A n ovel glycoside hydrolase (GH) family 46 chitosanase (SaCsn46A) from
Streptomyces avermitilis
was cloned and functionally expressed in
Escherichia coli
Rosetta (DE3) strains. SaCsn46A consists of ...271 amino acids, which includes a 34-amino acid signal peptide. The protein sequence of SaCsn46A shows maximum identity (83.5%) to chitosanase from
Streptomyces sp
. SirexAA-E. Then, the mature enzyme was purified to homogeneity through Ni-chelating affinity chromatography with a recovery yield of 78% and the molecular mass of purified enzyme was estimated to be 29 kDa by SDS-PAGE. The recombinant enzyme possessed a temperature optimum of 45 °C and a pH optimum of 6.2, and it was stable at pH ranging from 4.0 to 9.0 and below 30 °C. The
K
m
and
V
max
values of this enzyme were 1.32 mg/mL, 526.32 U/mg/min, respectively (chitosan as substrate). The enzyme activity can be enhanced by Mg
2+
and especially Mn
2+
, which could enhance the activity about 3.62-fold at a 3-mM concentration. The enzyme can hydrolyze a variety of polysaccharides which are linked by β-1,4-glycosidic bonds such as chitin, xylan, and cellulose, but it could not hydrolyze polysaccharides linked by α-1,4-glycosidic bonds. The results of thin-layer chromatography and HPLC showed that the enzyme exhibited an endo-type cleavage pattern and could hydrolyze chitosan to glucosamine (GlcN) and (GlcN)
2
. This study demonstrated that SaCsn46A is a promising enzyme to produce glucosamine and chitooligosaccharides (COS) from chitosan.
l-arginine, a semi essential amino acid, is an important amino acid in food flavoring and pharmaceutical industries. Its production by microbial fermentation is gaining more and more attention. In ...previous work, we obtained a new l-arginine producing Corynebacterium crenatum (subspecies of Corynebacterium glutamicum) through mutation breeding. In this work, we enhanced l-arginine production through improvement of the intracellular environment. First, two NAD(P)H-dependent H2O2-forming flavin reductases Frd181 (encoded by frd1 gene) and Frd188 (encoded by frd2) in C. glutamicum were identified for the first time. Next, the roles of Frd181 and Frd188 in C. glutamicum were studied by overexpression and deletion of the encoding genes, and the results showed that the inactivation of Frd181 and Frd188 was beneficial for cell growth and l-arginine production, owing to the decreased H2O2 synthesis and intracellular reactive oxygen species (ROS) level, and increased intracellular NADH and ATP levels. Then, the ATP level was further increased by deletion of noxA (encoding NADH oxidase) and amn (encoding AMP nucleosidase), and overexpression of pgk (encoding 3-phosphoglycerate kinase) and pyk (encoding pyruvate kinase), and the l-arginine production and yield from glucose were significantly increased. In fed-batch fermentation, the l-arginine production and yield from glucose of the final strain reached 57.3g/L and 0.326g/g, respectively, which were 49.2% and 34.2% higher than those of the parent strain, respectively. ROS and ATP are important elements of the intracellular environment, and l-arginine biosynthesis requires a large amount of ATP. For the first time, we enhanced l-arginine production and yield from glucose through reducing the H2O2 synthesis and increasing the ATP supply.
•Two new H2O2-forming flavin reductases from C. glutamicum are identified.•Inactivation of the flavin reductases facilitates L-arginine production.•Increasing the ATP supply facilitates L-arginine production.
Pseudomonas
are ubiquitous bacteria that can live under a wide range of environmental conditions; some species are successfully used in the fermentation industry.
