There are four nitrogen atoms in l-arginine molecule and the nitrogen content is 32.1%. By now, metabolic engineering for l-arginine production strain improvement was focused on carbon flux optimization. In previous work, we obtained an l-arginine-producing Corynebacterium crenatum SDNN403 (ARG) through screening and mutation breeding. In this paper, a strain engineering strategy focusing on nitrogen supply and ammonium assimilation for l-arginine production was performed. Firstly, the effects of nitrogen atom donor (l-glutamate, l-glutamine and l-aspartate) addition on l-arginine production of ARG were studied, and the addition of l-glutamine and l-aspartate was beneficial for l-arginine production. Then, the glutamine synthetase gene glnA and aspartase gene aspA from E. coli were overexpressed in ARG for increasing the l-glutamine and l-aspartate synthesis, and the l-arginine production was effectively increased. In addition, the l-glutamate supply re-emerged as a limiting factor for l-arginine biosynthesis. Finally, the glutamate dehydrogenase gene gdh was co-overexpressed for further enhancement of l-arginine production. The final strain could produce 53.2 g l−1 of l-arginine, which was increased by 41.5% compared to ARG in fed-batch fermentation.

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.

AbstractBACKGROUNDThe 3-ketosteroid-Δ1-dehydrogenase (KSDD) expressed in Escherichia coli was mainly in the form of inclusion bodies and the KSDD enzyme activity was at a low level. Therefore, the regulation of process conditions and supplementation of natural osmolytes were carried out to improve the soluble expression of KSDD in E. coli.RESULTSEffects of temperature, inducer concentration, and osmolytes (K-glutamate, proline, and betaine) supplementation on cell growth and soluble KSDD production in recombinant Escherichia coli were investigated. With the decrease of cultivation temperature, lower isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, and betaine addition, high intracellular soluble KSDD production was realized in the recombinant E. coli BL21/pET28a-ksdd and the KSDD enzyme activity reached 11.7 U mg−1, which is the highest KSDD activity ever reported. In addition, the whole-cell biocatalyst of this recombinant strain was used for bioconversion of androst-4-ene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD). By optimization of the transformation conditions and applying a fed-batch strategy, a final ADD yield of 5.7 g L−1 was achieved with a productivity of 0.158 g (L h)−1, which is the highest reported productivity using a microbial process. More importantly, no by-products were detected during the whole bioconversion process.CONCLUSIONThese results provide the basis for industrial scale production of ADD. © 2016 Society of Chemical Industry

Traditionally, testosterone (TS), an important hormone drug and precursor for the synthesis of other steroids, was chemically produced. Recently, TS has been prepared through side-chain degradation of some sterols (cholesterol or phytosterol) using microbial fermentation methods. However, the TS production is at a low level, and the biotransformation process is long and with many by-products formed. NADPH-dependent 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3) from human testis catalyzes the conversion of 4-androstene-3,17-dione (AD) to TS. In this work, we optimized the gene codons of human 17β-HSD3 and realized its functional expression in Pichia pastoris GS115. The engineered P. pastoris/17β-HSD3 cells exhibited good selectivity for the efficient transformation of AD to TS. Moreover, Saccharomyces cerevisiae glucose-6-phosphate dehydrogenase (G6PDH) was introduced to strengthen the NADPH regeneration system into the pathway from AD to TS by P. pastoris/17β-HSD3. By optimization of the transformation conditions from AD to TS and applying the fed-batch strategy, the co-expressed system P. pastoris/17β-HSD3-G6PDH produced TS of 11.6 g L−1, which is the highest reported yield using a bioconversion method. Compared with the ever highest reported production (≤1.7 g L−1), our production was improved by about 7-fold. More importantly, no by-products were detected during the whole bioconversion process. This study indicated that the recombinant P. pastoris harboring 17β-HSD3 and G6PDH could be a promising candidate to produce TS in the pharmaceutical industry. The P. pastoris system co-expression target enzyme and the cofactor regeneration enzyme may be helpful for enhancing the production of other steroids.

