, indicating the superior performance of the strain developed by genome breeding. When compared with the traditional 30 degrees C fermentation, the 40 degrees C fermentation allowed an increase in yield of about 20% with a concomitant decrease in final growth level, suggesting a significant transition of carbon flux distribution in glucose metabolism. DNA array analysis of metabolic changes between the 30 degrees C and 40 degrees C fermentations identified several differentially expressed genes in central carbon metabolism although we could not find stringent control-like global induction of amino-acid-biosynthetic genes in the 40 degrees C fermentation. Among these changes, two candidates were picked out as the potential causes of the increased production at 40 degrees C; decreased expression of the citrate synthase gene gltA and increased expression of malE, the product of which involves regeneration of pyruvate and NADPH.     

Direct production of cadaverine from soluble starch using <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> coexpressing α-amylase and lysine decarboxylase

Here, we demonstrated the one-step production of cadaverine from starch using a Corynebacterium glutamicum strain coexpressing Streptococcus bovis 148 α-amylase (AmyA) and Escherichia coli K-12 lysine decarboxylase (CadA). We constructed the E. coli–C. glutamicum shuttle vector, which produces CadA under the control of the high constitutive expression (HCE) promoter, and transformed this vector into C. glutamicum CSS secreting AmyA. The engineered C. glutamicum expressed both CadA and AmyA, which retained their activity. We performed cadaverine fermentation using 50 g/l soluble starch as the sole carbon source without pyridoxal-5’-phosphate, which is the coenzyme for CadA. C. glutamicum coexpressing AmyA and CadA successfully produced cadaverine from soluble starch and the yield of cadaverine was 23.4 mM after 21 h. CadA expression levels under the control of the HCE promoter were assumed to be sufficient to convert l-lysine to cadaverine, as there was no accumulation of l-lysine in the culture medium during fermentation. Thus, we demonstrated that C. glutamicum has great potential to produce cadaverine from biomass resources.     

Production,partial purification and characterization of α-<Emphasis Type="SmallCaps">l</Emphasis>-rhamnosidase from <Emphasis Type="Italic">Penicillium ulaiense</Emphasis>

Penicillium ulaiense is a post-harvest pathogenic fungus that attacks citrus fruits. The objective of this work was to study this microorganism as an α-l-rhamnosidase producer and to characterize it from P. ulaiense. The enzyme under study is used for different applications in food and beverage industries. α-l-Rhamnosidase was produced in a stirred-batch reactor using rhamnose as the main carbon source. The kinetic parameters for the growth of the fungi and for the enzyme production were calculated from the experimental values. A method for partial purification, including (NH₄)₂SO₄ precipitation, incubation at pH 12 and DEAE-sepharose chromatography yielded an enzyme with very low β-glucosidase activity. The pH and temperature optima were 5.0 and 60°C, respectively. The Michaelis–Menten constants for the hydrolysis of p-nitrophenyl-α-l-rhamnoside were V _max = 26 ± 4 IU ml⁻¹ and K _m  = 11 ± 2 mM. The enzyme showed good thermostability up to 60°C and good operational stability in white wine. Co²⁺ affected positively the activity; EDTA, Mn²⁺, Mg²⁺, dithiotreitol and Cu²⁺ reduced the activity by different amounts, and Hg²⁺ completely inhibited the enzyme. The enzyme showed more activity on p-nitrophenyl-α-l-rhamnoside than on naringin. According to these results, this enzyme has potential for use in the food and pharmacy industries since P. ulaiense does not produce mycotoxins.     

