Rational regulation of reaction specificity of nitrilase for efficient biosynthesis of 2-chloronicotinic acid through a single site mutation

Author(s):  
An-Di Dai ◽  
Xiao-Ling Tang ◽  
Zhe-Ming Wu ◽  
Jiang-Tao Tang ◽  
Ren-Chao Zheng ◽  
...  

Nitrilase-catalyzed hydrolysis of 2-chloronicotinonitrile (2-CN) is a promising approach for efficient synthesis of 2-chloronicotinic acid (2-CA). Development of nitrilase with ideal catalytic properties is crucial for the biosynthetic route with industrial potentail. Herein, a nitrilase from Rhodococcus zopfii ( Rz NIT), which showed much higher hydration activity than hydrolysis activity, was designed for efficient hydrolysis of 2-CN. Two residues (N165 and W167) significantly affecting the reaction specificity were precisely identified. By tuning these two residues, a single mutation of W167G with abolished hydration activity and 20-fold improved hydrolysis activity was obtained. Molecular dynamics simulation and molecular docking revealed that the mutation generated a larger binding pocket, causing the substrate 2-CN bound more deeply in the pocket and the formation of delocalized π bond between the residues W190 and Y196, which reduced the negative influence of steric hindrance and electron effect caused by chlorine substituent. With mutant W167G as biocatalyst, 100 mM 2-CN was exclusively converted into 2-CA within 16 h. The study provides useful guidance in nitrilase engineering for simultaneous improvement of reaction specificity and catalytic activity, which are highly desirable in value-added carboxylic acids production from nitriles hydrolysis. Importance 2-CA is an important building block for agrochemicals and pharmaceuticals with rapid increase in demand in recent years. It is currently manufactured from 3-cyanopyridine by chemical methods. However, during the final step of 2-CN hydrolysis under high temperature and strong alkaline conditions, by-product 2-CM was generated except for the target product, leading to low yield and tedious separation steps. Nitrilase-mediated hydrolysis is regarded as a promising alternative for 2-CA production, which proceeds under mild conditions. Nevertheless, nitrilase capable of efficient hydrolysis of 2-CN was not reported till now, since the enzymes showed either extremely low activity or surprisingly high hydration activity towards 2-CN. Herein, the reaction specificity of Rz NIT was precisely tuned through a single site mutation. The mutant exhibited remarkably enhanced hydrolysis activity without formation of by-products, providing a robust biocatalyst for 2-CA biosynthesis with industrial potential.

2020 ◽  
Vol 33 (1) ◽  
Author(s):  
Jie Gao ◽  
Mi Xiao ◽  
Yan Zhang ◽  
Liang Gao

AbstractTopology Optimization (TO) is a powerful numerical technique to determine the optimal material layout in a design domain, which has accepted considerable developments in recent years. The classic Finite Element Method (FEM) is applied to compute the unknown structural responses in TO. However, several numerical deficiencies of the FEM significantly influence the effectiveness and efficiency of TO. In order to eliminate the negative influence of the FEM on TO, IsoGeometric Analysis (IGA) has become a promising alternative due to its unique feature that the Computer-Aided Design (CAD) model and Computer-Aided Engineering (CAE) model can be unified into a same mathematical model. In the paper, the main intention is to provide a comprehensive overview for the developments of Isogeometric Topology Optimization (ITO) in methods and applications. Finally, some prospects for the developments of ITO in the future are also presented.


Catalysts ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1343
Author(s):  
Mpho S. Mafa ◽  
Brett I. Pletschke ◽  
Samkelo Malgas

Lignocellulose has economic potential as a bio-resource for the production of value-added products (VAPs) and biofuels. The commercialization of biofuels and VAPs requires efficient enzyme cocktail activities that can lower their costs. However, the basis of the synergism between enzymes that compose cellulolytic enzyme cocktails for depolymerizing lignocellulose is not understood. This review aims to address the degree of synergism (DS) thresholds between the cellulolytic enzymes and how this can be used in the formulation of effective cellulolytic enzyme cocktails. DS is a powerful tool that distinguishes between enzymes’ synergism and anti-synergism during the hydrolysis of biomass. It has been established that cellulases, or cellulases and lytic polysaccharide monooxygenases (LPMOs), always synergize during cellulose hydrolysis. However, recent evidence suggests that this is not always the case, as synergism depends on the specific mechanism of action of each enzyme in the combination. Additionally, expansins, nonenzymatic proteins responsible for loosening cell wall fibers, seem to also synergize with cellulases during biomass depolymerization. This review highlighted the following four key factors linked to DS: (1) a DS threshold at which the enzymes synergize and produce a higher product yield than their theoretical sum, (2) a DS threshold at which the enzymes display synergism, but not a higher product yield, (3) a DS threshold at which enzymes do not synergize, and (4) a DS threshold that displays anti-synergy. This review deconvolutes the DS concept for cellulolytic enzymes, to postulate an experimental design approach for achieving higher synergism and cellulose conversion yields.


