A NaBH4 Coupled Ninhydrin-Based Assay for the Quantification of Protein/Enzymes During the Enzymatic Hydrolysis of Pretreated Lignocellulosic Biomass

Abstract

Accurate protein quantification is necessary in many of the steps during the enzymatic hydrolysis of pretreated lignocellulosic biomass, from the fundamental determination of enzyme kinetics to techno-economic assessments, such as the use of enzyme recycling strategies, evaluation of enzyme costs, and the optimization of various process steps. In the work described here, a modified, more rapid ninhydrin-based protein quantification assay was developed to better quantify enzyme levels in the presence of lignocellulosic biomass derived compounds. The addition of sodium borohydride followed by acid hydrolysis at 130 °C greatly reduced interference from monosaccharides and oligosaccharides and decreased the assay time 6-fold. The modified ninhydrin assay was shown to be more accurate as compared to various traditional colorimetric protein assays when commercial cellulase enzyme mixtures were quantified under typical pretreated lignocellulosic biomass enzymatic hydrolysis conditions. The relatively short assay time and microplate-reading capability of the modified assay indicated that the method could likely be used for high-throughput protein determination.

Limitation of cellulose accessibility and unproductive binding of cellulases by pretreated sugarcane bagasse lignin

“…Protein concentration was determined by a ninhydrin-based method [53]. Filter paper activity of cellulase preparations was measured according to Ghose [54].…”

“…The tubes were incubated at 50 °C in, 120 rpm, to simulate the hydrolysis conditions. After 3 h, the mixture was centrifuged at 10,000 rpm for 10 min and the protein concentration in the supernatant was measured by the ninhydrin assay [53]. The amount of protein adsorbed to the lignin was the difference between the protein concentration of the added cellulase solution and the supernatant after incubation.…”

Developing fast enzyme recycling strategy through elucidating enzyme adsorption kinetics on alkali and acid pretreated corn stover

“…To measure adsorption kinetics, the protein content in the supernatant of the enzymatic hydrolysate was determined along the time course of cellulose hydrolysis. The enzyme protein concentration of the supernatant of each sample was measured by a modified Ninhydrin method [ 32 ], with bovine serum albumin (BSA) as the standard (0–800 μg/mL). In detail, 200 μL diluted sample was mixed with 100 μL 13.3 g/L NaBH 4 in a pressure resistant glass tube and incubate at room temperature for 1 h, followed by adding 600 μL 9 M HCl and shaking the tube to remove bubbles.…”

Enhancing Enzyme-Mediated Cellulose Hydrolysis by Incorporating Acid Groups Onto the Lignin During Biomass Pretreatment

“…The tubes were incubated in a rotating incubator at 50 • C and the released sugars measured using a glucose analyzer (YSI 2700 Select Biochemistry Analyzer) after 6, 24, and 48 h of hydrolysis. Prior to enzymatic hydrolysis, the protein content of the Cellic CTec 3 preparation was measured using the ninhydrin essay, according to Mok et al (2015).…”

“…Vials containing 1% (w/v) lignin and enzymes (0.5 mg/mL) were incubated for 3 h, followed by centrifugation and the supernatant collected. The protein content of the supernatants was measured by the ninhydrin method according to Mok et al (2015), using BSA as the protein standard. The amount of enzyme adsorbed onto the lignin was determined as the difference between the initial enzyme loading and the free enzyme present in the supernatant.…”