From Natural Methylation to Versatile Alkylations Using Halide Methyltransferases

Halide methyltransferases (HMTs) enable the enzymatic synthesis of S-adenosyl-l -methionine (SAM) from S-adenosyl-l -homocysteine (SAH) and methyl iodide. Characterisation of a range of naturally occurring HMTs and subsequent protein engineering led to HMT variants capable of synthesising ethyl, propyl, and allyl analogues of SAM. Notably, HMTs do not depend on chemical synthesis of methionine analogues, as required by methionine adenosyltransferases (MATs). However, at the moment MATs have a much broader substrate scope than the HMTs. Herein we provide an overview of the discovery and engineering of promiscuous HMTs and how these strategies will pave the way towards a toolbox of HMT variants for versatile chemo- and regioselective biocatalytic alkylations.     

Synthesis of 2‐monoglycerides by alcoholysis of palm oil and tuna oil using immobilized lipases

Commercial immobilized lipases were used for the synthesis of 2‐monoglycerides (2‐MG) by alcoholysis of palm and tuna oils with ethanol in organic solvents. Several parameters were studied, i.e., the type of immobilized lipases, water activity, type of solvents and temperatures. The optimum conditions for alcoholysis of tuna oil were at a water activity of 0.43 and a temperature of 60 °C in methyl‐tert‐butyl ether for ～12 h. Although immobilized lipase preparations from Pseudomonas sp. and Candida antarctica fraction B are not 1, 3‐regiospecific enzymes, they were considered to be more suitable for the production of 2‐MG by the alcoholysis of tuna oil than the 1, 3‐regiospecific lipases (Lipozyme RM IM from Rhizomucor miehei and lipase D from Rhizopus delemar). With Pseudomonas sp. lipase a yield of up to 81% 2‐MG containing 80% PUFA (poly‐unsaturated fatty acids) from tuna oil was achieved. The optimum conditions for alcoholysis of palm oil were similar as these of tuna oil alcoholysis. However, lipase D immobilized on Accurel EP100 was used as catalyst at 40 °C with shorter reaction times (<12 h). This lead to a yield of ～60% 2‐MG containing 55.0‐55.7% oleic acid and 18.7‐21.0% linoleic acid.     

RollingStream OSS及NMS实现IPTV运营级管理平台

网络电视（IPTV）是目前业界最热门的话题,作为数字电视的多元化表现形式,IPTV也肩负着对传统电视收看方式的变革推动.本文以哈尔滨IPTV商用业务的市场发展情况作为引导,突出说明一个稳定的、可商用IPTV平台需要有强大的系统运营支撑平台及网络管理平台的支持,同时表明RollingStream IPTV商用系统的可维护性及可管理性.     

Two-step enzymatic reaction for the synthesis of pure structured triacylglycerides

Structured triacylglycerides with medium-chain fatty acids (caprylic acid) in sn1- and sn3-positions and a long-chain unsaturated fatty acid (oleic or linoleic acid) in the sn2-position of glycerol (MLM) were synthesized by lipase catalysis in a two-step process. First, pure 2-monoacylglycerides (2-MG) were synthesized by alcoholysis of triacylglycerides (triolein, trilinolein, or peanut oil) in organic solvents with 1,3-regiospecific lipases (from Rhizomucor miehei, Rhizopus delemar, and Rhizopus javanicus). The 2-MG were purified by crystallization and obtained in up to 71.8% yield. These 2-MG were esterified in a second reaction with caprylic acid in n-hexane to form almost pure MLM. For 2-MG obtained from peanut oil, the final product contained more than 90% caprylic acid in the sn1- and sn3-positions, whereas the sn2-position was composed of 98.5% unsaturated long-chain fatty acids. Reaction conditions for both steps were optimized with respect to source and immobilization of lipase, water activity, and solvent.     