Reactive oxygen species (ROS) is an inherent consequence to all aerobically living organisms that might lead to the cells being lethal and susceptible to oxidative stress. Bacillus pumilus is characterized by high-resistance oxidative stress that stimulated our interest to investigate the heterologous expression and characterization of heme-catalase as potential biocatalyst. Results indicated that recombinant enzyme significantly exhibited the high catalytic activity of 55,784 U/mg expressed in Bacillus subtilis 168 and 98.097 µmol/min/mg peroxidatic activity, the apparent Km of catalytic activity was 59.6 ± 13 mM with higher turnover rate (Kcat = 322.651 × 103 s−1). The pH dependence of catalatic and peroxidatic activity was pH 7.0 and pH 4.5 respectively with temperature dependence of 40 °C and the recombinant heme-catalase exhibited a strong Fe2+ preference. It was further revealed that catalase KatX2 improved the resistance oxidative stress of B. subtilis. These findings suggest that this B. pumilus heme-catalase can be considered among the industrially relevant biocatalysts due to its exceptional catalytic rate and high stability and it can be a potential candidate for the improvement of oxidative resistance of industrially produced strains.

Mycobacterium neoaurum ST-095 and its mutant M. neoaurum JC-12, capable of transforming phytosterol to androst-1,4-diene-3,17-dione (ADD) and androst-4-ene-3,17-dione (AD), produce very different molar ratios of ADD/AD. The distinct differences were related to the enzyme activity of 3-ketosteroid-Δ1-dehydrogenase (KSDD), which catalyzes the C1,2 dehydrogenation of AD to ADD specifically. In this study, by analyzing the primary structure of KSDDI (from M. neoaurum ST-095) and KSDDII (from M. neoaurum JC-12), we found the only difference between KSDDI and KSDDII was the mutation of Val366 to Ser366. This mutation directly affected KSDD enzyme activity, and this result was confirmed by heterologous expression of these two enzymes in Bacillus subtilis. Assay of the purified recombinant enzymes showed that KSDDII has a higher C1,2 dehydrogenation activity than KSDDI. The functional difference between KSDDI and KSDDII in phytosterol biotransformation was revealed by gene disruption and complementation. Phytosterol transformation results demonstrated that ksddI and ksddII gene disrupted strains showed similar ADD/AD molar ratios, while the ADD/AD molar ratios of the ksddI and ksddII complemented strains were restored to their original levels. These results proved that the different ADD/AD molar ratios of these two M. neoaurum strains were due to the differences in KSDD. Finally, KSDD structure analysis revealed that the Val366Ser mutation could possibly play an important role in stabilizing the active center and enhancing the interaction of AD and KSDD. This study provides a reliable theoretical basis for understanding the structure and catalytic mechanism of the Mycobacteria KSDD enzyme.

Corynebacterium crenatum SYPA5-5, an l-arginine high-producer obtained through multiple mutation-screening steps, had been deregulated by the repression of ArgR that inhibits l-arginine biosynthesis at genetic level. Further study indicated that feedback inhibition of SYPA5-5 N-acetylglutamate kinase (CcNAGK) by l-arginine, as another rate-limiting step, could be deregulated by introducing point mutations. Here, we introduced two of the positive mutations (H268N or R209A) of CcNAGK into the chromosome of SYPA5-5, however, resulting in accumulation of large amounts of the intermediates (l-citrulline and l-ornithine) and decreased production of l-arginine. Genetic and enzymatic levels analysis involved in l-arginine biosynthetic pathway of recombinants SYPA5-5-NAGKH268N (H-7) and SYPA5-5-NAGKR209A (R-8) showed that the transcription levels of argGH decreased accompanied with the reduction of argininosuccinate synthase and argininosuccinase activities, respectively, which led to the metabolic obstacle from l-citrulline to l-arginine. Co-expression of argGH with exogenous plasmid in H-7 and R-8 removed this bottleneck and increased l-arginine productivity remarkably. Compared with SYPA5-5, fermentation period of H-7/pDXW-10-argGH (H-7-GH) reduced to 16 h; meanwhile, the l-arginine productivity improved about 63.6 %. Fed-batch fermentation of H-7-GH in 10 L bioreactor produced 389.9 mM l-arginine with the productivity of 5.42 mM h−1. These results indicated that controlling the transcription of argGH was a key factor for regulating the metabolic flux toward l-arginine biosynthesis after deregulating the repression of ArgR and feedback inhibition of CcNAGK, and therefore functioned as another regulatory mode for l-arginine production. Thus, deregulating all these three regulatory modes was a powerful strategy to construct l-arginine high-producing C. crenatum.

