Pullulanase is a commonly used starch-debranching enzyme with broad application in food, chemical and pharmaceutical industries. Since the starch-debranching process requires a high temperature, a ...thermostable pullulanase is desirable. In this study, based on the strategy of surficial residue replacement and disulfide bond introduction, a mutant pullulanase (Pul
AC
) derived from the pullulanase (Pul
A
) of
Anoxybacillus
sp. WB42 with higher thermostability and activity was isolated. The surficial residue Lys419 from the wild-type Pul
A
was replaced by arginine, and two disulfide bonds were introduced between Thr245 and Ala326 and Trp651 and Val707. The specific activity and
k
cat
/
K
m
value of the Pul
AC
reached 98.20 U/mg and 12.22 mL/mg/s respectively, 1.5 times greater than that of wild-type Pul
A
. The optimum temperature of the mutant Pul
AC
was 65 °C. The Pul
AC
retained more than 85% activity after incubation at 65 °C for 30 min, which is much higher than the activity maintained by wild-type Pul
A
. Due to its high optimum temperature, thermostability, and specific activity, the mutant Pul
AC
reported here could play an important role in improving hydrolytic efficiency in the starch-debranching process.
Nattokinase (NK), which is a member of the subtilisin family, is a potent fibrinolytic enzyme that might be useful for thrombosis therapy. Extensive work has been done to improve its production for ...the food industry. The aim of our study was to enhance NK production by tandem promoters in Bacillus subtilis WB800.
Six recombinant strains harboring different plasmids with a single promoter (P
, P
, P
, P
, P
or P
) were constructed, and the analysis of the fibrinolytic activity showed that P
and P
exhibited a higher expression activity than that of the others. The NK yield that was mediated by P
and P
reached 140.5 ± 3.9 FU/ml and 110.8 ± 3.6 FU/ml, respectively. These promoters were arranged in tandem to enhance the expression level of NK, and our results indicated that the arrangement of promoters in tandem has intrinsic effects on the NK expression level. As the number of repetitive P
or P
increased, the expression level of NK was enhanced up to the triple-promoter, but did not increase unconditionally. In addition, the repetitive core region of P
or P
could effectively enhance NK production. Eight triple-promoters with P
and P
in different orders were constructed, and the highest yield of NK finally reached 264.2 ± 7.0 FU/ml, which was mediated by the promoter P
-P
-P
. The scale-up production of NK that was promoted by P
-P
-P
was also carried out in a 5-L fermenter, and the NK activity reached 816.7 ± 30.0 FU/mL.
Our studies demonstrated that NK was efficiently overproduced by tandem promoters in Bacillus subtilis. The highest fibrinolytic activity was promoted by P
-P
-P
, which was much higher than that had been reported in previous studies. These multiple tandem promoters were used successfully to control NK expression and might be useful for improving the expression level of the other genes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The HSR station area not only assumes the function of the city’s external communication, but also is an important public space, which plays an important role in the urban development. Based on the ...relevant theories, this paper analyzes and evaluates the status quo of the site functions in the Jinanxi Station area by combining the on-site survey and POI data. It is concluded that the site functions in the Jinanxi Station area are formed relatively slowly, with strong characteristics of circle structure and single residential function, showing unbalanced characteristics, but it will have huge space for future development. So in order to achieve an integrated development between station and city, it should avoid a single residential function, and achieve diversified functional development, require planning guidance and government guidance as well as further support and orderly promotion of policy funds.
Pullulanase is a commonly used debranching enzyme in the starch processing industry. Because the starch liquefaction process requires high temperature, a thermostable pullulanase is desired. Here, a ...novel hyperthermostable type II pullulanase gene (pul PY) was cloned from Pyrococcus yayanosii CH1, isolated from a deep-sea hydrothermal site. PulPY was optimally active at pH 6.6 and 95 °C, retaining more than 50% activity after incubation at 95 °C for 10 h. The thermostability was significantly higher than those of most pullulanases reported previously. To further improve its activity and thermostability, the N-terminal and C-terminal domains of PulPY were truncated. The optimum temperature of the combined truncation mutant Δ28N + Δ791C increased to 100 °C with a specific activity of 32.18 U/mg, which was six times higher than that of wild-type PulPY. PulPY and the truncation mutant enzyme could realize the combined use of pullulanase with α-amylase during the starch liquefaction process to improve hydrolysis efficiency.
