A set of simple methods for detection and extraction of laminarinase

Ananthamurthy Koteshwara,Subrahmanyam Volety Mallikarjuna,Nancy V Philip,Raghu Chandrashekhar Hariharapura,Jesil Mathew Aranjani

doi:10.1038/s41598-021-81807-2

Ananthamurthy Koteshwara, Subrahmanyam Volety Mallikarjuna + Show 3 more

Open Access

https://doi.org/10.1038/s41598-021-81807-2

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Journal: Scientific Reports	Publication Date: Jan 28, 2021
Citations: 7	License type: open-access

Affiliation: Manipal Academy of Higher Education

Abstract

A carefully designed ammonium sulfate precipitation will simplify extraction of proteins and is considered to be a gold standard among various precipitation methods. Therefore, optimization of ammonium sulfate precipitation can be an important functional step in protein purification. The presence of high amounts of ammonium sulphate precludes direct detection of many enzymatically active proteins including reducing sugar assays (e.g. Nelson-Somogyi, Reissig and 3,5-dinitrosalicylic acid methods) for assessing carbohydrases (e.g. laminarinase (β (1–3)-glucanohydrolase), cellulases and chitinases). In this study, a simple method was developed using laminarin infused agarose plate for the direct analysis of the ammonium sulphate precipitates from Streptomyces rimosus AFM-1. The developed method is simple and convenient that can give accurate results even in presence of ammonium sulfate in the crude precipitates. Laminarin is a translucent substrate requiring the use of a stain to visualize the zones of hydrolysis in a plate assay. A very low-cost and locally available fluorescent optical fabric brightener Tinopal CBS-X has been used as a stain to detect the zones of hydrolysis. We also report simple methods to prepare colloidal chitin and cell free supernatant in this manuscript.

Highlights

A carefully designed ammonium sulfate precipitation will simplify extraction of proteins and is considered to be a gold standard among various precipitation methods
A type of β-glucan, is the longterm carbon storage polysaccharide found in marine brown macroalgae and consists of β-(1–3) linked glucose molecules interspersed with β-(1–6) intrachain linkages, and 6-O-branching[1]
3,5-dinitrosalicylic acid (DNS), powdered shrimp chitin, yeast extract powder, malt extract powder and other ingredients used in the preparation of colloidal chitin minimal salts (CCMS) broth were purchased from HiMedia laboratories Pvt

Summary

Introduction

A carefully designed ammonium sulfate precipitation will simplify extraction of proteins and is considered to be a gold standard among various precipitation methods. Optimization of ammonium sulfate precipitation can be an important functional step in protein purification. The presence of high amounts of ammonium sulphate precludes direct detection of many enzymatically active proteins including reducing sugar assays (e.g. Nelson-Somogyi, Reissig and 3,5-dinitrosalicylic acid methods) for assessing carbohydrases (e.g. laminarinase (β (1–3)-glucanohydrolase), cellulases and chitinases). A simple method was developed using laminarin infused agarose plate for the direct analysis of the ammonium sulphate precipitates from Streptomyces rimosus AFM-1. AS precipitation is often used as a preliminary step in majority of protein purification schemes. A large bulk of literature describe AS precipitation as the preliminary step in protein purification. Based on a survey of the literature, the strategies for the extraction and purification of laminarinase can broadly be classified into the following categories:

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