The α-acetolactate decarboxylase (ALDC) can reduce diacetyl fleetly to promote mature beer. A safe strain Bacillus subtilis WB600 for high-yield production of ALDC was constructed with the ALDC gene saald from Staphylococcus aureus L3-15. SDS-PAGE analysis revealed that S. aureus α-acetolactate decarboxylase (SaALDC) was successfully expressed in recombinant B. siutilis strain. The enzyme SaALDC was purified using Ni-affinity chromatography and showed a maximum activity at 45 °C and pH 6.0. The values of Km and Vmax were 17.7 μM and 2.06 mM min−1, respectively. Due to the unstable property of SaALDC at low pH conditions that needed in brewing process, site-directed mutagenesis was proposed for improving the acidic stability of SaALDC. Homology comparative modeling analysis showed that the mutation (K52D) gave rise to the negative-electrostatic potential on the surface of protein while the numbers of hydrogen bonds between the mutation site (N43D) and the around residues increased. Taken together the effect of mutation N43D-K52D, recombinant SaALDCN43D-K52D showed dramatically improved acidic stability with prolonged half-life of 3.5 h (compared to the WT of 1.5 h) at pH 4.0. In a 5-L fermenter, the recombinant B. subtilis strain that could over-express SaALDCN43D-K52D exhibited a high yield of 135.8 U mL−1 of SaALDC activity, about 320 times higher comparing to 0.42 U mL−1 of S. aureus L3-15. This work proposed a  strategy for improving the acidic stability of SaALDC in the  B. subtilis host.

l-Ornithine, a non-protein amino acid, is usually extracted from hydrolyzed protein as well as produced by microbial fermentation. Here, we focus on a highly efficient whole-cell biocatalyst for the production of l-ornithine. The gene argI, encoding arginase, which catalyzes the hydrolysis of l-arginine to l-ornithine and urea, was cloned from Bacillus amyloliquefaciens B10-127 and expressed in GRAS strain Bacillus subtilis 168. The recombinant strain exhibited an arginase activity of 21.9 U/mg, which is 26.7 times that of wild B. subtilis 168. The optimal pH and temperature of the purified recombinant arginase were 10.0 and 40 °C, respectively. In addition, the recombinant arginase exhibited a strong Mn2+ preference. When using whole-cell biocatalyst-based bioconversion, a hyper l-ornithine production of 356.9 g/L was achieved with a fed-batch strategy in a 5-L reactor within 12 h. This whole-cell bioconversion study demonstrates an environmentally friendly strategy for l-ornithine production in industry.

Production of gamma-aminobutyric acid (GABA) from crop biomass such as cassava in high concentration is desirable, but difficult to achieve. A safe biotechnological route was investigated to produce GABA from cassava powder by C. glutamicum G01 and L. plantarum GB01-21. Liquefied cassava powder was first transformed to glutamic acid by simultaneous saccharification and fermentation with C. glutamicum G01, followed by biotransformation of glutamic acid to GABA with resting cells of L. plantarum GB01-21 in the reaction medium. After optimizing the reaction conditions, the maximum concentration of GABA reached 80.5 g/L with a GABA productivity of 2.68 g/L/h. This is the highest yield ever reported of GABA production from cassava-derived glucose. The bioprocess provides the added advantage of employing nonpathogenic microorganisms, C. glutamicum and L. plantarum, in microbial production of GABA from cassava biomass, which can be used in the food and pharmaceutical industries.

