A novel enzymatic method for synthesis of glycopeptides carrying natural eukaryotic N-glycans

2017 ◽  
Vol 53 (65) ◽  
pp. 9075-9077 ◽  
Author(s):  
Yangyang Xu ◽  
Zhigang Wu ◽  
Peiru Zhang ◽  
He Zhu ◽  
Hailiang Zhu ◽  
...  
Keyword(s):  
Enzymatic Reactions ◽  
Enzymatic Method ◽  
Complex Type

Production of glycopeptides carrying complex-type N-glycans via three steps of enzymatic reactions.

scholarly journals A library of structurally homogeneous human N-glycans synthesized from microbial oligosaccharide precursors

2017 ◽  
Author(s):  
Brian S. Hamilton ◽  
Joshua D. Wilson ◽  
Marina A. Shumakovich ◽  
Adam C. Fisher ◽  
James C. Brooks ◽  
...  
Keyword(s):  
Enzymatic Method ◽  
Molecular Tools ◽  
Wild Type ◽  
Complex Type ◽  
Purification Scheme ◽  
Major Bottleneck ◽  
Health And Disease ◽  
Glycosylation Pathway ◽  
User Friendly ◽  
Oligosaccharide Structures

AbstractSynthesis of homogenous glycans in quantitative yields represents a major bottleneck to the production of molecular tools for glycoscience, such as glycan microarrays, affinity resins, and reference standards. Here, we describe a combined biological/enzymatic method termed bioenzymatic synthesis that is capable of efficiently converting microbially-derived precursor oligosaccharides into structurally uniform human-type N-glycans. Unlike starting material obtained by chemical synthesis or direct isolation from natural sources, which can be time consuming and costly to generate, bioenzymatic synthesis involves precursors derived from renewable sources including wild-type Saccharomyces cerevisiae glycoproteins and lipid-linked oligosaccharides from glycoengineered Escherichia coli. Following deglycosylation of these biosynthetic precursors, the resulting microbial oligosaccharides are subjected to a greatly simplified purification scheme followed by structural remodeling using commercially available and recombinantly produced glycosyltransferases including key N- acetylglucosaminyltransferases (e.g., GnTI, GnTII, and GnTIV) involved in early remodeling of glycans in the mammalian glycosylation pathway. Using this approach, preparative quantities of hybrid and complex-type N-glycans including asymmetric multi-antennary structures were generated all without the need of a specialized skillset. Collectively, our results reveal bioenzymatic synthesis to be a user-friendly methodology for rapidly supplying homogeneous oligosaccharide structures that can be used to understand the human glycome and probe the biological roles of glycans in health and disease.

Development of 96 Multiple Injection-GC-MS Technique and Its Application in Protein Engineering of Natural and Non-Natural Enzymatic Reactions

2019 ◽  
Author(s):  
Anja Knorrscheidt ◽  
Pascal Püllmann ◽  
Eugen Schell ◽  
Dominik Homann ◽  
Erik Freier ◽  
...  
Keyword(s):  
Microtiter Plate ◽  
Axial Ligand ◽  
Cell System ◽  
Enzymatic Reactions ◽  
The Novel ◽  
Internal Standards ◽  
E Coli ◽  
Enzyme Screening ◽  
Plate Analysis ◽  
Microtiter Plate Analysis

Directed evolution requires the screening of enzyme libraries in biological matrices. Available assays are mostly substrate or enzyme specific. Chromatographic techniques like LC and GC overcome this limitation, but require long analysis times. The herein developed multiple injections in a single experimental run (MISER) using GC coupled to MS allows the injection of samples every 33 s resulting in 96-well microtiter plate analysis within 50 min. This technique is implementable in any GC-MS system with autosampling. Since the GC-MS is far less prone to ion suppression than LCMS, no chromatographic separation is required. This allows the utilisation of an internal standards and the detection of main and side-product. To prove the feasibility of the system in enzyme screening, two libraries were assessed: i) YfeX library in an E. coli whole cell system for the carbene-transfer reaction on indole revealing the novel axial ligand tryptophan, ii) a library of 616 chimeras of fungal unspecific peroxygenase (UPO) in S. cerevisiae supernatant for hydroxylation of tetralin resulting in novel constructs. The data quality and representation are automatically assessed by a new R-script.

Perbandingan Karakteristik Surfaktan Metil Ester Sulfonat dan Sodium Lauril Sulfonat sebagai Bahan Emulsifier

2016 ◽  
Vol 9 (2) ◽  
pp. 167-176
Author(s):  
Eldha Sampepana ◽  
Paluphy Eka Yustini ◽  
Aditya Rinaldi ◽  
Amiroh Amiroh
Keyword(s):  
Fatty Acids ◽  
Surface Tension ◽  
Methyl Ester ◽  
Interfacial Tension ◽  
Palm Oil ◽  
Emulsion Stability ◽  
Raw Material ◽  
Enzymatic Method