Characterization of a family 54 α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase from <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

A glycosyl hydrolase family 54 (GH54) α-l-arabinofuranosidase gene (abfA) of Aureobasidium pullulans was amplified by polymerase chain reaction from genomic DNA and a 498-amino-acid open reading frame deduced from the DNA sequence. Modeling of the highly conserved A. pullulans AbfA protein sequence on the crystal structure of Aspergillus kawachii AkabfB showed that the catalytic amino acid arrangement and overall structure were highly similar including the N-terminal catalytic and C-terminal arabinose binding domains. The abfA gene was expressed in Saccharomyces cerevisiae, and the heterologous enzyme was purified. The protein was monomeric, migrating at 49 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and eluting at 36 kDa upon gel filtration. AbfA showed maximal activity at 55°C and between pH 3.5 and pH 4. The enzyme had a K _m value for p-nitrophenyl-α-l-arabinofuranoside of 3.7 mM and a V _max of 34.8 μmol min⁻¹ mg protein⁻¹. Arabinose acted as a noncompetitive inhibitor with a K _i of 38.4 mM. The enzyme released arabinose from maize fiber, oat spelt arabinoxylan, and wheat arabinoxylan, but not from larch wood arabinogalactan or α-1,5-debranched arabinan. AbfA displayed low activity against α-1,5-l-arabino-oligosaccharides. The enzyme acted synergistically with endo-β-1,4-xylanase in the breakdown of wheat arabinoxylan. Binding of AbfA to xylan from several sources confirmed the presence of a functional carbohydrate-binding module. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Glutamate production from β-glucan using endoglucanase-secreting <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

We demonstrate glutamate production from β-glucan using endoglucanase (EG)-expressing Corynebacterium glutamicum. The signal sequence torA derived from Escherichia coli K12, which belongs to the Tat pathway, was suitable for secreting EG of Clostridium thermocellum using C. glutamicum as a host. Using the torA signal sequence, endoglucanase from Clostridium cellulovorans 743B was successfully expressed, and the secreted EG produced 123 mg of reducing sugar from 5 g of β-glucan at 30 °C for 72 h, which is the optimal condition for C. glutamicum growth. Subsequently, glutamate fermentation from β-glucan was carried out with the addition of Aspergillus aculeatus β-glucosidase produced by recombinant Aspergillus oryzae. Using EG-secreting C. glutamicum, 178 mg/l of glutamate was produced from 15 g of β-glucan. This is the first report of glutamate fermentation from β-glucan using endoglucanase-secreting C. glutamicum.     

A family GH51 α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase from <Emphasis Type="BoldItalic">Pleurotus ostreatus</Emphasis>: identification,recombinant expression and characterization

An α-l-arabinofuranosidase produced by Pleurotus ostreatus (PoAbf) during solid state fermentation on tomato pomace was identified and the corresponding gene and cDNA were cloned and sequenced. Molecular analysis showed that the poabf gene carries 26 exons interrupted by 25 introns and has an open reading frame encoding a protein of 646 amino acid residues, including a signal peptide of 20 amino acid residues. The amino acid sequence similar to the other α-l-arabinofuranosidases indicated that the enzyme encoded by poabf can be classified as a family 51 glycoside hydrolase. Heterologous recombinant expression of PoAbf was carried out in the yeasts Pichia pastoris and Kluyveromyces lactis achieving the highest production level of the secreted enzyme (180 mg L⁻¹) in the former host. rPoAbf produced in P. pastoris was purified and characterized. It is a glycosylated monomer with a molecular weight of 81,500 Da in denaturing conditions. Mass spectral analyses led to the localization of a single O-glycosylation site at the level of Ser160. The enzyme is highly specific for α-l-arabinofuranosyl linkages and when assayed with p-nitrophenyl α-l-arabinofuranoside it follows Michaelis–Menten kinetics with a K _M of 0.64 mM and a k _cat of 3,010 min⁻¹. The optimum pH is 5 and the optimal temperature 40°C. It is worth noting that the enzyme shows a very high stability in a broad range of pH. The more durable activity showed by rPoAbf in comparison to the other α-l-arabinofuranosidases enhances its potential for biotechnological applications and increases interest in elucidating the molecular bases of its peculiar properties.     

