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Building analytical test kits; molecular biology, the next step forward for megazyme

B.V. McCleary and A. M. Kennedy

Megazyme International Ireland Limited, Bray Business Park, Bray, County Wicklow, Ireland.

Introduction

Megazyme was established in North Rocks, Sydney in 1999. The specific aim of the company was to produce a range of test kits and reagents to assist in the measurement of enzymes and carbohydrates that affect the quality of plant products both industrially, and in the final food product. Initially, products included a range of polysaccharides purified from plant materials for use as substrates in specific enzyme assays, and highly purified enzymes for use in research and analytical applications. Particular effort was directed towards the development of simple-to-use test kits for measurement of enzymes and polysaccharides that affect the processing and final quality of plant derived food products. Assays have been developed for oligo- and poly-saccharides such as fructo-oligosaccharides, raffinose, β-glucan, starch, starch damage, resistant starch and fructan, to name just a few. To simplify the measurement of enzymes, a range of dyed polysaccharides were made and these are supplied either as soluble dyed substrates or as insoluble, cross-linked dyed polysaccharides (which were disintegrated into gel particles, dried and incorporated into tablet form).

In 2002, we decided to develop genetic engineering capabilities within Megazyme to allow the production of specific, high purity enzymes via molecular biology. This development was essential to allow the further development of analytical kits within Megazyme. The initial aim was to evaluate the test kits for the wine and food industries developed by Boehringer-Mannheim about 20-30 years ago, and to determine if there was room for improvement in view of the dramatic developments in biochemistry over the past two decades. More specifically, we decided to look at ways of improving assay specificity, sensitivity and reagent stability and overcoming inhibition problems. A second objective was to identify areas where analytical techniques were not available and to develop appropriate methodology.

Background

In the initial overview, we considered that a need existed for improved analytical methodology for a range of polysaccharides and oligosaccharides, leading to the development of methods for total starch, damaged starch, gelatinized starch, resistant starch, amylose/amylopectin content of starch, barley and oat b-glucan, yeast b-glucan, galactomannan, glucomannan, arabinan, fructan, fructo-oligosaccharides and galactosyl-sucrose oligosaccharides. To date, we have been unable to develop a suitable method for the measurement of arabinoxylan from wheat and rye flour. Procedures for the measurement of key enzymes associated with cereal quality and/or involved in the processing of cereals and plant products are summarized in Table 1. Several of these methods for the measurement of carbohydrates and enzymes are industry standards, and

Table 1. Assay procedures for enzymes of importance in cereal quality and processing.

Substrate

Enzyme

Substrate/Assay developed

Starch

α-amylase

Amylazyme/Red Starch

   

Ceralpha/β-Limit dextrin

 

β-amylase

Betamyl reagent

 

Pullulanase/limit dextrinase

Limit-dextrizyme tablets

   

Red Pullulan

 

amyloglucosidase

AMG Assay Reagent

     

β-Glucan (barley & oat)

endo-1,3:1,4-β-D-glucanase

Pure barley β-glucan

   

Azo-Barley Glucan

   

Beta-Glucazyme Tablets

 

endo-1,4-β-D-glucanase

Cellazyme C Tablets

   

Cellazyme T Tablets

   

Azo-CM-Cellulose

     

Arabinoxylan

endo-1,4-β-D-xylanase

Pure arabinoxylan

   

Xylazyme AX tablets

   

Azo-xylan (birchwood)

     

Galactomannan

endo-1,4-β-D-mannanase

Beta-Mannazyme Tablets

Glucomannan

endo-1,4-β-D-mannanase

Azo-Carob Galactomannan

     

Arabinan

endo-1,5-α-L-arabinanase

Arabinazyme Tablets

Galactan

endo-1,4-β-D-galactanase

Galactazyme Tablets

Rhamnogalacturonan

Rhamnogalacturonan hydrolase and RG-lyase

Rhamnozyme Tablets

some of the methods have been subjected to interlaboratory evaluation under the auspices of AOAC International, AACC International, RACI, EBC, ASBC and ICC. The current status of acceptance of methods in USA is shown in Table 2.

Table 2. Acceptance of Megazyme developed methods in USA.