Steroid medication is used extensively in clinical applications and comprises a large and vital part of the pharmaceutical industry. However, the difficulty of separating 4-androstene-3,17-dione (AD) from 1,4-androstadiene-3,17-dione (ADD) restricts the application of the microbial transformation of phytosterols in the industry. A novel atmospheric and room temperature plasma (ARTP) treatment, which employs helium as the working gas, was used to generate Mycobacterium neoaurum mutants producing large amounts of AD. After treatment of cultures with ARTP, four mutants were selected using a novel screening method with a color assay. Among the mutants, M. neoaurum ZADF-4 was considered the best candidate for industrial application. When the fermentation medium contained 15 g/L phytosterols and was cultivated on a rotary shaker at 160 r/min at 30°C for 7 d, (6.28±0.11) g/L of AD and (0.82±0.05) g/L of ADD were produced by the ZADF-4 mutant, compared with (4.83±0.13) g/L of AD and (2.34±0.06) g/L of ADD by the original strain, M. neoaurum ZAD. Compared with ZAD, the molar yield of AD increased from 48.3% to 60.3% in the ZADF-4 mutant. This result indicates that ZADF-4 may have potential for industrial production of AD.获得一株高产雄甾-4-烯-3,17-二酮 (AD) 的Mycobacterium neoaurum突变株。获得了一株3-甾酮-Δ1-脱氢酶 (KSDD) 酶活缺陷型的高产AD的诱变菌株Mycobacterium neoaurumZADF-4, 并采用菌落显色法筛选KSDD酶活缺陷型M. neoaurum 突变株。(1) 诱变方法: 采用常压室温等离子体 (ARTP) 诱变技术来处理出发菌株M. neoaurum ZAD。ARTP诱变条件如下: 功率40W, 气流量12.5 L/min, 辐射距离1 cm, 样品体积10 μl, 辐射时间为60、90、120、150和180 s; 致死率统计优化后, 最适辐射时间为150 s, 致死率为90%~96%。 (2) 筛选方法: 将ARTP 诱变处理后的菌株点种在硝酸纤维滤膜上, 30°C培养2d, 然后将长有菌落的滤膜小心取出并漂浮在4 mg/ml二氯靛酚 (DCPIP) 溶液 (0.1 mmol/L磷酸缓冲液pH 7.0), 30°C培养1 d直到全部菌落染成蓝色。然后将该滤膜取出, 漂浮在250 mmol/L AD溶液 (2%甲醇和50 mmol/L Tris pH 7.0缓冲液), 室温放置15 min左右, 观察菌落颜色变化。KSDD在底物AD存在时会脱氢产生雄甾-1,4-二烯-3,17-二酮 (ADD) 和H+, H+可以使被DCPIP染成蓝色的菌株褪色。因此, 酶活缺陷型的菌株会仍保持蓝色, 而酶活高的菌株会褪色为黄色 (图3)。 (3) 对获得的潜在的高产AD菌株进行进一步的酶活检测以及产量验证, 以期获得最优的突变株。获得了4株具有潜在的高产AD能力的菌株, 其中, 最优的突变株ZADF-4的KSDD 酶活相较于出发菌株ZAD下降了81.2% (图4), 活性胶也证明其KSDD酶活相较于出发菌株下降明显 (图5)。薄层色谱法 (TLC) 和高效液相色谱法 (HPLC) 实验证明突变株ZADF-4中, AD的产量有了明显的提高 (图6和图7), 提高到了(6.28±0.11) g/L, AD/ADD提高到8:1, AD的摩尔产率达到60.3% (表1)。对出发菌株ZAD和突变株ZADF-4的ksdd基因进行克隆和序列比对, 发现ZADF-4 的ksdd 序列在5’端缺失9个核苷酸 (atgttctac), 导致3个氨基酸 (MFY) 的缺失; 还发生了两个点突变, 其中一个是无义突变 (g.15a>6t), 另一个是有义突变 (g.413c>404t), 并引起了相应位置上的氨基酸变化 (p.138S>135L)。上述的基因突变及其引起的氨基酸序列的变化可能是引起M. neoaurum ZADF-4中KSDD酶活降低及AD产量提高的主要原因。