2019 ◽  
Author(s):  
YunFeng Zhang ◽  
Qixing Liang ◽  
Chuanzhi Zhang ◽  
Juan Zhang ◽  
Guocheng Du ◽  
...  

Abstract Background: Trypsin has a plenty application in food and pharmaceutical manufacture. While, the commercial trypsin is usually extracted from pork pancreas, which has the risk of infectious and immunogenicity. Therefore, the microbial Streptomyces griseus trypsin (SGT) is a prior alternation because it processes efficient hydrolysis activity without the aforementioned risk. The remarkable hydrolysis efficiency of SGT caused its autolysis, and five autolysis sites R21, R32, K122, R153, and R201 were identified from its' autolysate. Results: The tbcf (K101A, R201V) mutant was screened by directed selection approach for improved activity in flask culture (60.85 ± 3.42 U·mL -1 , increased 1.5-fold). From the molecular dynamics simulation, the K101A/R201V mutation shortened the distant between catalytical residues D102 and H57 from 6.5 Å vs 7.0 Å, which afforded the improved specific activity 1527.96 ± 62.81 U·mg -1 . Further, the production of trypsin was increased 302.8% (689.47 ± 6.78 U·mL −1 ) in 3-L bio-reactor, with co-overexpression of chaperones SSO2 and UBC1 in Pichia pastoris. Conclusions: The SGT protein could be an adequate trypsin for insulin production. When working with hydrolysates analysis and direction selection, the tbcf (K101A, R201V) mutant increased 1.5-fold activity. Further, the production of trypsin was improved 3-fold by overexpressing chaperone protein in Pichia pastoris . The future study should be emphasized on the application of SGT in insulin manufacture and pharmaceutical.


2022 ◽  
Vol 9 (1) ◽  
pp. 17
Author(s):  
Malinee Sriariyanun ◽  
Nichaphat Kitiborwornkul ◽  
Prapakorn Tantayotai ◽  
Kittipong Rattanaporn ◽  
Pau-Loke Show

Ionic liquid (IL) pretreatment of lignocellulose is an efficient method for the enhancement of enzymatic saccharification. However, the remaining residues of ILs deactivate cellulase, therefore making intensive biomass washing after pretreatment necessary. This study aimed to develop the one-pot process combining IL pretreatment and enzymatic saccharification by using low-toxic choline acetate ([Ch][OAc]) and IL-tolerant bacterial cellulases. Crude cellulases produced from saline soil inhabited Bacillus sp. CBD2 and Brevibacillus sp. CBD3 were tested under the influence of 0.5–2.0 M [Ch][OAc], which showed that their activities retained at more than 95%. However, [Ch][OAc] had toxicity to CBD2 and CBD3 cultures, in which only 32.85% and 12.88% were alive at 0.5 M [Ch][OAc]. Based on the specific enzyme activities, the sugar amounts produced from one-pot processes using 1 mg of CBD2 and CBD3 were higher than that of Celluclast 1.5 L by 2.0 and 4.5 times, respectively, suggesting their potential for further application in the biorefining process of value-added products.


2019 ◽  
Vol 15 (2) ◽  
pp. 200-205
Author(s):  
Maryam Husin ◽  
Nurnadiah Rahim ◽  
Mohd Radzi Ahmad ◽  
Ahmad Zafir Romli ◽  
Zul Ilham

The waste seeds of Leucaena leucocephala (LLS) used in this study were unused residues obtained after oil and polysaccharides extraction. The microcrystalline cellulose (MCC) was isolated from LLS by acid treatment. MCC produced was, then, further converted to glucose by using sulphuric acid at 121 °C by varying the acid concentration and reaction time. The sugar composition was analyzed by using the phenol-sulfuric acid method and pre-column derivatization HPLC technique. The yield of glucose ranging from 70–85% could be obtained from MCC hydrolyzates, depending on the hydrolysis factors, which corresponding to around 57-75% of the percentage conversion of MCC to glucose.Cellulose isolated from LLS was, therefore, potentially suitable to be utilized in liquid biofuels and other value-added chemicals such as bioethanol, 5-hydroxymethylfurfural(HMF), and levulinic acid.


2004 ◽  
Vol 10 (S02) ◽  
pp. 1498-1499 ◽  
Author(s):  
Derek L Beahm ◽  
Guido Gaietta ◽  
Anjana Chandrasekhar ◽  
Galen M Hand ◽  
Amy Smock ◽  
...  

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.


Author(s):  
Yuxi Song ◽  
Kaiyue Zhang ◽  
Xiangrong Li ◽  
Chuanwei Yan ◽  
Qinghua Liu ◽  
...  

Aqueous all-iron flow battery is a promising alternative for large-scale energy storage applications due to low cost and high safety. However, inferior Fe plating/stripping reversibility and hydrolysis of Fe2+ at...


2017 ◽  
Vol 542 ◽  
pp. 1-9 ◽  
Author(s):  
Guiyu Zhang ◽  
Yeqian Wen ◽  
Zhihua Liu ◽  
Songmei Zhang ◽  
Gang Li

Sign in / Sign up

Export Citation Format

Share Document