δ-(<Emphasis Type="SmallCaps">l</Emphasis>-α-aminoadipyl)-<Emphasis Type="SmallCaps">l</Emphasis>-cysteinyl-<Emphasis Type="SmallCaps">d</Emphasis>-valine synthetase (ACVS): discovery and perspectives

The δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV) tripeptide is the first dedicated intermediate in the biosynthetic pathway leading to the penicillin and cephalosporin classes of β-lactam natural products in bacteria and fungi. It is synthesized nonribosomally by the ACV synthetase (ACVS) enzyme, which has been purified and partially characterized from many sources. Due to its large size and instability, many details regarding the reaction mechanism of ACVS are still not fully understood. In this review we discuss the chronology and associated methodology that led to the discovery of ACVS, some of the main findings regarding its activities, and some recent/current studies being conducted on the enzyme. In addition, we conclude with perspectives on what can be done to increase our understating of this very important protein in the future.     

Surface-<Emphasis Type="Italic">engineered Saccharomyces cerevisiae</Emphasis> displaying α-acetolactate decarboxylase from <Emphasis Type="Italic">Acetobacter aceti</Emphasis> ssp <Emphasis Type="Italic">xylinum</Emphasis>

Objectives

To convert α-acetolactate into acetoin by an α-acetolactate decarboxylase (ALDC) to prevent its conversion into diacetyl that gives beer an unfavourable buttery flavour.

Results

We constructed a whole Saccharomyces cerevisiae cell catalyst with a truncated active ALDC from Acetobacter aceti ssp xylinum attached to the cell wall using the C-terminal anchoring domain of α-agglutinin. ALDC variants in which 43 and 69 N-terminal residues were absent performed equally well and had significantly decreased amounts of diacetyl during fermentation. With these cells, the highest concentrations of diacetyl observed during fermentation were 30 % less than those in wort fermented with control yeasts displaying only the anchoring domain and, unlike the control, virtually no diacetyl was present in wort after 7 days of fermentation.

Conclusions

Since modification of yeasts with ALDC variants did not affect their fermentation performance, the display of α-acetolactate decarboxylase activity is an effective approach to decrease the formation of diacetyl during beer fermentation.

     

Efficient bioconversion of <Emphasis Type="SmallCaps">l</Emphasis>-glutamate to γ-aminobutyric acid by <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> resting cells

This work investigated the efficient bioconversion process of l-glutamate to GABA by Lactobacillus brevis TCCC 13007 resting cells. The optimal bioconversion system was composed of 50 g/L 48 h cultivated wet resting cells, 0.1 mM pyridoxal phosphate in glutamate-containing 0.6 M citrate buffer (pH 4.5) and performed at 45 °C and 180 rpm. By 10 h bioconversion at the ratio of 80 g/L l-glutamic acid to 240 g/L monosodium glutamate, the final titer of GABA reached 201.18 g/L at the molar bioconversion ratio of 99.4 %. This process presents a potential for industrial and commercial applications and also offers a promising feasibility of continuous GABA production coupled with fermentation. Besides, the built kinetics model revealed that the optimum operating conditions were 45 °C and pH 4.5, and the bioconversion kinetics at low ranges of substrate concentration (0 < S < 80 g/L) was assumed to follow the classical Michaelis–Menten equation.     

Efficient production of α-ketoglutarate in the <Emphasis Type="Italic">gdh</Emphasis> deleted <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> by novel double-phase pH and biotin control strategy

Production of l-glutamate using a biotin-deficient strain of Corynebacterium glutamicum has a long history. The process is achieved by controlling biotin at suboptimal dose in the initial fermentation medium, meanwhile feeding NH₄OH to adjust pH so that α-ketoglutarate (α-KG) can be converted to l-glutamate. In this study, we deleted glutamate dehydrogenase (gdh1 and gdh2) of C. glutamicum GKG-047, an l-glutamate overproducing strain, to produce α-KG that is the direct precursor of l-glutamate. Based on the method of l-glutamate fermentation, we developed a novel double-phase pH and biotin control strategy for α-KG production. Specifically, NH₄OH was added to adjust the pH at the bacterial growth stage and NaOH was used when the cells began to produce acid; besides adding an appropriate amount of biotin in the initial medium, certain amount of additional biotin was supplemented at the middle stage of fermentation to maintain a high cell viability and promote the carbon fixation to the flux of α-KG production. Under this control strategy, 45.6 g/L α-KG accumulated after 30-h fermentation in a 7.5-L fermentor and the productivity and yield achieved were 1.52 g/L/h and 0.42 g/g, respectively.     