Haloarchaea is an important group of polyhydroxyalkanoate (PHA)-accumulating organisms. However, few promising haloarchaeal species for economical and efficient PHA production have been reported. Here, we first discovered that Halogranum amylolyticum TNN58 could efficiently accumulate poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with a high 3-hydroxyvalerate (3HV) fraction using glucose as carbon source. Briefly, transmission electron microscopy (TEM) analysis revealed the presence of a large number of PHA granules in the cells. Gas chromatography–mass spectrometry (GC-MS) and proton nuclear magnetic resonance (1H NMR) analyses showed that PHAs synthesized from glucose was PHBV. Moreover, the 3HV content reached 20.1 mol%, which is the highest 3HV fraction thus far reported, as for PHBV produced by the wild-type strains grown on unrelated carbon courses. Fermentation experiments suggested that nitrogen-limited MG medium was better than nutrient-rich NOMG and AS168 medium for PHBV production. Additionally, glucose was the most suitable carbon source among the tested carbon sources. Interestingly, PHBV accumulation was almost paralleled by cell growth and glucose consumption. By applying the fed-batch process in fermentor, the PHBV production and cell dry weight were increased by approximately eight and four times, respectively, as compared with those of the batch process in shaking flasks. The classical PHA synthase genes were successfully cloned via consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) and high-efficiency thermal asymmetric interlaced (hiTAIL) PCR methods. This finding suggested that H. amylolyticum shows promising potential in the low-cost biotechnological production of PHBV after further process optimization.

Polyhydroxyalkanoates (PHAs) are a class of natural biopolyesters accumulated intracellularly by many microorganisms. These polymers have attracted particular attention as green plastic in biomedical and industrial applications due to their good biodegradability and biocompatibility. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is one of the most common members of PHAs. However, there is no report comparing the properties of PHBV from different groups of producers, e.g., bacteria and haloarchaea. In this study, two types of PHBV copolymers were synthesized in Halogranum amylolyticum and Ralstonia eutropha, respectively, by feeding different carbon sources. They possessed a similar concentration of 3HV monomers (21 mol%) and were named PHBV-H (produced by H. amylolyticum) and PHBV-B (produced by R. eutropha) based on their source. Interestingly, they exhibited different behaviors especially in thermal stability, melting temperature, crystallinity percentage, and mechanical properties. Furthermore, the films of PHBV-H and PHBV-B possessed different surface properties, such as surface roughness, wettability, and surface free energy. The value of hemolysis on the PHBV-H film was lower in comparison with the PHBV-B film, although both values were within the limit of 5 % permissible for biomaterials. Notably, few inactivated platelets adhered to the surface of the PHBV-H film, whereas numerous activated platelets were seen on film PHBV-B. These results indicated that PHBV-H was a better potential component of blood-contact biomaterials than PHBV-B. Our study clearly revealed that the properties of PHAs are source dependent and haloarchaeal species provide a new opportunity for the production of desired PHAs.

Lactobacillus plantarum CCTCC M209102 efficiently produces γ-aminobutyric acid (GABA) from L-glutamate, in which glutamate decarboxylase and pyridoxal kinase are involved in the transformation. Pyridoxal kinase catalyzes ATP-dependent phosphorylation of pyridoxal to produce pyridoxal-5′-phosphate, which is the cofactor required for glutamate decarboxylase to biotransform GABA from L-glutamate. Corynebacterium glutamicum G01 is a good producer of L-glutamate from glucose. However, it cannot yield GABA from L-glutamate due to the absence of glutamate decarboxylase and pyridoxal kinase. In this work, to realize the efficient one-step preparation of GABA from glucose without exogenous pyridoxal-5′-phosphate, the metabolic module from L-glutamate to GABA based on glutamate decarboxylase and pyridoxal kinase in L. plantarum was grafted into C. glutamicum. To further improve the GABA production, the pathways to by-product pools of L-arginine, L-proline and L-lysine were blocked using the insertional mutation technique. The engineered C. glutamicum APLGGP carrying argB::tacgad, proB::tacgad and dapA::tacplk could efficiently convert glucose into GABA in one-step without an exogenous co-factor. In fed-batch cultures, the recombinant C. glutamicum APLGGP produced 70.6 g L−1 GABA at 30 °C and 70 h through a two-stage pH control strategy. To our knowledge, this is the highest reported GABA production using glucose as a substrate, and this designed C. glutamicum should be an excellent candidate for producing GABA on an industrial scale. This work is expected to pave the way to redesign the bioreactor for efficient one-step biosynthesis of GABA from glucose without an exogenous co-factor.