Conversion of a Mono‐ and Diacylglycerol Lipase into a Triacylglycerol Lipase by Protein Engineering

Despite the fact that most lipases are believed to be active against triacylglycerides, there is a small group of lipases that are active only on mono‐ and diacylglycerides. The reason for this difference in substrate scope is not clear. We tried to identify the reasons for this in the lipase from Malassezia globosa. By protein engineering, and with only one mutation, we managed to convert this enzyme into a typical triacylglycerol lipase (the wild‐type lipase does not accept triacylglycerides). The variant Q282L accepts a broad spectrum of triacylglycerides, although the catalytic behavior is altered to some extent. From in silico analysis it seems that specific hydrophobic interactions are key to the altered substrate specificity.     

Suppression of Water as a Nucleophile in Candida antarctica Lipase B Catalysis

A water tunnel in Candida antarctica lipase B that provides the active site with substrate water is hypothesized. A small, focused library created in order to prevent water from entering the active site through the tunnel was screened for increased transacylation over hydrolysis activity. A single mutant, S47L, in which the inner part of the tunnel was blocked, catalysed the transacylation of vinyl butyrate to 20 mM butanol 14 times faster than hydrolysis. The single mutant Q46A, which has a more open outer end of the tunnel, showed an increased hydrolysis rate and a decreased hydrolysis to transacylation ratio compared to the wild‐type lipase. Mutants with a blocked tunnel could be very useful in applications in which hydrolysis is unwanted, such as the acylation of highly hydrophilic compounds in the presence of water.     

An improved assay for the determination of phospholipase C activity

Phospholipases C (PLC, EC 3.1.4.3) are enzymes that specifically hydrolyze the C‐O‐P bond in phospholipids, yielding sn‐1,2(2,3)‐diacylglycerides and the phosphate residue bearing the corresponding headgroup. The biochemical characterization of PLC requires methods for the reliable determination of their activity. Here, an assay is described in which the phosphate residue released by the PLC is cleaved with an alkaline phosphatase. The phosphate formed is then extracted with n‐butanol and quantified as phosphomolybate complex. The applicability of this method is demonstrated for a concentration range from 10 nM to 10 mM for a range of phospholipids bearing different headgroups in an aqueous and a two‐phase system. The method has the additional advantage that the crude enzyme can be used without the need for purification.     

Microbial Synthesis of Medium‐Chain α,ω‐Dicarboxylic Acids and ω‐Aminocarboxylic Acids from Renewable Long‐Chain Fatty Acids

Biotransformation of long‐chain fatty acids into medium‐chain α,ω‐dicarboxylic acids or ω‐aminocarboxylic acids could be achieved with biocatalysts. This study presents the production of α,ω‐dicarboxylic acids (e.g., C₉, C₁₁, C₁₂, C₁₃) and ω‐aminocarboxylic acids (e.g., C₁₁, C₁₂, C₁₃) directly from fatty acids (e.g., oleic acid, ricinoleic acid, lesquerolic acid) using recombinant Escherichia coli‐based biocatalysts. ω‐Hydroxycarboxylic acids, which were produced from oxidative cleavage of fatty acids via enzymatic reactions involving a fatty acid double bond hydratase, an alcohol dehydrogenase, a Baeyer–Villiger monooxygenase and an esterase, were then oxidized to α,ω‐dicarboxylic acids by alcohol dehydrogenase (ADH, AlkJ) from Pseudomonas putida GPo1 or converted into ω‐aminocarboxylic acids by a serial combination of ADH from P. putida GPo1 and an ω‐transaminase of Silicibacter pomeroyi. The double bonds present in the fatty acids such as ricinoleic acid and lesquerolic acid were reduced by E. coli‐native enzymes during the biotransformations. This study demonstrates that the industrially relevant building blocks (C₉ to C₁₃ saturated α,ω‐dicarboxylic acids and ω‐aminocarboxylic acids) can be produced from renewable fatty acids using biocatalysis.