Functional and biophysical characterization of a hyperthermostable GH51 α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase from <Emphasis Type="Italic">Thermotoga petrophila</Emphasis>

A hyperthermostable glycoside hydrolase family 51 (GH51) α-l-arabinofuranosidase from Thermotoga petrophila RKU-1 (TpAraF) was cloned, overexpressed, purified and characterized. The recombinant enzyme had optimum activity at pH 6.0 and 70°C with linear α-1,5-linked arabinoheptaose as substrate. The substrate cleavage pattern monitored by capillary zone electrophoresis showed that TpAraF is a classical exo-acting enzyme producing arabinose as its end-product. Far-UV circular dichroism analysis displayed a typical spectrum of α/β barrel proteins analogously observed for other GH51 α-l-arabinofuranosidases. Moreover, TpAraF was crystallized in two crystalline forms, which can be used to determine its crystallographic structure.     

Purification and characterization of a highly thermostable α-<Emphasis Type="SmallCaps">l</Emphasis>-Arabinofuranosidase from <Emphasis Type="Italic">Geobacillus caldoxylolyticus</Emphasis> TK4

The gene encoding an α-l-arabinofuranosidase from Geobacillus caldoxylolyticus TK4, AbfATK4, was isolated, cloned, and sequenced. The deduced protein had a molecular mass of about 58 kDa, and analysis of its amino acid sequence revealed significant homology and conservation of different catalytic residues with α-l-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. A histidine tag was introduced at the N-terminal end of AbfATK4, and the recombinant protein was expressed in Escherichia coli BL21, under control of isopropyl-β-D-thiogalactopyranoside-inducible T7 promoter. The enzyme was purified by nickel affinity chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 236 kDa, suggesting a homotetrameric structure. AbfATK4 was active at a broad pH range (pH 5.0–10.0) and at a broad temperature range (40–85°C), and it had an optimum pH of 6.0 and an optimum temperature of 75–80°C. The enzyme was more thermostable than previously described arabinofuranosidases and did not lose any activity after 48 h incubation at 70°C. The protein exhibited a high level of activity with p-nitrophenyl-α-l-arabinofuranoside, with apparent K _m and V _max values of 0.17 mM and 588.2 U/mg, respectively. AbfATK4 also exhibited a low level of activity with p-nitrophenyl-β-d-xylopyranoside, with apparent K _m and V _max values of 1.57 mM and 151.5 U/mg, respectively. AbfATK4 released l-arabinose only from arabinan and arabinooligosaccharides. No endoarabinanase activity was detected. These findings suggest that AbfATK4 is an exo-acting enzyme.     

Production of ε-poly-<Emphasis Type="SmallCaps">l</Emphasis>-lysine by <Emphasis Type="Italic">Streptomyces</Emphasis> sp. using resin-based,in situ product removal

Resin-based, in situ product removal (ISPR) was used to increase production of ε-poly-l-lysine (PL) by Streptomyces sp. GIM8. D152 resin was selected over Amberlite IRC-50, Amberlite IRC-76 and Amberlite IR-120 to develop ISPR using adsorption capacity and desorption ratio as bases. The yield of PL in response to external PL was unaffected in shake-flask culture; however, the production of PL increased to 2.9 from 0.8 g l⁻¹ shake-flasks using ISPR. In a 5 l fermentor, 23.4 g PL l⁻¹ was achieved compared to 3.76 g PL l⁻¹, in the controls by attaching two bags of D152 resin to the probes and baffles of the fermentor.     

