Altering the substrate specificity of Candida rugosa LIP4 by engineering the substrate-binding sites

Candida rugosa (formerly Candida cylindracea) lipase (CRL) is an important industrial enzyme that is widely used in biotechnological applications such as the production of fatty acids and the synthesis of various esters. CRL comprises at least seven isozymes (LIP1-LIP7), which share a similar amino acid sequence but with different specificities for substrates. Previously, LIP4 was reported to have higher esterase activity toward long acyl-chain ester and lower lipase activity toward triglycerides. A296 and V344 of LIP4 were predicted to play decisive roles in its substrate specificity. In this study, site-specific saturation mutagenesis has been employed to study the substrate specificity of LIP4. Point mutations were separately introduced into A296 and V344 positions using degenerate primer sets containing 32 codons to generate two libraries of variants. LIP4 variants were heterologously expressed in the yeast Pichia pastoris. A specific plate assay was used to identify lipase-producing P. pastoris clones in a medium containing tributyrin. LIP4 variants with high activity toward short fatty acyl-chain triglyceride (tributyrin) were screened. Specificity analysis and biochemical characterization indicated that the recombinant variants A296I, V344Q, and V344H had properties remarkably different from those of wild-type LIP4. All three variant enzymes had significantly higher specific activities toward tributyrin than LIP4. In addition to short-chain triglyceride, A296I and V344Q also improved hydrolytic activities of triglycerides toward medium- and long-chain triglycerides tested. The results suggested that A296 played an important role in lipase activity and high-temperature dependence of LIP4, whereas it had no effect on the chain-length specificity in lipolytic reaction. The V344 residue had a significant effect on the substrate chain-length specificity of LIP4.     

Synthesis and characterization of canola oil-stearic acid-based trans-free structured lipids for possible margarine application

Incorporation of stearic acid into canola oil to produce trans-free structured lipid (SL) as a healthy alternative to partially hydrogenated fats for margarine formulation was investigated. Response surface methodology was used to study the effects of lipozyme RM IM from Rhizomucor miehei and Candida rugosa lipase isoform 1 (LIP1) and two acyl donors, stearic acid and ethyl stearate, on the incorporation. Lipozyme RM IM and ethyl stearate gave the best result. Gram quantities of SLs were synthesized using lipozyme RM IM, and the products were compared to SL made by chemical catalysis and fat from commercial margarines. After short-path distillation, the products were characterized by GC and RPHPLC-MS to obtain fatty acid and triacylglycerol profiles, 13C NMR spectrometry for regiospecific analysis, X-ray diffraction for crystal forms, and DSC for melting profile. Stearic acid was incorporated into canola oil, mainly at the sn-1,3 positions, for the lipase reaction, and no new trans fatty acids formed. Most SL products did not have adequate solid fat content or beta' crystal forms for tub margarine, although these may be suitable for light margarine formulation.     

Use of Fourier‐Transform Infrared Spectroscopy to Quantify Immunoglobulin G Concentrations in Alpaca Serum

Background

Rapid, economical, and quantitative assays for measurement of camelid serum immunoglobulin G (IgG) are limited. In camelids, failure of transfer of maternal immunoglobulins has a reported prevalence of up to 20.5%. An accurate method for quantifying serum IgG concentrations is required.

Objective

To develop an infrared spectroscopy‐based assay for measurement of alpaca serum IgG and compare its performance to the reference standard radial immunodiffusion (RID) assay.

Animals

One hundred and seventy‐five privately owned, healthy alpacas.

Methods

Eighty‐two serum samples were collected as convenience samples during routine herd visits whereas 93 samples were recruited from a separate study. Serum IgG concentrations were determined by RID assays and midinfrared spectra were collected for each sample. Fifty samples were set aside as the test set and the remaining 125 training samples were employed to build a calibration model using partial least squares (PLS) regression with Monte Carlo cross validation to determine the optimum number of PLS factors. The predictive performance of the calibration model was evaluated by the test set.

Results

Correlation coefficients for the IR‐based assay were 0.93 and 0.87, respectively, for the entire data set and test set. Sensitivity in the diagnosis of failure of transfer of passive immunity (FTPI) ([IgG] <1,000 mg/dL) was 71.4% and specificity was 100% for the IR‐based method (test set) as gauged relative to the RID reference method assay.

Conclusions and Clinical Importance

This study indicated that infrared spectroscopy, in combination with chemometrics, is an effective method for measurement of IgG in alpaca serum.     

THE SIMULATION OF ANIMAL RETURNS IN GRASSLAND EXPERIMENTATION

An experiment was carried out on a grass/white-clover sward at North Wyke to evaluate the use of mixtures of inorganic and organic nutrient sources to simulate the nutrient effect of animal excreta, both with and without overall PK fertilizer. Grazing with sheep and the return of excreta from caged sheep both increased herbage yields, as did artificial return treatments. Artificial return treatments giving 75% of the nutrient level returned by excreta gave yields comparable with grazing. The effect of the simulated return treatments was not influenced by PK application. Grazing gave a very different botanical composition to that from the treatments in wbich natural or simulated excreta were returned to cut swards. Mixtures simulating excreta may replace the nutrient effect of the grazing animal, but not its physical effect, in some forms of grassland experimentation. They are not recommended for fertilizer trials.     

Hypoadrenocorticism in young related Nova Scotia duck tolling retrievers.

Five cases of hypoadrenocorticism in young related Nova Scotia duck tolling retrievers are discussed. Two were littermates and the others shared one or more common ancestors. No specific mode of inheritance was determined. Clinical and laboratory findings typical of hypoadrenocorticism were observed. Further documentation will be required to confirm a breed predisposition.     

Serological response in broiler chicks to different commercial Newcastle disease and infectious bronchitis vaccines.

Broiler chicks were administered vaccines against Newcastle disease and infectious bronchitis (both Arkansas and Massachusetts strains) at 2 weeks of age as either primary or secondary vaccinations. The vaccine was administered as a spray at 2 weeks of age to chicks that had received Newcastle disease vaccine alone, bronchitis vaccine alone, both vaccines in combination, or no vaccine at day 1 in the hatchery. The Newcastle disease hemagglutination-inhibition response was significantly lower in chicks receiving Newcastle disease vaccine as a secondary vaccine at 2 weeks than in those receiving the vaccine as a primary vaccination at that age. In contrast, the bronchitis hemagglutination-inhibition response was significantly higher in chicks receiving bronchitis vaccine as a secondary vaccination at 2 weeks than in those receiving the vaccine as a primary vaccination at that age.