ABSTRACTNAD-dependentl- andd-lactate dehydrogenases coexist inLactobacillusgenomes and may convert pyruvic acid intol-lactic acid andd-lactic acid, respectively. Our findings suggest that the relative catalytic efficiencies ofldhL- andldhD-encoded products are crucial for the optical purity of lactic acid produced byLactobacillusstrains.

Extracellular proteins are of paramount importance in the cell−cell interactions of anammox biofilms. However, the inherent aggregation mechanisms of anammox have largely remained elusive. Herein, using a quartz sand extraction protocol and follow-up iTRAQ-based quantitative proteomics, we identified 367 extracellular proteins from initial colonizers, mature biofilm, and detached biofilm. The extracellular proteins were mainly membrane-associated. Most of the recovered proteins (226, 72.5%) originated from the phylum Planctomycetes. In summary, 215 and 190 of the 367 proteins recovered were up-and/ or downregulated at least 1.2-fold during the biofilm formation and detachment periods, respectively. These differentially expressed proteins were dominantly involved in metal ion binding, which was regarded as strong evidence for their abilities to enhance ionic bridges in extracellular polymeric substances (EPS). Scanning electron microscopy−energy-dispersive X-ray spectroscopy (SEM-EDX) analysis of the biofilms further showed substantial levels of calcium and iron minerals. Critically, representative Sec-dependent secretory proteins affiliated with coccoid Planctomycetes, rod-shaped Proteobacteria, and filamentous Chloroflexi (11, 4, and 2 with differential expression, respectively) were found to have typical and abundant inner βsheet structures, wherein hydrophobic moieties can promote anammox aggregation. Overall, these findings highlight links between differentially expressed protein functions and morphologic traits of anammox consortia during biofilm development.

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b D-Lactate was identified as one of the few available organic acids that supported the growth of Gluconobacter oxydans 621H in this study. Interestingly, the strain used D-lactate as an energy source but not as a carbon source, unlike other lactate-utilizing bacteria. The enzymatic basis for the growth of G. oxydans 621H on D-lactate was therefore investigated. Although two putative NAD-independent D-lactate dehydrogenases, GOX1253 and GOX2071, were capable of oxidizing D-lactate, GOX1253 was the only enzyme able to support the D-lactate-driven growth of the strain. GOX1253 was characterized as a membrane-bound dehydrogenase with high activity toward D-lactate, while GOX2071 was characterized as a soluble oxidase with broad substrate specificity toward D-2-hydroxy acids. The latter used molecular oxygen as a direct electron acceptor, a feature that has not been reported previously in D-lactate-oxidizing enzymes. This study not only clarifies the mechanism for the growth of G. oxydans on D-lactate, but also provides new insights for applications of the important industrial microbe and the novel D-lactate oxidase.

The corn starch industry produces a large amount of corn steep water, leading to high organic waste load. Lactate could be separated from corn steep water at a low cost, which supports the value-added utilization of corn steep water. However, the racemic characteristic of lactate from corn steep water restricts the development of this promising process. In this study, using D-lactate oxidase (D-LOX) from Gluconobacter oxydans, L-lactate oxidase (L-LOX) from Pediococcus sp., pyruvate decarboxylase from Zymomonas mobilis, and catalase from bovine liver, we synthesized an in vitro enzymatic system, including different enzymatic cascades, for the production of valuable platform chemicals from lactate separated from the corn steep water. Pyruvate was produced as an important intermediate and further converted into C2 (acetaldehyde) and C4 (acetoin) platform chemicals at high yields using optimized concentrations of pyruvate decarboxylase. The in vitro enzymatic system not only provides a novel technology platform for the production of optical lactate and lactate derivatives but also supports the sustainable development of corn starch industry.

An NAD-dependent d-lactate dehydrogenase (d-nLDH) of Lactobacillus bulgaricus ATCC 11842 was rationally re-designed for asymmetric reduction of a homologous series of α-keto carboxylic acids such as phenylpyruvic acid (PPA), α-ketobutyric acid, α-ketovaleric acid, β-hydroxypyruvate. Compared with wild-type d-nLDH, the Y52L mutant d-nLDH showed elevated activities toward unnatural substrates especially with large substitutes at C-3. By the biocatalysis combined with a formate dehydrogenase for in situ generation of NADH, the corresponding (R)-α-hydroxy carboxylic acids could be produced at high yields and highly optical purities. Taking the production of chiral (R)-phenyllactic acid (PLA) from PPA for example, 50 mM PPA was completely reduced to (R)-PLA in 90 min with a high yield of 99.0% and a highly optical purity (>99.9% e.e.) by the coupling system. The results presented in this work suggest a promising alternative for the production of chiral α-hydroxy carboxylic acids.

It is advantageous for rhizosphere-dwelling microorganisms to utilize organic acids such as lactate. Pseudomonas putida KT2440 is one of the most widely studied rhizosphere-dwelling model organisms. The P. putida KT2440 genome contains an NAD-dependent d-lactate dehydrogenase encoding gene, but mutation of this gene does not play a role in d-lactate utilization. Instead, it was found that d-lactate utilization in P. putida KT2440 proceeds via a multidomain NAD-independent d-lactate dehydrogenase with a C-terminal domain containing several Fe-S cluster-binding motifs (Fe-S d-iLDH) and glycolate oxidase, which is widely distributed in various microorganisms. Both Fe-S d-iLDH and glycolate oxidase were identified to be membrane-bound proteins. Neither Fe-S d-iLDH nor glycolate oxidase is constitutively expressed but both of them can be induced by either enantiomer of lactate in P. putida KT2440. This study shows a case in which an environmental microbe contains two types of enzymes specific for d-lactate utilization.

Oxidase‐catalyzed kinetic resolution is important for the production of enantiopure 2‐hydroxycarboxylic acids (2‐HAs), which are versatile building blocks for the synthesis of many significant compounds. However, in contrast to that of (R)‐2‐HAs, the production of (S)‐2‐HA is challenging because of the lack of related oxidases. Herein, suitable enzymes were screened systematically through the analysis of numerous putative d‐lactate oxidase sequences and identification of several required properties. Finally, a d‐lactate oxidase from Gluconobacter oxydans 621H with advantageous characteristics, such as good solubility, broad substrate spectrum, and high stereoselectivity, was selected to resolve 2‐HAs into (S)‐2‐HAs. A variety of (S)‐2‐HAs was produced successfully using this d‐lactate oxidase with excellent enantiomeric excess values (>99 %). The presented screening criteria and approach for target biocatalysis suggested a guideline for the production of optically active chemicals such as (S)‐2‐HAs.