Analyte

AOAC International

AACC International

β-Glucan (barley and oat)

Official Method 995.16

Method 32-23

Total starch

Official Method 996.11

Method 76-13

Resistant starch

Official Method 2002.02

Method 32-40

Damaged starch

-

Method 76-31

Fructan

Official Method 999.03

Method 32-32

α-Amylase (Ceralpha)

Official Method 2002.01

Method 22.02

α-Amylase (Amylazyme)

-

Method 22-05

Molecular biology; the way forward

Establishment of genetic engineering and molecular biology capabilities within Megazyme represented a major investment in terms of new personnel and laboratory/equipment facilities. To justify this investment, we identified various areas where these capabilities could be exploited. These included production of enzymes for expansion of the current range of test kits into the wine and food additives areas and for the development of specific antigens in the development of kits for the measurement of allergenic proteins. In terms of test kits, the obvious target was the range of food and beverage test kite developed by Boehringer-Mannheim approximately 20-30 years ago. We considered that with all of the recent developments in biochemistry and specifically, molecular biology, there should be scope for updating this analytical technology. The specific targets were enzyme specificity, stability; reaction rates; inhibition problems; assay format and the degree of robustness of the total of the kit components. Developments in this area to date are summarized in Table 3.

Table 3. Improved test kits developed by Megazyme using enzymes derived via molecular biology; with stated improvements.

Analyte

Improvements in kit technology

Acetaldehyde

Reagents stable for > 2 years after preparation

Acetic acid

Reagents stable for > 2 years after preparation

New assay format also available based on Acetate Kinase

Ammonia

Tablet format for increased stability

Ammonia rapid

Very rapid. GlDH not inhibited by wine tannins

L-Arginine/Urea/Ammonia

Novel rapid method for these analytes in wine

L-Ascorbic acid

Very rapid. Reagents stable after preparation

Aspartame

Novel Method. Simple format. Rapid

Ethanol

Reagents stable for > 2 years after preparation

D-Fructose/D-Glucose

Rapid reaction. Mega-CalcTM calculator available

D-Fructose/D-Glucose MegaQuantTM Format

Simple procedure in which the reagents are provided as tablets and a colorimeter is used

Galactomannan

Novel procedure. Rapid determination of D-galactose

Glucomannan

Novel enzymic procedure. Rapid and specific

D-Gluconic acid

Very rapid reaction

D-Glucose (HK format)

Very rapid reaction. Mega-CalcTM calculator available

L-Glutamic acid

Rapid reaction. Improved stability of reagents

Glycemic carbohydrates and dietary fibre

Novel procedure. Improved methodology for dietary fibre determination. Integrated format for GCH

Glycerol

Tablet format for improved stability. Rapid reaction

D-3-Hydroxybutyric acid

Very rapid reaction. Greater stability of prepared reagents

D-Isocitric acid

Very rapid reaction. Greater stability of prepared reagents

D-Lactic acid

Very rapid reaction. Greater stability of prepared reagents

D-/L-Lactic acid

Very rapid reaction. Greater stability of prepared reagents

L-Lactic acid

Very rapid reaction. Greater stability of prepared reagents

Table 3. Improved test kits developed by Megazyme using enzymes derived via molecular biology; with stated improvements (continued).

Analyte

Improvements in kit technology

Lactose/D-Galactose

Extremely rapid reaction (Patent pending)

D-Malic acid n

Very rapid reaction. Greater stability of prepared reagents

L-Malic acid

Very rapid reaction. Greater stability of prepared reagents

L-Malic acid MegaQuantTM Format

Simple procedure in which the reagents are provided as tablets and a colorimeter is used

D-Mannitol/L-Arabitol

Novel kit. Rapid reaction

D-Mannose/D-Fructose/

D-Glucose

Novel kit. Very stable reagents

Primary Amino Nitrogen

Novel kit. Very stable reagents

Raffinose/D-Galactose

Very rapid reaction. Very stable reagents

D-Sorbitol/Xylitol

Rapid reaction. Greater stability of prepared reagents

Succinic acid

Very rapid reaction. Greater stability of prepared reagents

Sucrose/D-Fructose/

D-Glucose

Rapid reaction. Greater stability of prepared reagents

Trehalose/D-Glucose

Novel kit. Very rapid reaction

Urea/Ammonia Rapid

Very rapid. GlDH not inhibited by wine tannins

Methods under development

Methods are currently under development for a range of other analytes including sulphite, carbon dioxide, succinic acid and citric acid, and genes for enzymes required for the development of several other methods are currently being cloned. The next stage in kit development at Megazyme will involve the production of specific antigens (peptide fractions) for use in antibody based tests.

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