Endogeneous β-<Emphasis Type="SmallCaps">d</Emphasis>-xylosidase and α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase activity in flax seed mucilage

Flax seed mucilage (FM) contains a mixture of highly doubly substituted arabinoxylan as well as rhamnogalacturonan I with unusual side group substitutions. Treatment of FM with a GH11 Bacillus subtilis XynA endo 1,4-β-xylanase (BsX) gave limited formation of reducing ends but when BsX and FM were incubated together on different wheat arabinoxylan substrates and birchwood xylan, significant amounts of xylose were released. Moreover, arabinose was released from both water-extractable and water-unextractable wheat arabinoxylan. Since no xylose or arabinose was released by BsX addition alone on these substrates, nor without FM or BsX addition, the results indicate the presence of endogenous β-d-xylosidase and α-l-arabinofuranosidase activities in FM. FM also exhibited activity on both p-nitrophenyl α-l-arabinofuranoside (pNPA) and p-nitrophenyl β-d-xylopyranoside (pNPX). Based on K _M values, the FM enzyme activities had a higher affinity for pNPX (K _M 2 mM) than for pNPA (K _M 20 mM).     

Insoluble but enzymatically active <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

The gene encoding thermostable α-amylase from Bacillus licheniformis consisting of 483 amino acid residues (mature protein) was cloned and expressed in Escherichia coli under the control of T7 promoter. The analysis of the soluble and insoluble fractions after lyzing the host cells revealed that recombinant α-amylase was produced in insoluble aggregates. Despite being produced in the insoluble aggregates the recombinant enzyme was highly active with a specific activity of 408 U/mg.     

Efficient production of ε-poly-<Emphasis Type="SmallCaps">l</Emphasis>-lysine from agro-industrial by-products by <Emphasis Type="Italic">Streptomyces</Emphasis> sp. M-Z18

ε-Poly-l-lysine (ε-PL)—a natural food preservative with wide antimicrobial activity and high food safety—is increasingly attracting widespread attention. However, the high cost of raw materials severely impairs its economy and utilization. In this study, agro-industrial by-products, i.e., fish meal coupled with corn steep liquor, were employed as alternative organic nitrogen sources for industrial ε-PL production by Streptomyces sp. M-Z18. An economical medium was then developed by using an artificial neural network. Amino acids analyses showed that the improved medium was rich in glutamate, arginine, lysine and aspartate, which not only elevated the acid tolerance capability of the mycelia but also enhanced cell growth and ε-PL production. Subsequently, a cost-effective and efficient strategy for ε-PL production was established on fermenter scale, based on the improved medium and two-stage pH control. Notably, ε-PL production and productivity reached 35.24 g/L and 4.85 g/L day in fed-batch fermentation. Further profit assessment at the 10 m³ scale indicated that application of this strategy resulted in a net profit increase of 9,057 USD. Therefore, the proposed strategy has great potential for industrial production of ε-PL.     

Co-expression of <Emphasis Type="SmallCaps">l</Emphasis>-glutamate oxidase and catalase in <Emphasis Type="Italic">Escherichia coli</Emphasis> to produce α-ketoglutaric acid by whole-cell biocatalyst

Objectives

To improve the production of α-ketoglutaric acid (α-KG) from l-glutamate by whole-cell biocatalysis.

Results

A novel and highly active l-glutamate oxidase, SmlGOX, from Streptomyces mobaraensis was overexpressed and purified. The recombinant SmlGOX was approx. 64 kDa by SDS-PAGE. SmlGOX had a maximal activity of 125 ± 2.7 U mg^?1 at pH 6.0, 35 ^oC. The apparent K_m and V_max values of SmlGOX were 9.3 ± 0.5 mM and 159 ± 3 U mg^?1, respectively. Subsequently, a co-expression plasmid containing the SmlGOX and KatE genes was constructed to remove H₂O₂, and the protein levels of SmlGOX were improved by codon optimization. Finally, by optimizing the whole-cell transformation conditions, the production of α-KG reached 77.4 g l^?1 with a conversion rate from l-glutamate of 98.5% after 12 h.

Conclusions

An efficient method for the production of α-KG was established in the recombinant Escherichia coli, and it has a potential prospect in industrial application.