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.

This study focused on the cloning, overexpression, and characterization of the gene encoding l-asparaginase (ansZ) from a nonpathogenic strain of Bacillus subtilis B11–06. The recombinant enzyme showed high thermostability and low affinity to l-glutamine. The ansZ gene, encoding a putative l-asparaginase II, was amplified by PCR and expressed in B. subtilis 168 using the shuttle vector pMA5. The activity of the recombinant enzyme was 9.98 U/mL, which was significantly higher than that of B. subtilis B11–06. The recombinant enzyme was purified by a two-step procedure including ammonium sulfate fractionation and hydrophobic interaction chromatography. The optimum pH and temperature of the recombinant enzyme were 7.5 and 40 °C, respectively. The enzyme was quite stable at a pH range of 6.0–9.0 and exhibited about 14.7 and 9.0% retention of activity following 2 h incubation at 50 or 60 °C, respectively. The Km for l-asparagine was 0.43 mM, and the Vmax was 77.51 μM/min. Results of this study also revealed the potential industrial application of this enzyme in reducing acrylamide formation during the potato frying process.

•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.

•Site directed mutagenesis was applied to increase thermostability and enzyme activity of BsAII.•One mutant showed 83.10% and 17.5 °C increase in specific activity and half-inactivation temperature, respectively.•The structure changes might contribute to the improved activity.•The increased hydrophilicity and surface charge redistribution helped stabilize the conformation of the enzyme.l-Asparaginase (l-asparagine amidohydrolase, EC 3.5.1.1) catalyzes the hydrolysis of l-asparagine to l-aspartic acid and ammonia. It can be used to reduce the formation of acrylamide, which is carcinogenic to humans in foods, via removal of the precursor, asparagine, from the primary ingredients. However, low activity and poor thermostability of l-asparaginase restrict its application in food industry. In this study, we successfully improved thermostability and catalytic efficiency of l-asparaginase II (BsAII) from Bacillus subtilis B11-06 by site-directed mutagenesis. According to sequences alignment and homologous modeling, residues G107, T109 and S166 which were adjacent to the catalytic cavity were selected and substituted by Asp, Gln/Ser and Ala, respectively, to construct mutants G107D, T109Q, T109S and S166A. The BsAII mutant of G107D (G107Dansz) displayed superior performance in thermal tolerance and higher activity than the wild-type enzyme (towards l-asparagine). Comparative analysis of hydrogen bond interactions, surface electrostatic potential and structure of substrate binding pocket between G107Danszand BsAII indicated that the substitution of G107, which was adjacent to catalytic cavity with Asp, resulted in small conformational changes and surface electrostatic potential redistribution and contributed to the improved protein stability and catalytic efficiency.

•Development of a multi-enzymatic desymmetrization of l-norvaline from dl-norvaline.•The catalytic system obtained various l-amino acids with varied optical purities.•The system produced l-norvaline with a high yield, enantiomeric purity and titer.Perindopril is an effective antihypertensive drug in strong demand used to treat hypertension. l-norvaline is a vital intermediate of Perindopril production mainly produced by chemical synthesis with low purity. We developed an environmentally friendly method to produce l-norvaline with high purity based on a desymmetrization process. d-Norvaline was oxidized to the corresponding keto acid by d-amino acid oxidase from the substrate dl-norvaline. Asymmetric hydrogenation of the keto acid to l-norvaline was carried out by leucine dehydrogenase with concomitant oxidation of NADH to NAD+. A NADH regeneration system was introduced by overexpressing a formate dehydrogenase. The unwanted H2O2 by-product generated during d-norvaline oxidation was removed by adding catalase. A total of 54.09 g/L of l-norvaline was achieved, with an enantiomeric excess over 99% under optimal conditions, with a 96.7% conversion rate. Our desymmetrization method provides an environmental friendly strategy for the production of enantiomerically pure l-norvaline in the pharmaceutical industry.Download high-res image (91KB)Download full-size image