•The His6-tagged pullulanase was immobilized on Ni (II)-modified MNPs.•The catalytic properties of the immobilized pullulanases was mainly dependent on the orientation of pullulanase.•The reusability ...of immobilized pullulanases was independent from other catalytic properties.•Fe3O4@PEI-BDDE-PEA400-IDA-Ni+2/H6-PUL retained 60% of initial activity after 18 consecutive cycles with a total reaction time of 9h.
Chelating of pullulanases onto nickel (II)-modified magnetic nanoparticles results in one-step purification and immobilization of pullulanase, and facilitates the commercial application of pullulanase in industrial scale. To improve the catalytic behavior, especially the operational stability, of the nanocatalyst in consecutive batch reactions, we prepared various iminodiacetic acid-modified magnetic nanoparticles differed in surface polarity and spacer length, on which the His6-tagged pullulanases were chelated via nickel ions, and then studied the correlation between the MNPs surface property and the corresponding catalyst behavior. When pullulanases were chelated onto the surface-modified MNPs, the thermostability of all pullulanase derivatives were lower than that of free counterpart, being not relevant to the protein orientation guided by the locality of the His6-tag, but related to the MNPs basal surface polarity and the grafted spacer length. After chelating of pullulanases onto MNPs, there were changes observed in the pH-activity profile and the apparent Michaelis constant toward pullulan. The changing tendencies were mainly dependent on the His6-tagged pullulanase orientation, and the changing extents were tuned by the spacer length. The reusability of pullulanase immobilized by N-terminal His6-tag was higher than that of pullulanase immobilized by C-terminal His6-tag. Moreover, the reusability of the immobilized pullulanase tested increased till grafting polyether amine-400 as spacer-arm, therefore the N-terminal His6-tagged pullulanase chelating MNPs grafted polyether amine-400 gave the best reusability, which retained 60% of initial activity after 18 consecutive cycles with a total reaction time of 9h. Additionally, the correlation analysis of the catalyst behaviors indicated that the reusability was independent from other catalytic properties such as thermostability and substrate affinity. All the results revealed that the catalyst behavior can be mainly controlled by the His6-tagged pullulanase orientation than by the MNPs surface property which can tune the catalyst function.
Promoter evolution by synthetic promoter library (SPL) is a powerful approach to development of functional synthetic promoters to synthetic biology. However, it requires much tedious and ...time-consuming screenings because of the plethora of different variants in SPL. Actually, a large proportion of mutants in the SPL are significantly lower in strength, which contributes only to fabrication of a promoter library with a continuum of strength. Thus, to effectively obtain the evolved synthetic promoter exhibiting higher strength, it is essential to develop novel strategies to construct mutant library targeting the pivotal region rather than the arbitrary region of the template promoter. In this study, a strategy termed stepwise evolution targeting the spacer of core promoter (SETarSCoP) was established in Bacillus subtilis to effectively evolve the strength of bacterial promoter.
The native promoter, P
, from B. subtilis, which exhibits higher strength than the strong promoter P43, was set as the parental template. According to the comparison of conservation of the spacer sequences between - 35 box and - 10 box among a set of strong and weak native promoter, it revealed that 7-bp sequence immediately upstream of the - 10 box featured in the regulation of promoter strength. Based on the conservative feature, two rounds of consecutive evolution were performed targeting the hot region of P
. In the first round, a primary promoter mutation library (pPML) was constructed by mutagenesis targeting the 3-bp sequence immediately upstream of the - 10 box of the P
. Subsequently, four evolved mutants from pPML were selected to construction of four secondary promoter mutation libraries (sPMLs) based on mutagenesis of the 4-bp sequence upstream of the first-round target. After the consecutive two-step evolution, the mutant P
was identified and verified to be a highly evolved synthetic promoter. The strength of P
was higher than P
by approximately 3 times. Moreover, P
also exhibited broad suitability for different cargo proteins, such as β-glucuronidase and nattokinase. The proof-of-principle test showed that SETarSCoP successfully evolved both constitutive and inducible promoters.
Comparing with the commonly used SPL strategy, SETarSCoP facilitates the evolution process to obtain strength-evolved synthetic bacterial promoter through fabrication and screening of small-scale mutation libraries. This strategy will be a promising method to evolve diverse bacterial promoters to expand the toolbox for synthetic biology.
