Sucrose isomerase (SI), catalyzing sucrose to isomaltulose, has been widely used in isomaltulose production, but its poor thermostability is still resisted in sustainable batches production. Here, ...protein engineering and one-step immobilized cell strategy were simultaneously coupled to maintain steady state for long-term operational stabilities. First, rational design of
Pantoea dispersa
SI (PdSI) for improving its thermostability by predicting and substituting the unstable amino acid residues was investigated using computational analysis. After screening mutagenesis library, two single mutants (PdSIV280L and PdSIS499F) displayed favorable characteristics on thermostability, and further study found that the double mutant PdSIV280L/S499F could stabilize PdSIWT better. Compared with PdSIWT, PdSIV280L/S499F displayed a 3.2°C-higher
T
m
, and showed a ninefold prolonged half-life at 45°C. Subsequently, a one-step simplified immobilization method was developed for encapsulation of PdSIV280L/S499F in food-grade
Corynebacterium glutamicum
cells to further enhance the recyclability of isomaltulose production. Recombinant cells expressing combinatorial mutant (RCSI2) were successfully immobilized in 2.5% sodium alginate without prior permeabilization. The immobilized RCSI2 showed that the maximum yield of isomaltulose by batch conversion reached to 453.0 g/L isomaltulose with a productivity of 41.2 g/l/h from 500.0 g/L sucrose solution, and the conversion rate remained 83.2% after 26 repeated batches.
Cofactor regeneration is indispensable to avoid the addition of large quantities of cofactor NADH or NAD
in oxidation-reduction reactions. Water-forming NADH oxidase (Nox) has attracted substantive ...attention as it can oxidize cytosolic NADH to NAD
without concomitant accumulation of by-products. However, its applications have some limitations in some oxidation-reduction processes when its optimum pH is different from its coupled enzymes. In this study, to modify the optimum pH of BsNox, fifteen relevant candidates of site-directed mutations were selected based on surface charge rational design. As predicted, the substitution of this asparagine residue with an aspartic acid residue (N22D) or with a glutamic acid residue (N116E) shifts its pH optimum from 9.0 to 7.0. Subsequently, N20D/N116E combined mutant could not only downshift the pH optimum of BsNox but also significantly increase its specific activity, which was about 2.9-fold at pH 7.0, 2.2-fold at pH 8.0 and 1.2-fold at pH 9.0 that of the wild-type. The double mutant N20D/N116E displays a higher activity within a wide range of pH from 6 to 9, which is wider than the wide type. The usability of the BsNox and its variations for NAD
regeneration in a neutral environment was demonstrated by coupling with a glutamate dehydrogenase for α-ketoglutaric acid (α-KG) production from L-glutamic acid (L-Glu) at pH 7.0. Employing the variation N20D/N116E as an NAD+ regeneration coenzyme could shorten the process duration; 90% of L-Glu were transformed into α-KG within 40 min vs. 70 min with the wild-type BsNox for NAD
regeneration. The results obtained in this work suggest the promising properties of the BsNox variation N20D/N116E are competent in NAD
regeneration applications under a neutral environment.
N
-acetyl-
l
-glutamate kinase (NAGK) catalyzes the second step of
l
-arginine biosynthesis and is inhibited by
l
-arginine in
Corynebacterium crenatum
. To ascertain the basis for the arginine ...sensitivity of CcNAGK, residue E19 which located at the entrance of the Arginine-ring was subjected to site-saturated mutagenesis and we successfully illustrated the inhibition-resistant mechanism. Typically, the E19Y mutant displayed the greatest deregulation of
l
-arginine feedback inhibition. An equally important strategy is to improve the catalytic activity and thermostability of CcNAGK. For further strain improvement, we used site-directed mutagenesis to identify mutations that improve CcNAGK. Results identified variants I74V, F91H and K234T display higher specific activity and thermostability. The
l
-arginine yield and productivity of the recombinant strain
C. crenatum
SYPA-EH3 (which possesses a combination of all four mutant sites, E19Y/I74V/F91H/K234T) reached 61.2 and 0.638 g/L/h, respectively, after 96 h in 5 L bioreactor fermentation, an increase of approximately 41.8% compared with the initial strain.
d-tagatose is a low-calorie alternative to sucrose natural monosaccharide that is nearly as sweet. As a ketohexose, d-tagatose has disease-relieving and health-promoting properties. Due to its ...scarcity in nature, d-tagatose is mainly produced through chemical and biological methods. Compared to traditional chemical methods, biological methods use whole cells and isolated enzymes as catalysts under mild reaction conditions with few by-products and no pollution. Nowadays, biological methods have become a very important topic in related fields due to their high efficiency and environmental friendliness. This paper introduces the functions and applications of d-tagatose and systematically reviews its production, especially by l-arabinose isomerase (L-AI), using biological methods. The molecular structures and catalytic mechanisms of L-AIs are also analyzed. In addition, the properties of L-AIs from different microbial sources are summarized. Finally, we overview strategies to improve the efficiency of d-tagatose production by engineering L-AIs and provide prospects for the future bioproduction of d-tagatose.