Pseudomonas plecoglossicida
JUIM01 ...strain is an important industrial 2-ketogluconate (2KGA) producer. In
Pseudomonas
,
kguT
gene encodes a putative 2KGA transporter KguT, and there is no detailed research on the role of KguT. In this work, the role of KguT in
P. plecoglossicida
JUIM01 was investigated. The results showed that the KguT of
P. plecoglossicida
JUIM01 plays an essential role in the transport of 2KGA into cells; inactivation of KguT could prevent the assimilation of 2KGA as carbon source by the cells and had no negative effect on 2KGA biosynthesis. Under fermentation conditions, the
kguT
deletion strain had the same capability of 2KGA production with
P. plecoglossicida
JUIM01 and avoided the assimilation of the product 2KGA by cells after the depletion of glucose. These results can provide references for
Pseudomonas
study and fermentation production of 2KGA.
Trehalose (α-D-glucopyranosyl-1,1-α-D-glucopyranoside) is a stable and nonreducing disaccharide; can be used as sweetener, stabilizer, and humectant; and has many applications in the food, ...pharmaceutical, and cosmetic industries. Trehalose production from maltose catalyzed by trehalose synthase (TreS) is simple and economically feasible for industrial-scale application. Reducing the cost and enhancing the efficiency of TreS synthesis and the conversion of maltose to trehalose is critical for trehalose production. In this study, the homologous TreS was constitutively overexpressed in Corynebacterium glutamicum ATCC13032 by removing the repressor gene lacIq fragment in the plasmid, and TreS expression could be exempt from the inducer addition and induction process. For cell permeabilization, Triton X-100 was used as a permeabilization agent, and the treatment time was 3 h. In the conversion system, the permeabilized cells of recombinant C. glutamicum were used as biocatalysts, 300 g/L maltose was used as a substrate, and 173.7 g/L trehalose was produced within 12 h under 30 °C and pH 7.0 conditions. In addition, the whole-cell biocatalysts showed promising reusability. This study provides a safe, convenient, practical, and low-cost pathway for the production of trehalose.
2-Ketogluconate (2KGA) is an organic acid that is important for pharmaceutical, cosmetic, and environmental applications.
JUIM01 strain is an important industrial 2KGA producer in China. In this ...paper, we found that
JUIM01 could convert glucose to 2KGA extracellularly, and the formed 2KGA was subsequently consumed after glucose was exhausted during the fermentation process. Experiments of glucose and 2KGA supplementation during fermentation process revealed that, only when glucose was exhausted, the strain started to consume the product 2KGA. Then, the mechanism of this phenomenon was investigated at transcription and protein levels, and the results indicated that
JUIM01 possesses carbon catabolite repression of 2KGA metabolism by glucose. Next, increasing the supply of glucose could attenuate 2KGA consumption and enhance the 2KGA yield from glucose. Finally, fed-batch fermentation of
JUIM01 resulted in 205.67 g/L of 2KGA with a productivity of 6.86 g/L/h and yield of 0.953 g/g glucose. These results can provide references for the industrial fermentation production of 2KGA and other fermentation products.
L-citrulline plays important roles in many physiological processes, and its application range is expanding rapidly. Corynebacterium glutamicum strains have the potential to be efficient L-citrulline ...producers. In this study, we performed optimization of L-citrulline biosynthesis operon in C. glutamicum ATCC13032 for L-citrulline production. Chromosomal integration of the integral argBEc gene from Escherichia coli (encoding natively insensitive N-acetylglutamate kinase), the deletion of the argR gene (encoding repressor ArgR), and the deletion of the argG gene (encoding argininosuccinate synthase) were achieved simultaneously by one-step genome modification and by obtaining the L-citrulline-producing strain. Then, plasmid-based overexpression of the optimized L-citrulline operon was carried out and the L-citrulline production was further improved. In fed-batch fermentation, the L-citrulline production and yield from glucose of the final strain reached 26.7 g/L and 0.18 g/g, respectively. These results indicate that optimization of L-citrulline operon in C. glutamicum is effective to construct the L-citrulline over-producing strain.
A 30-kDa novel tyrosinase was purified to homogeneity. The Km for L-Dopa and L-tyrosine were determined as 0.42 and 0.25 mM. The 1231 bp (base pair) melC gene and its 167 bp promoter Pskmel were ...obtained by thermal asymmetric interlaced polymerase chain reaction based on the amino acids fragment obtained from MS results of the purified enzyme. The protein sequence of tyrosinase shows maximum identity (84%) to tyrosinase from Streptomyces galbus. The melC was introduced into S. kathirae. The melanin production and the transcriptional level of melC in recombinant S. kathirae pIJPskmelmelC were about 2.1-fold and 2-fold higher than the wild-type strain, respectively. The melanin concentration was maximized at 28.8 g L−1.
