Thermostability enhancement and insight of L-asparaginase from Mycobacterium sp. via consensus-guided engineering

Chi, Huibing; Zhu, Xiaoyu; Shen, Juan; Lu, Zhaoxin; Lu, Fengxia; Lyu, Yunbin; Ping, Zhu

doi:10.1007/s00253-023-12443-1

Cited by 12 publications

(18 citation statements)

References 71 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We obtained the I191F/H279D double mutant, which shows an increased catalytic activity at neutral pH, increased thermodynamic stability and almost undetectable propensity to aggregation under physiological condition of ionic strength and pH. These effects are similar to those obtained on other enzymes using analogous strategies (24, 38–42). A comparative analysis of the effects of each single mutation indicates that both increase the melting temperature.…”

Section: Discussionsupporting

confidence: 78%

Engineered Oxalate decarboxylase boosts activity and stability for biological applications

Dindo,

Conter,

Uechi

et al. 2024

Preprint

View full text Add to dashboard Cite

Oxalate decarboxylase (OxDC) from Bacillus subtilis is a Mn-dependent hexameric enzyme which converts oxalate to carbon dioxide and formate. Recently, OxDC has attracted the interest of the scientific community, due to its biotechnological and medical applications for the treatment of hyperoxalurias, a group of pathologic conditions associated with excessive oxalate urinary excretion due to either increased endogenous production or increased exogenous absorption. The fact that OxDC displays optimum pH in the acidic range, represents a big limitation for most biotechnological applications involving processes occurring at neutral pH, where the activity and stability of the enzyme are remarkably reduced. Here, through bioinformatics-guided protein engineering, followed by combinatorial mutagenesis and analyses of activity and thermodynamic stability, we identified a double mutant of OxDC endowed with enhanced catalytic efficiency and stability under physiological conditions. The obtained engineered form of OxDC offers a potential tool for improved intestinal oxalate degradation in hyperoxaluria patients.

show abstract

Section: Discussionsupporting

confidence: 78%

Engineered Oxalate decarboxylase boosts activity and stability for biological applications

Dindo,

Conter,

Uechi

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

“…The existence of l -asparaginase was further confirmed by using l -asparaginase-specific antibody, and the enzymatic product from tomato has two subunits with molecular weights of 50 and 30 kDa respectively. The optimum conditions for l -asparaginase activity from tomato are shown to vary with other sources. − …”

Section: Resultsmentioning

confidence: 99%

l-Asparaginase from Solanum lycopersicum as a Nutraceutical for Acute Lymphoblastic Leukemia

Khabade,

Sirigiri,

Ram

2024

ACS Omega

View full text Add to dashboard Cite

L-Asparaginase (E.C. 3.5.1.1) is an indispensable analeptic anticancer enzyme used as an amalgam with additional cancer medicines for the cure of acute lymphoblastic leukemia (ALL). The presence of LAparaginase in tomato was confirmed byWestern blotting and DNA sequencing. The L-Asparaginase gene from tomato has been deposited in the NCBI database with accession number: OR736141. Crude enzyme was extracted from the fruit pulp of Solanum lycopersicum, and the activity was determined by the Nesslerization method. Further, the crude extract was subjected to purification, and kinetic parameters were studied. The percentage yield was calculated to be 6.457, and the purification fold was 0.086. The enzyme showed maximum activity at optimum pH 7.0, optimum temperature 37 °C, and incubation time of 05 min. The Michaelis constant "K m " and maximum velocity "V max " values were determined by the Lineweaver−Burk plot, which showed a low K m value of 0.66 and V max of 3.846 IU. Cytotoxic studies were carried out for crude and purified L-asparaginase. Purified L-Asparaginase has exhibited anticancer activity against the ALL model system, K-562 cell line, comparable to that of the anticancer compound vinblastine. Hence, L-Asparaginase from the fruit extract of tomato could be used as a nutraceutical to support cancer treatment in acute lymphoblastic leukemia.

show abstract

“…As scASNases are more similar to the type I than to type II asparaginases (Figure 2 ), it is likely that the lower substrate affinity is a feature that is shared among all members of this group of enzymes. Since problems caused by low substrate affinity were frequently mentioned when new type I ASNases were described (Ashok et al, 2019 ; Chi et al, 2023 ; Guo et al, 2017 ), this limitation is also present for scASNases. Although there have been no successful attempts to reduce the K m of typical type I ASNases that have been reported, such experiments have not been performed with scASNases.…”

Section: Discussionmentioning

confidence: 99%

RrA, an enzyme from Rhodospirillum rubrum, is a prototype of a new family of short‐chain L‐asparaginases

Zhang,

Czapinska,

Bochtler

et al. 2024

Protein Science

View full text Add to dashboard Cite

L‐Asparaginases (ASNases) catalyze the hydrolysis of L‐Asn to L‐Asp and ammonia. Members of the ASNase family are used as drugs in the treatment of leukemia, as well as in the food industry. The protomers of bacterial ASNases typically contain 300–400 amino acids (typical class 1 ASNases). In contrast, the chain of ASNase from Rhodospirillum rubrum, reported here and referred to as RrA, consists of only 172 amino acid residues. RrA is homologous to the N‐terminal domain of typical bacterial class 1 ASNases and exhibits millimolar affinity for L‐Asn. In this study, we demonstrate that RrA belongs to a unique family of cytoplasmic, short‐chain ASNases (scASNases). These proteins occupy a distinct region in the sequence space, separate from the regions typically assigned to class 1 ASNases. The scASNases are present in approximately 7% of eubacterial species, spanning diverse bacterial lineages. They seem to be significantly enriched in species that encode for more than one class 1 ASNase. Here, we report biochemical, biophysical, and structural properties of RrA, a member of scASNases family. Crystal structures of the wild‐type RrA, both with and without bound L‐Asp, as well as structures of several RrA mutants, reveal topologically unique tetramers. Moreover, the active site of one protomer is complemented by two residues (Tyr21 and Asn26) from another protomer. Upon closer inspection, these findings clearly outline scASNases as a stand‐alone subfamily of ASNases that can catalyze the hydrolysis of L‐Asn to L‐Asp despite the lack of the C‐terminal domain that is present in all ASNases described structurally to date.

show abstract

Thermostability enhancement and insight of L-asparaginase from Mycobacterium sp. via consensus-guided engineering

Cited by 12 publications

References 71 publications

Engineered Oxalate decarboxylase boosts activity and stability for biological applications

Engineered Oxalate decarboxylase boosts activity and stability for biological applications

l-Asparaginase from Solanum lycopersicum as a Nutraceutical for Acute Lymphoblastic Leukemia

RrA, an enzyme from Rhodospirillum rubrum, is a prototype of a new family of short‐chain L‐asparaginases

Contact Info

Product

Resources

About