Nitrile hydratase (NHase) from Rhodococcus rhodochrous J1 is widely used for industrial production of acrylamide and nicotinamide. However, the two types of NHases (L-NHase and H-NHase) from R. ...rhodochrous J1 were only slightly expressed in E. coli by routine methods, which limits the comprehensive and systematic characterization of the enzyme properties. We successfully expressed the two types of recombinant NHases in E. coli by codon-optimization, engineering of Ribosome Binding Site (RBS) and spacer sequences. The specific activity of the purified L-NHase and H-NHase were 400 U/mg and 234 U/mg, respectively. The molecular mass of L-NHase and H-NHase was identified to be 94 kDa and 504 kDa, respectively, indicating that the quaternary structure of the two types of NHases was the same as those in R. rhodochrous J1. H-NHase exhibited higher substrate and product tolerance than L-NHase. Moreover, higher activity and shorter culture time were achieved in recombinant E. coli, and the whole cell catalyst of recombinant E. coli harboring H-NHase has equivalent efficiency in tolerance to the high-concentration product relative to that in R. rhodochrous J1. These results indicate that biotransformation of nitrile by R. rhodochrous J1 represents a potential alternative to NHase-producing E. coli.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Base editors (BEs) are widely used as revolutionary genome manipulation tools for cell evolution. To screen the targeted individuals, it is often necessary to expand the editing window to ensure ...highly diverse variant library. However, current BEs suffer from a limited editing window of 5–6 bases, corresponding to only 2–3 amino acids. Here, by engineering the CRISPR‒Cas12b, the study develops dCas12b‐based CRISPRi system, which can efficiently repress gene expression by blocking the initiation and elongation of gene transcription. Further, based on dCas12b, a new‐generation of BEs with an expanded editing window is established, covering the entire protospacer or more. The expanded editing window results from the smaller steric hindrance compared with other Cas proteins. The universality of the new BE is successfully validated in Bacillus subtilis and Escherichia coli. As a proof of concept, a spectinomycin‐resistant E. coli strain (BL21) and a 6.49‐fold increased protein secretion efficiency in E. coli JM109 are successfully obtained by using the new BE. The study, by tremendously expanding the editing window of BEs, increased the capacity of the variant library exponentially, greatly increasing the screening efficiency for microbial cell evolution.
This study designed a new‐generation base editor (BE) based on CRISPR/Cas12b. The BE can induce C‐to‐T substitution within an expanded editing window in different microorganisms, providing a powerful targeted mutagenesis tool for microbial synthetic biology.
Phenylalanine hydroxylase from
(CvPAH) is a monomeric enzyme that converts phenylalanine to tyrosine. It shares high amino acid identity and similar structure with a subunit of human phenylalanine ...hydroxylase that is a tetramer, resulting in the latent application in medications. In this study, semirational design was applied to CvPAH to improve the catalytic ability based on molecular dynamics simulation analyses. Four Nterminal truncated variants and one single point variant were constructed and characterized. The D267P variant showed a 2.1-fold increased thermal stability compared to the wild type, but lower specific activity was noted compared with the wild type. The specific activity of all truncated variants was a greater than 25% increase compared to the wild type, and these variants showed similar or slightly decreased thermostability with the exception of the N-Δ9 variant. Notably, the N-Δ9 variant exhibited a 1.2-fold increased specific activity, a 1.3-fold increased thermostability and considerably increased catalytic activity under the neutral environment compared with the wild type. These properties of the N-Δ9 variant could advance medical and pharmaceutical applications of CvPAH. Our findings indicate that the N-terminus might modulate substrate binding, and are directives for further modification and functional research of PAH and other enzymes.
An efficient enzymatic process was developed to produce optically pure D-phenylalanine through asymmetric resolution of the racemic DL-phenylalanine using immobilized phenylalanine ammonia-lyase ...(RgPAL) from Rhodotorula glutinis JN-1. RgPAL was immobilized on a modified mesoporous silica support (MCM-41-NH-GA). The resulting MCM-41-NH-GA-RgPAL showed high activity and stability. The resolution efficiency using MCM-41-NH-GA-RgPAL in a recirculating packed-bed reactor (RPBR) was higher than that in a stirred-tank reactor. Under optimal operational conditions, the volumetric conversion rate of L-phenylalanine and the productivity of D-phenylalanine reached 96.7 mM h⁻¹ and 0.32 g L⁻¹ h⁻¹, respectively. The optical purity (eeD) of D-phenylalanine exceeded 99%. The RPBR ran continuously for 16 batches, the conversion ratio did not decrease. The reactor was scaled up 25-fold, and the productivity of D-phenylalanine (eeD>99%) in the scaled-up reactor reached 7.2 g L⁻¹ h⁻¹. These results suggest that the resolution process is an alternative method to produce highly pure D-phenylalanine.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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