Melanin production in Streptomyces kathirae was enhanced by introducing homogenous melC controlled by the Pskmel promoter.
•PoPMuSiC algorithm was applied to predict more thermostable tyrosinase.•Site-directed mutagenesis was applied to obtain more thermostable tyrosinase.•Mutants showed 3-fold and 10°C increase in ...half-life and optimal temperature.•The additional hydrogen bonds may be of value to improve the thermostability.•The improved thermostability may be due to the newly formed favorable interaction.
This study aimed to improve the thermostability of a newly cloned tyrosinase from Streptomyces kathirae SC-1. The POPMuSiC algorithm was applied to predict the folding free energy change (ΔDG) of amino acid substitution. Site-directed mutagenesis was used to construct mutants (Q7K, G234P, and Q7K/G234P), and the mutant, and wild-type enzymes were expressed in Escherichia coli (DE3). Compared to the wild-type tyrosinase, all three mutant enzymes showed improved thermal properties. The mutant with combined substitution (Q7K/G234P) showed the most pronounced shifts in temperature optima, about 10°C upward, and the half-life for thermal inactivation at 60°C, and melting temperatures were increased by 3 times and approximately 10°C, respectively. Finally, the mechanisms responsible for the increased thermostability were analyzed through comparative analysis of structure models. The structure-based rational design strategies in this study may also provide further insight into the thermostability of other industrial enzymes and suggest further potential industrial applications.
In this study, a novel strain of Pichia jadinii, HBY61, capable of the biocatalysis of 4-hydroxy-2-butanone (4H2B) to (R)-1,3-BD was isolated. HBY61 produced (R)-1,3-BD with high activity and ...absolute stereochemical selectivity (100 % e.e). Glucose and beef extract were found to be the key factors governing the fermentation, and their optimal concentrations were determined to be 84.2 and 43.7 g/L, respectively. The optimal bioconversion conditions of 4H2B catalyzed by HBY61 were pH 7.4, 30 °C, and 250 rpm with 6 % (v/v) glucose as the co-substrate. Accordingly, when 45 g/L of 4H2B was divided into three equal parts and added successively into the system at set time intervals, the maximum (R)-1,3-BD concentration reached 38.3 g/L with high yield (85.1 %) and strict 100 % enantioselectivity. Compared with previously reported yields for the biocatalytic production of (R)-1,3-BD, the use of strain HBY61 provided a high yield with excellent stereoselectivity.
BsCsn46A, a GH46 family chitosanase from Bacillus subtilis, has great potential for industrial chitooligosaccharide production due to its high activity and stability. In this study, a special amino ...acid Pro121 was identified not fit in the helix structure, which was located in the opposite side of the active center in BsCsn46A, by the PoPMuSiC algorithm. Then, saturation mutagenesis was performed to explore the role of the site amino acid 121. Compared with the wild type, the specific activity of P121N, P121C, and P121V was increased by 1.69-, 1.97-, and 2.15-fold, respectively. In particular, the specific activity of P121N was increased without loss of thermostability, indicating that replacing the structural stiffness of proline in the helical structure could significantly improve the chitosanase activity. The K m values of P121N, P121C, and P121V decreased significantly, indicating that the affinity between the enzyme–substrate complex was enhanced. Through molecular docking, it was found that the increase of hydrogen bonds and van der Waals force between the enzyme–substrate complex and the removal of unfavorable bonds might be the main reason for the change of enzyme properties. In addition, the optimal temperature of the three mutants changed from 60 to 55 °C. These results indicate that the site 121 plays a critical role in the catalytic activity and enzymatic properties of chitosanase. To our knowledge, the results provide novel data on chitosanase activity and identify an excellent candidate of industrial chitosanase.
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