Surfactant which is used as raw emulsifier in an industry activity such as Sodium Lauryl Sulfonate is a raw material import, it is petroleum derivative which is not renewable and may cause pollution to the environment, because it is not degraded and are carcinogenic. The purpose of the research is to compare the characteristics of the Quaternary methyl ester sulfonat (MES) and Sodium Lauryl Sulfonat (SLS) as emulsifier. First, make the MES by filtering and eliminating fatty acids of palm oil, then process the MES with enzymatic method become methyl ester, then react it in sulfonation and metanolization process, and also neutralized with NaOH. Next, the MES experiment is compared with SLS and existing MES in the market. The results show that surfactants MES experiment has value hidrofil lipofil balance (HLB) interfacial tension and emulsion stability greater than MES in the market and SLS. And the surface tension of MES experiment is larger than MES in the market, but smaller compared to SLS.ABSTRAKSurfaktan yang digunakan sebagai bahan baku emulsifer dalam aktivitas suatu industri pada saat ini seperti Sodium Lauril Sulfonat  merupakan bahan baku import yang merupakan turunan dari minyak bumi, dengan sifat tidak dapat diperbaharui dan dapat menimbulkan pencemaran terhadap lingkungan karena tidak mudah terdegradasi serta bersifat karsinogenik. Metil ester sulfonat dari bahan minyak sawit merupakan surfaktan dengan sifat mudah terdegradasi yang perlu diketahui karakteristiknya. Penelitian bertujuan untuk membandingkan karakteristik surfaktan metil ester sulfonat (MES) dan Sodium Lauril Sulfonat (SLS) sebagai bahan emulsifier. Mula-mula dilakukan pembuatan MES dengan cara menyaring dan menghilangkan asam lemak minyak sawit terlebih dahulu, kemudian diolah menjadi metil ester secara enzimatis, lalu direaksikan secara sulfonasi dan metanolisis, serta dinetralkan dengan NaOH. Selanjutnya MES hasil percobaan dibandingkan dengan SLS dan MES yang ada dipasaran. Hasil penelitian menunjukkan bahwa surfaktan MES memiliki nilai hidrofil lipofil balance (HLB) tegangan antar muka dan stabilitas emulsi lebih besar apabila dibandingkan dengan MES di pasaran dan SLS, kecuali nilai stabilitas emulsi antara MES dan SLS sama. Dan tegangan permukaan MES hasil percobaan, lebih besar dibandingkan dengan MES dipasaran, dan lebih kecil dibandingkan dengan SLS. Kata kunci :   Metil  ester sulfonat, hidrofil lipofil balance, emulsifier, sodium lauril sulfonat , stabilitas emulsi 

Modular Simulation to Determine the Optimal Operating Policy of a Batch Reactor for the Enzymatic Fructose Reduction to Mannitol with the in situ Continuous Enzymatic Regeneration of the NAD Cofactor

2017 ◽  
Vol 68 (9) ◽  
pp. 2196-2203 ◽  
Author(s):  
Mara Crisan ◽  
Gheorghe Maria
Keyword(s):  
Batch Reactor ◽  
Production Capacity ◽  
Optimization Procedure ◽  
Enzymatic Reactions ◽  
Mannitol Dehydrogenase ◽  
Optimal Operating ◽  
Operating Policy ◽  
Enzymatic Reduction ◽  
Optimal Operating Policy

Novel coupled enzymatic systems reported important applications in the industrial bio-catalysis. Multi-enzymatic reactions can successfully replace complex chemical syntheses, using milder reaction conditions, and generating less waste. For such systems acting simultaneously, the model-based engineering calculations (design, reactor operation optimization) are difficult tasks, because they must account for interacting reactions, differences in enzymes optimal activity domains and deactivation kinetics. The determination of the optimal operating mode (enzyme ratios, enzyme feeding policy, temperature, pH) often turns into a difficult multi-objective optimization problem with multiple constraints to be solved for every particular system. The paper focuses on applying a modular screening procedure that can identify the optimal operating policy of an enzymatic reactor, which minimizes the enzyme consumption, given the process kinetic model, and an imposed production capacity. Following an optimization procedure, the process effectiveness is evaluated in a systematic approach, by including simple batch reactor (BR), batch with intermittent addition of the key-enzyme following certain optimal policies (BRP). Exemplification is made for the case of the enzymatic reduction of D-fructose to mannitol by using suspended MDH (mannitol dehydrogenase) and NADH (Nicotinamide adenine dinucleotide) cofactor, with the in-situ continuous regeneration of the cofactor by the expense of formate degradation in the presence of suspended FDH (Formate dehydrogenase).

Synthesis of Rare Pentoses Using Microbial and Enzymatic Reactions

2016 ◽  
Vol 20 (14) ◽  
pp. 1456-1464 ◽  
Author(s):  
Zijie Li ◽  
Xiao-Dong Gao ◽  
Li Cai
Keyword(s):  
Enzymatic Reactions

Enzymatic-Ultraviolet Method for Measuring Lactose in Milk: Collaborative Study

1984 ◽  
Vol 67 (3) ◽  
pp. 637-640 ◽  
Author(s):  
David O Biltcliffe ◽  
Dick H Kleyn ◽  
J Richard Trout ◽  
◽  
D Azzara ◽  
...  
Keyword(s):  
Food Industry ◽  
Collaborative Study ◽  
Skim Milk ◽  
Gravimetric Method ◽  
T Test ◽  
Enzymatic Method ◽  
Commercial Laboratory ◽  
Aoac Method ◽  
Standard Deviations

Abstract Collaborators in 8 dairy and food industry laboratories performed one lactose determination on each of 8 unknown samples of milk, lowfat milk, or skim milk, as 3 pairs of blind duplicates. Two known samples were provided to gain experience prior to analysis of the unknown samples. All of the above samples were also analyzed for lactose content by the official AOAC gravimetric method (16.507) by a commercial laboratory. From the overall mean of results on all samples, determinations by the enzymatic method averaged 0.49% lower than by the AOAC method. This difference was significant by the t-test (P = 0.05), which indicated a lack of agreement between the compared methods in determining lactose content. Standard deviations were similar for the 3 sets of blind duplicates which ranged between 3.67 and 4.55% lactose content. F-values revealed that variations between means obtained by laboratories differed significantly as compared with variations within laboratory means. The method has been adopted official first action.

Sign in / Sign up

Export Citation Format

Share Document