With the intensification of energy crisis, considerable attention has been paid to the application and research of lithium-ion batteries. A significant progress has also been made in the research of ...lithium-ion battery capacity evaluation using electrochemical and electrical parameters. In this study, the effect of mechanical characteristic parameter (i.e., stack stress) on battery capacity is investigated using the experimental combined numerical approach. The objective of the proposed approach is to evaluate the capacity based on the initial applied stress, the real-time stress, charging open circuit voltage, and discharging open circuit voltage. Experiments were designed and the data is fed into evolutionary approach of genetic programming. Based on analysis, the accuracy of the proposed GP model is fairly high while the maximum percentage of error is about 5%. In addition, a negative correlation exists between the initial stress and battery capacity while the capacity increases with real-time stress.
l-asparaginase (EC 3.5.1.1) hydrolyzes l-asparagine to produce l-aspartate and ammonia and is widely found in microorganisms, plants, and some rodent sera. l-asparaginase used for industrial ...production should have good thermostability. We heterologously expressed l-asparaginase from
, selected nine loci for site-directed mutagenesis by rational design, and obtained two mutants with significantly improved thermostability. The optimal temperature of mutants S302I and S302M was 50 °C. After incubating the mutant and wild-type enzymes at 45 °C for 35 h, the residual activity of the wild-type enzyme (WT) was only about 10%. In contrast, the residual activity of S302I and S302M was more than 50%. After combination mutagenesis,
168-pMA5-A344E/S302I was constructed using the food-safe host strain
168. Additionally, a 5' untranslated region (UTR) modification strategy was adopted to enhance the expression level of
-derived l-asparaginase in
. In a 5-L fermenter scale-up experiment, the enzyme activity of recombinant
168-pMA5-UTR-A344E/S302I reached 521.9 U·mL
by fed-batch fermentation.
The low productivity in long fermentation duration and high-salt working conditions limit the application of L-glutaminase in soy sauce brewing. In this study, a novel L-glutaminase (LreuglsA) with ...eminent salt tolerance was mined and achieved more than 70% activity with 30% NaCl. To improve the robustness of the enzyme at different fermentation strategies, mutation LreuglsAH105K was built by a computer-aided design, and the recombinant protein expression level, an essential parameter in industrial applications, was increased 5.61-fold with the synthetic biology strategy by improving the mRNA stability. Finally, the LreuglsAH105K functional expression box was contributed to Bacillus subtilis 168 by auxotrophic complementation, and the production in a 5-L bioreactor was improved to 2516.78 ± 20.83 U mL−1, the highest production ever reported. When the immobilized cells were applied to high-salt dilute-state soy sauce brewing, the L-glutamate level was increased by 45.9%. This work provides insight into the salt-tolerant enzyme for improving the efficiency of industrial applications.
Styrene monooxygenase (SMO) catalyzes the first step of aromatic alkene degradation yielding the corresponding epoxides. Because of its broad spectrum of substrates, the enzyme harbors a great ...potential for an application in medicine and chemical industries.
In this study, we achieved higher enzymatic activity and better stability towards styrene by enlarging the ligand entrance tunnel and improving the hydrophobicity through error-prone PCR and site-saturation mutagenesis. It was found that Asp305 (D305) hindered the entrance of the FAD cofactor according to the model analysis. Therefore, substitution with amino acids possessing shorter side chains, like glycine, opened the entrance tunnel and resulted in up to 2.7 times higher activity compared to the wild-type enzyme. The half-lives of thermal inactivation for the variant D305G at 60 °C was 28.9 h compared to only 3.2 h of the wild type SMO. Moreover, overexpression of SMO in Pseudomonas putida KT2440 with NADH regeneration was carried out in order to improve biotransformation efficiency for epoxide production. A hexadecane/buffer (v/v) biphasic system was applied in order to minimize the inactivation effect of high substrate concentrations on the SMO enzyme. Finally, SMO activities of 190 U/g CDW were measured and a total amount of 20.5 mM (S)-styrene oxide were obtained after 8 h.
This study offers an alternative strategy for improved SMO expression and provides an efficient biocatalytic system for epoxide production via engineering the entrance tunnel of the enzyme's active site.
Determinations of active earth pressures are commonly performed two-dimensionally (2D) based on completely saturated and/or dry assumptions, though the soil in cases of geotechnical interest is ...mostly unsaturated and the earth pressures are usually of conspicuous three-dimensional (3D) features. In this paper, a novel finite prismoid element method (FPEM) for calculating the lateral earth pressures acting against the retaining wall is suggested. The main feature of the FPEM is that the whole backfill is discretized into numerous horizontally distributed prismoid elements that might characterized with different soil properties. For unsaturated backfills, the prismoid elements are characterized with various soil cohesions and unit soil weights. Upper bound solutions to active earth pressures under 2D and 3D conditions with and without suction are both calculated and compared with several other analytical ones, indicating the reliability and applicability of the proposed method. The responses of unsaturated backfills to surcharge loads on the crest are numerically studied and discussed. An illustrative example is reexamined to further demonstrate the practical use of the technique.
9α-Hydroxy-4-androstene-3,17-dione (9-OH-AD) is one of the significant intermediates for the preparation of β-methasone, dexamethasone, and other steroids. In general, the key enzyme that enables the ...biotransformation of 4-androstene-3,17-dione (AD) to 9-OH-AD is 3-phytosterone-9α-hydroxylase (KSH), which consists of two components: a terminal oxygenase (KshA) and ferredoxin reductase (KshB). The reaction is carried out with the concomitant oxidation of NADH to NAD
. In this study, the more efficient 3-phytosterone-9α-hydroxylase oxygenase (KshC) from the
sp. strain VKM Ac-1817D was confirmed and compared with reported KshA. To evaluate the function of KshC on the bioconversion of AD to 9-OH-AD, the characterization of KshC and the compounded system of KshB, KshC, and NADH was constructed. The optimum ratio of KSH oxygenase to reductase content was 1.5:1. An NADH regeneration system was designed by introducing a formate dehydrogenase, further confirming that a more economical process for biological transformation from AD to 9-OH-AD was established. A total of 7.78 g of 9-OH-AD per liter was achieved through a fed-batch process with a 92.11% conversion rate (mol/mol). This enzyme-mediated hydroxylation method provides an environmentally friendly and economical strategy for the production of 9-OH-AD.
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